Signal transducer and activator of transcription 3 (STAT3) loss-of-function mutations in humans result in Job’s syndrome, characterized by elevated IgE, bacterial infections, chronic mucocutaneous candidiasis, pulmonary aspergillosis due to structural lung disease, and connective tissue abnormalities. Whole exome sequencing (WES) was performed on a putatively immunocompetent patient with sino-orbital aspergillosis and his parents. Evaluation of STAT3 phosphorylation (pSTAT3) was performed by flow cytometry on peripheral blood mononuclear cells after IL-6 stimulation. A 37-year-old-male with a history of eosinophilic esophagitis developed a locally invasive sino-orbital infection with progressive cavernous sinus involvement associated with facial numbness, diplopia, and visual loss. Biopsy showed chronic inflammation and invasive fungal hyphae. Cultures grew an isolate of Aspergillus fumisynnematus that was identified by morphology and the sequences of β-tubulin and Mcm7. MICs (in µg/ml) for the isolate were: micafungin <=0.015, amphotericin 2, posaconazole 0.25, terbinafine 0.06, and voriconazole 0.25. The patient underwent multiple surgical debridements and was treated over time with various antifungals (amphotericin B, micafungin, terbinafine, voriconazole, posaconazole), adjunct cytokines (IFN-γ, GM-CSF), and hyperbaric oxygen. However, the infection progressed into the right middle cranial fossa and meninges and the patient died 1 year after symptoms began. WES revealed a de novo splice-site mutation in STAT3 (c.1140-3C>G). cDNA sequencing showed nonsense mediated decay of the affected allele. No mutations in CARD9 or NADPH oxidase subunits were found; a DHR test was normal. The patient had normal blood myeloid cell subsets. Serum IgE level was elevated at 833 IU/ml. After stimulation with IL-6, the patient’s memory CD4+ T cells and CD11c+ myeloid cells had reduced pSTAT3 levels compared with control cells. Cellular analysis of SOCS3, a STAT3-dependent downstream target, is underway to evaluate for functional STAT3 haploinsufficiency. A novel de novo STAT3 splice site mutation results in impaired pSTAT3, and is associated with elevated IgE, eosinophilic esophagitis, and sino-orbital aspergillosis without other common features of Job’s syndrome All authors: No reported disclosures.
Recently, there has been tremendous interest in the sinus microbiome and how it relates to disease. However, a lack of a standardized sample collection and DNA extraction methods makes comparison of results across studies nearly impossible. Furthermore, current techniques fail to identify which components of the microbiome are actually alive within the host at the time of sampling.To develop and optimize a method to differentiate which bacterial species in the human sinus microbiome are live versus dead.Duplicate samples from the middle meatus of patients with healthy sinus tissue and those patients with chronic rhinosinusitis were collected by using brushes (n = 12), swabs (n = 27), and tissue biopsy (n = 8) methods. One sample from each pair was either deoxyribonuclease I- or control-treated before DNA extraction. The relative bacterial versus human composition of each sample was determined. A 16S ribosomal RNA gene analysis was performed on a six-paired sample from patients with healthy sinus tissue.We found that swabs and brushes collected a higher percentage of bacterial DNA than did tissue biopsy. We also determined that as much as 50% of the bacteria collected in these samples was already dead at the time of collection. The 16S ribosomal RNA gene analysis found significant changes in the relative abundance of taxa identified in the live versus dead bacterial communities of healthy human sinuses.Our findings indicated that swabs provided the best quality microbiome samples and that a large portion of the bacteria identified in the sinus were deoxyribonuclease I sensitive. These results highlighted the need for improved techniques such as those presented here, which can differentiate between living and dead bacteria in a sample, a potentially critical distinction when examining changes in sinus innate immune function because both components play important, but distinct, functions. Further studies will determine how these living and dead bacterial populations shift in different disease states and after clinical intervention.
Abstract The ongoing SARS-CoV-2 pandemic and subsequent demand for viral testing has led to issues in scaling diagnostic lab efforts and in securing basic supplies for collection and processing of samples. This has motivated efforts by the scientific community to establish improved protocols that are more scalable, less resource intensive, and less expensive. One such developmental effort has resulted in an assay called “Swab-Seq”, so named because it was originally developed to work with dry nasal swab samples. The existing gold standard test consists of RNA extracted from a nasopharyngeal (NP) swab that is subjected to quantitative reverse transcription polymerase chain reaction (qRT-PCR). Swab-Seq adapts this method to a next-generation sequencing readout. By pairing this modification with extraction-free sampling techniques, Swab-Seq achieves high scalability, low cost per sample, and a reasonable turnaround time. We evaluated the effectiveness of this assay in a community surveillance setting by testing samples collected from both symptomatic and asymptomatic individuals using the traditional NP swab. In addition, we evaluated extraction-free sampling techniques (both saliva and saline mouth gargle samples). We found the assay to be as clinically sensitive as the qRT-PCR assay, adaptable to multiple sample types, and able to easily accommodate hundreds of samples at a time. We thus provide independent validation of Swab-Seq and extend its utility regarding sample type and sample stability. Assays of this type greatly expand the possibility of routine, noninvasive, repeated testing of asymptomatic individuals suitable for current and potential future needs.
