Regulation of nucleo-cytosolic 26S proteasome translocation by aromatic amino acids via mTOR is essential for cell survival under stress.

The proteasome is responsible for removal of ubiquitinated proteins. Although several aspects of its regulation (e.g., assembly, composition, and post-translational modifications) have been unraveled, studying its adaptive compartmentalization in response to stress is just starting to emerge. We found that following amino acid starvation, the proteasome is translocated from its large nuclear pool to the cytoplasm-a response regulated by newly identified mTOR-agonistic amino acids-Tyr, Trp, and Phe (YWF). YWF relay their signal upstream of mTOR through Sestrin3 by disrupting its interaction with the GATOR2 complex. The triad activates mTOR toward its downstream substrates p62 and transcription factor EB (TFEB), affecting both proteasomal and autophagic activities. Proteasome translocation stimulates cytosolic proteolysis which replenishes amino acids, thus enabling cell survival. In contrast, nuclear sequestration of the proteasome following mTOR activation by YWF inhibits this proteolytic adaptive mechanism, leading to cell death, which establishes this newly identified pathway as a key stress-coping mechanism.

Eosinophilic Pleocytosis in the Cerebrospinal Fluid following CAR-T Cell Therapy for Central Nervous System Lymphoma: A Case for Warning?

INTRODUCTION: Chimeric antigen receptor T (CAR-T) cell therapy, emerging as an efficient treatment option for patients with secondary central nervous system (CNS) lymphoma, is frequently complicated with immune effector cell-associated neurotoxicity syndrome (ICANS). CASE PRESENTATION: We report a case of a 64-year-old woman with transformed follicular lymphoma, developing high-grade ICANS with eosinophilic pleocytosis following third-line therapy with CAR-T cells (tisagenlecleucel). During bridging therapy, she declined neurologically and was diagnosed with secondary CNS lymphoma. She received methotrexate-cytarabine-thiotepa-rituximab regimen with clinical and radiological improvement. Post-CAR-T cell infusion she developed cytokine release syndrome grade II and ICANS grade III. Given the lack of response to steroids, anakinra was initiated with complete ICANS resolution. Cerebrospinal fluid (CSF) analysis, performed only on day +10 due to thrombocytopenia, revealed eosinophils, while infections were excluded. CONCLUSION: This report emphasizes the importance of CSF analysis in individuals with CAR-T-related neurotoxicity for elucidating the role of specific immune cells in such complications.

Immunophenotyping and viral studies in pityriasis lichenoides et varioliformis acuta lesions.

BACKGROUND: The underlying pathogenesis of pityriasis lichenoides et varioliformis acuta (PLEVA) remains unclear, although immunologic injury and viral etiology have been suggested. OBJECTIVE: To evaluate and expand the immunophenotype of PLEVA and to search for possible viral pathogens. METHODS: Formalin-fixed, paraffin-embedded specimens of 20 patients with PLEVA and 9 patients with common inflammatory dermatoses (ID) were studied for immunophenotyping and for human herpesvirus (HHV) 1 and 2, cytomegalovirus (CMV), HHV-8, parvovirus B19, and Epstein-Barr virus (EBV) immunohistochemistry. The presence of HHV-6, HHV-7, and enteroviruses was assayed molecularly. RESULTS: The numbers of CD8+ T cells and T-cell intracellular antigen-1 (TIA-1)+ cells were statistically significantly higher in PLEVA compared to the ID group. Immunohistochemistry for human HHV-1 and HHV-2, CMV and HHV-8, parvovirus B19, and in situ hybridization for EBV were all negative. There was molecular evidence for HHV-7 in only one PLEVA case (5%). Molecular studies for HHV-6 and enterovirus involvement were negative in all the PLEVA specimens. CONCLUSIONS: The predominant T-cell infiltrate in PLEVA is dominated by CD8+ cells, and by increased numbers of TIA1+ cells, which may indicate a cytotoxic T-cell damage to the epidermis. Viral presence was not detected.

Ser194Leu DSG2 mutation, associated with arrhythmogenic left ventricular cardiomyopathy and ventricular tachycardia.

INTRODUCTION: Arrhythmogenic cardiomyopathy (AC) is an inherited cardiomyopathy characterized by fibro-fatty replacement of cardiomyocytes, leading to life-threatening ventricular arrhythmia and heart failure. Pathogenic variants of desmoglein2 gene (DSG2) have been reported as genetic etiologies of AC. In contrast, many reported DSG2 variants are benign or variants of uncertain significance. Correct genetic variant classification is crucial for determining the best medical therapy for the patient and family members. METHODS: Pathogenicity of the DSG2 Ser194Leu variant that was identified by whole exome sequencing in a patient, who presented with ventricular tachycardia and was diagnosed with AC, was investigated by electron microscopy and immunohistochemical staining of endomyocardial biopsy sample. RESULTS: Electron microscopy demonstrated a widened gap in the adhering junction and a less well-organized intercalated disk region in the mutated cardiomyocytes compared to the control. Immunohistochemical staining in the proband diagnosed with AC showed reduced expression of desmoglein 2 and connexin 43 and intercalated disc distortion. Reduced expression of DSG2 and Connexin 43 were observed in cellular cytoplasm and gap junctions. Additionally, we detected perinuclear accumulation of DSG2 and Connexin 43 in the proband sample. CONCLUSION: Ser194Leu is a missense pathogenic mutation of DSG2 gene associated with arrhythmogenic left ventricular cardiomyopathy.

Heparanase Increases Podocyte Survival and Autophagic Flux after Adriamycin-Induced Injury.

The kidney glomerular filtration barrier (GFB) is enriched with heparan sulfate (HS) proteoglycans, which contribute to its permselectivity. The endoglycosidase heparanase cleaves HS and hence appears to be involved in the pathogenesis of kidney injury and glomerulonephritis. We have recently reported, nonetheless, that heparanase overexpression preserved glomerular structure and kidney function in an experimental model of Adriamycin-induced nephropathy. To elucidate mechanisms underlying heparanase function in podocytes-key GFB cells, we utilized a human podocyte cell line and transgenic mice overexpressing heparanase. Notably, podocytes overexpressing heparanase (H) demonstrated significantly higher survival rates and viability after exposure to Adriamycin or hydrogen peroxide, compared with mock-infected (V) podocytes. Immunofluorescence staining of kidney cryo-sections and cultured H and V podocytes as well as immunoblotting of proteins extracted from cultured cells, revealed that exposure to toxic injury resulted in a significant increase in autophagic flux in H podocytes, which was reversed by the heparanase inhibitor, Roneparstat (SST0001). Heparanase overexpression was also associated with substantial transcriptional upregulation of autophagy genes BCN1, ATG5, and ATG12, following Adriamycin treatment. Moreover, cleaved caspase-3 was attenuated in H podocytes exposed to Adriamycin, indicating lower apoptotic cell death in H vs. V podocytes. Collectively, these findings suggest that in podocytes, elevated levels of heparanase promote cytoprotection.

Pure Red Cell Aplasia following ABO-Mismatched Allogeneic Hematopoietic Stem Cell Transplantation: Resolution with Daratumumab Treatment.

Pure red cell aplasia (PRCA) can potentially occur after allogeneic hematopoietic stem cell transplantation (allo-HSCT) if recipient and donor ABO blood groups are mismatched, with the recipient having isoagglutinins against the donor blood group. Patient plasma cells that survive transplant conditioning produce anti-ABO isoagglutinins targeting donor erythroid precursors in the bone marrow and thus causing red cell aplasia. Therapeutic options include steroids, discontinuation of immunosuppression, plasmapheresis, donor lymphocyte infusion, rituximab, and bortezomib, all with limited benefit. Daratumumab utilized in the treatment of multiple myeloma is an anti-CD38 monoclonal antibody targeting plasma cells, which makes it a potentially efficient therapy for PRCA. The current case report presents a patient with post-allo-HSCT PRCA cured with daratumumab applied after failure of other therapies. Our findings demonstrate safety and high efficiency of daratumumab, suggesting its applicability as early treatment of post-allo-HSCT PRCA.

A Unique Presentation of Infantile-Onset Colitis and Eosinophilic Disease without Recurrent Infections Resulting from a Novel Homozygous CARMIL2 Variant.

PURPOSE: This study aimed to characterize the clinical phenotype, genetic basis, and consequent immunological phenotype of a boy with severe infantile-onset colitis and eosinophilic gastrointestinal disease, and no evidence of recurrent or severe infections. METHODS: Trio whole-exome sequencing (WES) was utilized for pathogenic variant discovery. Western blot (WB) and immunohistochemical (IHC) staining were used for protein expression analyses. Immunological workup included in vitro T cell studies, flow cytometry, and CyTOF analysis. RESULTS: WES revealed a homozygous variant in the capping protein regulator and myosin 1 linker 2 (CARMIL2) gene: c.1590C>A; p.Asn530Lys which co-segregated with the disease in the nuclear family. WB and IHC analyses demonstrated reduced protein levels in patient's cells compared with controls. Moreover, comprehensive immunological workup revealed severely diminished blood-borne regulatory T cell (Treg) frequency and impaired in vitro CD4+ T cell proliferation and Treg generation. CyTOF analysis showed significant shifts in the patient's innate and adaptive immune cells compared with healthy controls and ulcerative colitis patients. CONCLUSIONS: Pathogenic variants in CARMIL2 have been implicated in an immunodeficiency syndrome characterized by recurrent infections, occasionally with concurrent chronic diarrhea. We show that CARMIL2-immunodeficiency is associated with significant alterations in the landscape of immune populations in a patient with prominent gastrointestinal disease. This case provides evidence that CARMIL2 should be a candidate gene when diagnosing children with very early onset inflammatory and eosinophilic gastrointestinal disorders, even when signs of immunodeficiency are not observed.

Eculizumab Is Safe and Effective as a Long-term Treatment for Protein-losing Enteropathy Due to CD55 Deficiency.

OBJECTIVES: Loss of the complement inhibitor CD55 leads to a syndrome of early-onset protein-losing enteropathy (PLE), associated with intestinal lymphangiectasia and susceptibility to large-vein thrombosis. The in vitro and short-term treatment benefits of eculizumab (C5-inhibitor) therapy for CD55-deficiency have been previously demonstrated. Here we present the 18-months treatment outcomes for 3 CD55-deficiency patients with sustained therapeutic response. METHODS: Three CD55-deficiency patients received off-label eculizumab treatment. Clinical and laboratory treatment outcomes included frequency and consistency of bowl movements, weight, patient/parent reports of overall well-being, and serum albumin and total protein levels. Membrane attack complex deposition on leukocytes was tested by flow cytometry, before and during eculizumab treatment. RESULTS: Marked clinical improvement was noted in all 3 patients with resolution of PLE manifestations, that is, diarrhea, edema, malabsorption, overall well-being, growth, and quality of life. In correlation with the clinical observations, we observed progress in all laboratory outcome parameters, including increase in albumin and total protein levels, and up to 80% reduction in membrane attack complex deposition on leukocytes (P < 0.001). The progress persisted over 18 months of treatment without any severe adverse events. CONCLUSIONS: CD55-deficiency patients present with early-onset diarrhea, edema, severe hypoalbuminemia, abdominal pain, and malnutrition. Targeted therapy with the terminal complement inhibitor eculizumab has positive clinical and laboratory outcomes in PLE related to CD55 loss-of-function mutations, previously a life-threatening condition. Our results demonstrate the potential of genetic diagnosis to guide tailored treatment, and underscore the significant role of the complement system in the intestine.

Establishing the role of PLVAP in protein-losing enteropathy: a homozygous missense variant leads to an attenuated phenotype.

BACKGROUND: Intestinal integrity is essential for proper nutrient absorption and tissue homeostasis, with damage leading to enteric protein loss, that is, protein-losing enteropathy (PLE). Recently, homozygous nonsense variants in the plasmalemma vesicle-associated protein gene (PLVAP) were reported in two patients with severe congenital PLE. PLVAP is the building block of endothelial cell (EC) fenestral diaphragms; its importance in barrier function is supported by mouse models of Plvap deficiency. OBJECTIVE: To genetically diagnose two first-degree cousins once removed, who presented with PLE at ages 22 and 2.5 years. METHODS: Family-based whole exome sequencing was performed based on an autosomal recessive inheritance model. In silico analyses were used to predict variant impact on protein structure and function. RESULTS: We identified a rare homozygous variant (NM_031310.2:c.101T>C;p.Leu34Pro) in PLVAP, which co-segregated with the disease. Leu34 is predicted to be located in a highly conserved, hydrophobic, α-helical region within the protein's transmembrane domain, suggesting Leu34Pro is likely to disrupt protein function and/or structure. Electron microscopy and PLVAP immunohistochemistry demonstrated apparently normal diaphragm morphology, predicted to be functionally affected. CONCLUSIONS: Biallelic missense variants in PLVAP can cause an attenuated form of the PLE and hypertriglyceridaemia syndrome. Our findings support the role of PLVAP in the pathophysiology of PLE, expand the phenotypic and mutation spectrums and underscore PLVAP's importance in EC barrier function in the gut.

Evaluation of Microscopic Changes in Fallopian Tubes of BRCA Mutation Carriers by Morphometric Analysis of Histologic Slides: A Preliminary Pilot Study.

Mutations in BRCA genes increase the risk of ovarian cancer, yet no method for early diagnosis is available. Some serous ovarian tumors are hypothesized to stem from cells of the fallopian tube fimbria. Using a novel method of computerized morphometry of the fimbrial epithelium, this study aimed to detect morphologic differences in noncancerous fimbriae between BRCA mutation carriers and noncarriers, and between healthy and serous ovarian cancer patients. Twenty-four fimbriae from healthy women (13 BRCA+, 11 BRCA-) and 21 fimbriae from women with serous ovarian cancer (10 BRCA+, 11 BRCA-), all reported as "normal" by hematoxylin and eosin examination, were subjected to computerized histomorphometric analysis. A Fast Fourier Transformation was applied to images of fimbrial epithelium and the Fast Fourier Transformation 2-dimensional frequency maps were subsequently quantified for nuclear orientation and planar distribution by a cooccurrence matrix analysis. Additional analysis of nuclear contour was applied to the fimbriae of the healthy women. Among the healthy women, significant differences were found in morphometric characteristics between the BRCA mutation carriers and noncarriers. Among the women with ovarian cancer, no significant differences were found between BRCA mutation carriers and noncarriers. Between healthy women and those with ovarian cancer, significant differences were detected, regardless of BRCA mutational status. A novel method, which combined Fast Fourier Transformation with cooccurrence matrix analysis, demonstrated differences in morphometric characteristics in the fimbriae between healthy and ovarian cancer patients, and between BRCA mutation carriers and noncarriers. The clinical significance of these observations should be investigated.

Glycogenic hepatopathy.

BACKGROUND: Glycogenic hepatopathy (GH) is a disorder associated with uncontrolled diabetes mellitus, most commonly type 1, expressed as right upper quadrant abdominal pain, hepatomegaly and increased liver enzymes. The diagnosis may be difficult, because laboratory and imaging tests are not pathognomonic. Although GH may be suggested based on clinical presentation and imaging studies, the gold standard for diagnosis is a liver biopsy, showing a significant accumulation of glycogen within the hepatocytes. GH may be diagnosed also after elevated liver enzymes in routine blood tests. GH usually regresses after tight glycemic control. Progression to end-stage liver disease has never been reported. This review aims to increase the awareness to this disease, to suggest a pathway for investigation that may reduce the use of unnecessary tests, especially invasive ones. DATA SOURCES: A PubMed database search (up to July 1, 2017) was done with the words "glycogenic hepatopathy", "hepatic glycogenosis", "liver glycogenosis" and "diabetes mellitus-associated glycogen storage hepatopathy". Articles in which diabetes mellitus-associated liver glycogen accumulation was described were included in this review. RESULTS: A total of 47 articles were found, describing 126 patients with GH. Hepatocellular disturbance was more profound than cholestatic disturbance. No synthetic failure was reported. CONCLUSIONS: GH may be diagnosed conservatively, based on corroborating medical history, physical examination, laboratory tests, imaging studies and response to treatment, even without liver biopsy. In case of doubt about the diagnosis or lack of clinical response to treatment, a liver biopsy may be considered. There is no role for noninvasive tests like fibroscan or fibrotest for the diagnosis of GH or for differentiation of this situation from nonalcoholic fatty liver disease.

Intraoperative hybrid technique for excision of temporal bone paraganglioma: A case report.

BACKGROUND: Temporal bone paragangliomas are vascularized neoplasms. Although preoperative angioembolization serves as a valuable approach to reduce intraoperative blood loss, it comes with an elevated risk of cranial neuropathies, offers no assurance of complete hemostasis, and precludes real-time adjustments during surgery. METHODS: A 74-year-old patient presented with recurrent episodes of ear bleeding. On examination, a vascular lesion obstructed her external auditory canal. It had the clinical and radiological characteristics of a paraganglioma. Angiography revealed that it had three feeding vessels. RESULTS: The patient was successfully scheduled for hybrid, intraoperative angiography and temporary balloon occlusion of the feeding vessels supplying the lesion instead of preoperative angioembolization. CONCLUSIONS: Utilizing hybrid intraoperative angiography with temporary balloon occlusion during the surgical removal of temporal bone paragangliomas represents an innovative technique that reduces the risk of permanent cranial neuropathies while providing the capacity for real-time adjustments and improved hemostasis.

Algorithm-assisted diagnosis of Hirschsprung's disease - evaluation of robustness and comparative image analysis on data from various labs and slide scanners.

BACKGROUND: Differences in the preparation, staining and scanning of digital pathology slides create significant pre-analytic variability. Algorithm-assisted tools must be able to contend with this variability in order to be applicable in clinical practice. In a previous study, a decision support algorithm was developed to assist in the diagnosis of Hirschsprung's disease. In the current study, we tested the robustness of this algorithm while assessing for pre-analytic factors which may affect its performance. METHODS: The decision support algorithm was used on digital pathology slides obtained from four different medical centers (A-D) and scanned by three different scanner models (by Philips, Hamamatsu and 3DHISTECH). A total of 192 cases and 1782 slides were used in this study. RGB histograms were constructed to compare images from the various medical centers and scanner models and highlight the differences in color and contrast. RESULTS: The algorithm was able to correctly identify ganglion cells in 99.2% of cases, from all medical centers (All scanned by the Philips slide scanner) as well as 95.5% and 100% of the slides scanned by the 3DHISTECH and Hamamatsu brand slide scanners, respectively. The total error rate for center D was lower than the other medical centers (3.9% vs 7.1%, 10.8% and 6% for centers A-C, respectively), the vast majority of errors being false positives (3.45% vs 0.45% false negatives). The other medical centers showed a higher rate of false negatives in relation to false positives (6.81% vs 0.29%, 9.8% vs 1.2% and 5.37% vs 0.63% for centers A-C, respectively). The total error rates for the Philips, Hamamatsu and 3DHISTECH brand scanners were 3.9%, 3.2% and 9.8%, respectively. RGB histograms demonstrated significant differences in pixel value distribution between the four medical centers, as well as between the 3DHISTECH brand scanner when compared to the Philips and Hamamatsu brand scanners. CONCLUSIONS: The results reported in this paper suggest that the algorithm-based decision support system has sufficient robustness to be applicable for clinical practice. In addition, the novel method used in its development - Hierarchial-Contexual Analysis (HCA) may be applicable to the development of algorithm-assisted tools in other diseases, for which available datasets are limited. Validation of any given algorithm-assisted support system should nonetheless include data from as many medical centers and scanner models as possible.

PD1 ligand functionality a biomarker of response to anti PD1 treatment in patients with HNSCC.

Therapies targeting the PD-1/PD-L1 pathway have transformed head and neck squamous cell carcinoma (HNSCC) treatment. However, predicting the response to anti-PD-1 therapy remains a clinical challenge. This study evaluated the functional binding of PD-1 ligands in 29 HNSCC patients and compared it to the standard PD-L1 Combined Positive Score (CPS). The assessment of PD-1 ligands' functionality advances the current ability to predict the response of HNSCC patients to anti-PD-1 therapy.

A fusion protein encoding the second extracellular domain of CCR5 arrests chemokine-induced cosignaling and effectively suppresses ongoing experimental autoimmune encephalomyelitis.

CCR5 is a key CCR that is highly expressed on CD4(+) T cells. It binds three different ligands: CCL3 (MIP-alpha), CCL4 (MIP-beta), and CCL5 (RANTES). Recent studies suggested that the interaction between CCR5 and its ligands is essential not only for attracting these CCR5(+) T cells but also substantial for transuding cosignals for their activation. The current study explores, for the first time, the in vivo consequences of CCR5 as a costimulatory molecule. First, we show redundancy between CCR5 ligands not only in chemoattractive properties but also in their ability to induced cosignals via CCR5. This has motivated us to generate a soluble receptor-based fusion protein that would selectively bind and neutralize all three CCR5 ligands. We show in this study that a 30-aa-based CCR5-Ig fusion protein encoding the second extracellular domain of receptor selectively binds and neutralizes all three CCR5 ligands and, when administered during ongoing experimental autoimmune encephalomyelitis, rapidly suppressed the disease while arresting Ag-specific effector T cell functions. Finally, our results clearly show that although CCR5 ligands induced cosignaling for IL-2 production is directed by CCR5, other proinflammatory properties of these ligands, such as TNF-alpha, IL-17, and IFN-gamma production, are CCR5 independent and therefore likely to be mediated by the other receptors for these ligands. These findings imply that implementing a CCR5-Ig-based therapy would be advantageous over blockade of this receptor or of the use of mAbs for targeting a single CCR5 ligand.

A common presentation - turning out as an uncommon diagnosis: From hip pain to Langerhans cell histiocytosis.

Langerhans cell histiocytosis (LCH) is an uncommon clonal proliferation of myeloid progenitor cells, it is especially rare in adults. We present a case of multi-system LCH in a 53-year-old woman, the sole symptom of which was prolonged, non-resolving hip pain for 18 months prior to the diagnosis. Initial evaluation included imaging studies aimed at identifying a presumed local etiology. X-ray demonstrated non-specific arthritic changes on the left femur. Computed tomography (CT) and magnetic resonance imaging (MRI) scans identified a lytic lesion at the same location, warranting a systemic workup. After non-invasive investigations failed to reveal the underlying etiology, a biopsy was performed, revealing cores of Langerhans cells that stained positive for both CD1a and langerin. These findings verified the surprising, uncommon diagnosis of LCH. A comprehensive workup was conducted in order to determine the extent of the disease and its molecular nature - revealing a BRAFV600E-positive, high-risk, multi-system LCH with skeletal, lung and liver involvement.

Mechanism underlying painful radiculopathy in patients with lumbar disc herniation.

BACKGROUND: Painful lumbar radiculopathy is a neuropathic pain condition, commonly attributed to nerve root inflammation/compression by disc herniation. The present exploratory study searched for associations between pain intensity and inflammatory markers, herniated disc size, infection, psychological factors and pain modulation in patients with confirmed painful lumbar radiculopathy scheduled for spine surgery. METHODS: Prior to surgery, 53 patients underwent the following evaluation: pain intensity measured on a 0-10 numeric rating scale (NRS) and the Short-Form McGill Pain Questionnaire; sensory testing (modified DFNS protocol); pain processing including temporal summation and conditioned pain modulation (CPM); neurological examination; psychological assessment including Spielberger's Anxiety Inventory, Pain Sensitivity Questionnaire and the Pain Catastrophizing Scale. Pro-inflammatory cytokine levels (IL-1b, IL-6, IL-8, IL-17, TNFα, IFNg) and microbial infection (ELISA and rt-PCR) in blood and disc samples obtained during surgery. MRI scans assessments for disc herniation size/volume (MSU classification/ three-dimensional volumetric analysis). RESULTS: Complete data were available from 40 (75%) patients (15 female) aged 44.8 ± 16.3 years. Pain intensity (NRS) positively correlated with pain catastrophizing and CPM (r = 0.437, p = 0.006; r = 0.421, p = 0.007; respectively), but not with disc/blood cytokine levels, bacterial infection or MRI measures. CPM (p = 0.001) and gender (p = 0.029) were associated with average pain intensity (adjusted R2  = 0.443). CONCLUSIONS: This exploratory study suggests that pain catastrophizing, CPM and gender, seem to contribute to pain intensity in patients with painful lumbar radiculopathy. The role of mechanical compression and inflammation in determining the intensity of painful radiculopathy remains obscure. SIGNIFICANCE OF STUDY: Pain catastrophizing, CPM and gender rather than objective measures of inflammation and imaging seem to contribute to pain in patients with painful radiculopathy.

NGS in the clinical microbiology settings.

We hypothesized that targeted NGS sequencing might have an advantage over Sanger sequencing, especially in polymicrobial infections. The study included 55 specimens from 51 patients. We compared targeted NGS to Sanger sequencing in clinical samples submitted for Sanger sequencing. The overall concordance rate was 58% (32/55) for NGS vs. Sanger. NGS identified 9 polymicrobial and 2 monomicrobial infections among 19 Sanger-negative samples and 8 polymicrobial infections in 11 samples where a 16S gene was identified by gel electrophoresis, but could not be mapped to an identified pathogen by Sanger. We estimated that NGS could have contributed to patient management in 6/18 evaluated patients and thus has an advantage over Sanger sequencing in certain polymicrobial infections.