The next generation of ocular pathogen detection

Metagenomic next-generation sequencing is a powerful method for pathogen detection that combines advanced genome sequencing technology with cutting-edge bioinformatics to analyze microbial populations. Metagenomic next-generation sequencing has the potential to identify uncommon, unculturable, and even previously unidentified pathogens from a clinical isolate. Of particular interest to ophthalmology, this robust data extraction can occur from very small volume clinical samples. Here we discuss the opportunities and limitations of this technique and their current and future application to ophthalmic diagnostics

Retropharyngeal Abscess and Pott’s Disease Due to Tuberculosis: A Case Report

Introduction: Extrapulmonary mycobacterial infection can lead to vertebral spondylitis and osteomyelitis (Pott’s disease). Retropharyngeal abscess with concurrent spinal osteomyelitis is a rare presentation of tuberculosis in the US. Chart review on a patient was completed, and the relevant published literature was reviewed. Case Presentation: A previously healthy 34-year-old male originally from Sudan presented to an outside hospital with a 2-month history of neck pain, sore throat, odynophagia, fevers, and chills. MRI showed a retropharyngeal abscess and suspected cervical spine osteomyelitis. Acid-fast bacillus (AFB) smear was positive from a neck drain specimen, but sputum was negative. Chest imaging did not show findings consistent with pulmonary tuberculosis. He was treated with rifampin, isoniazid, pyrazinamide, and ethambutol (RIPE) along with moxifloxacin and linezolid due to concern for possible multidrug resistant tuberculosis and transferred to our center for further care. Culture isolated Mycobacterium tuberculosis. CT neck showed vertebral tuberculous osteomyelitis (Pott’s disease) of C1-C3 with a multiloculated retropharyngeal and prevertebral abscess (Figure 1). The patient was taken to the OR for posterior spinal fusion from occiput to C4 and transoral incision and drainage of the abscess. The post-operative course was uneventful, and moxifloxacin and linezolid were discontinued when Xpert MTB/RIF test revealed rifampin susceptibility. At follow-up the patient’s symptoms had resolved. Patient consent was obtained to utilize this case for educational purposes. Conclusions: This report presents the multidisciplinary treatment of this patient requiring infection control measures and antibiotic therapy by infectious disease, posterior spine fusion by orthopedic surgery, and retropharyngeal abscess drainage by otolaryngology

Abortive Lytic Reactivation of KSHV in CBF1/CSL Deficient Human B Cell Lines

Since Kaposi's sarcoma associated herpesvirus (KSHV) establishes a persistent infection in human B cells, B cells are a critical compartment for viral pathogenesis. RTA, the replication and transcription activator of KSHV, can either directly bind to DNA or use cellular DNA binding factors including CBF1/CSL as DNA adaptors. In addition, the viral factors LANA1 and vIRF4 are known to bind to CBF1/CSL and modulate RTA activity. To analyze the contribution of CBF1/CSL to reactivation in human B cells, we have successfully infected DG75 and DG75 CBF1/CSL knock-out cell lines with recombinant KSHV.219 and selected for viral maintenance by selective medium. Both lines maintained the virus irrespective of their CBF1/CSL status. Viral reactivation could be initiated in both B cell lines but viral genome replication was attenuated in CBF1/CSL deficient lines, which also failed to produce detectable levels of infectious virus. Induction of immediate early, early and late viral genes was impaired in CBF1/CSL deficient cells at multiple stages of the reactivation process but could be restored to wild-type levels by reintroduction of CBF1/CSL. To identify additional viral RTA target genes, which are directly controlled by CBF1/CSL, we analyzed promoters of a selected subset of viral genes. We show that the induction of the late viral genes ORF29a and ORF65 by RTA is strongly enhanced by CBF1/CSL. Orthologs of ORF29a in other herpesviruses are part of the terminase complex required for viral packaging. ORF65 encodes the small capsid protein essential for capsid shell assembly. Our study demonstrates for the first time that in human B cells viral replication can be initiated in the absence of CBF1/CSL but the reactivation process is severely attenuated at all stages and does not lead to virion production. Thus, CBF1/CSL acts as a global hub which is used by the virus to coordinate the lytic cascade

Human Herpesvirus 8 (HHV8) Sequentially Shapes the NK Cell Repertoire during the Course of Asymptomatic Infection and Kaposi Sarcoma

The contribution of innate immunity to immunosurveillance of the oncogenic Human Herpes Virus 8 (HHV8) has not been studied in depth. We investigated NK cell phenotype and function in 70 HHV8-infected subjects, either asymptomatic carriers or having developed Kaposi's sarcoma (KS). Our results revealed substantial alterations of the NK cell receptor repertoire in healthy HHV8 carriers, with reduced expression of NKp30, NKp46 and CD161 receptors. In addition, down-modulation of the activating NKG2D receptor, associated with impaired NK-cell lytic capacity, was observed in patients with active KS. Resolution of KS after treatment was accompanied with restoration of NKG2D levels and NK cell activity. HHV8-latently infected endothelial cells overexpressed ligands of several NK cell receptors, including NKG2D ligands. The strong expression of NKG2D ligands by tumor cells was confirmed in situ by immunohistochemical staining of KS biopsies. However, no tumor-infiltrating NK cells were detected, suggesting a defect in NK cell homing or survival in the KS microenvironment. Among the known KS-derived immunoregulatory factors, we identified prostaglandin E2 (PGE2) as a critical element responsible for the down-modulation of NKG2D expression on resting NK cells. Moreover, PGE2 prevented up-regulation of the NKG2D and NKp30 receptors on IL-15-activated NK cells, and inhibited the IL-15-induced proliferation and survival of NK cells. Altogether, our observations are consistent with distinct immunoevasion mechanisms that allow HHV8 to escape NK cell responses stepwise, first at early stages of infection to facilitate the maintenance of viral latency, and later to promote tumor cell growth through suppression of NKG2D-mediated functions. Importantly, our results provide additional support to the use of PGE2 inhibitors as an attractive approach to treat aggressive KS, as they could restore activation and survival of tumoricidal NK cells

Isoelectric focusing spectrotypes of anti-thyroglobulin autoantibodies in autoimmune thyroiditis: a PVG rat model

We have studied the isoelectric focusing (IEF) spectrotypes of autoantibodies against thyroglobulin in two rat models of Hashimoto's thyroiditis: (1) AUG strain rats immunized with autologous thyroglobulin in Freund's complete adjuvant; (2) PVG/c strain rats which have been thymectomized and sublethally irradiated. The IEF spectrotypes revealed differences between the two models. The anti-thyroglobulin response in immunized AUG rats is mainly oligoclonal or polyclonal, often with dominant clones in the spectrotype, similar to about 90% of Hashimoto's patients, whereas the response in the PVG/c rat is highly restricted. There were pronounced changes in spectrotype with time in the PVG/c, but not AUG, rats with considerable variation in the lifespan of individual clones. The maximum lifespan of an anti-self secreting B-cell clone in the PVG/c rat, determined by persistence of its clonotype, was at least 16 weeks

Analysis of the spectrotypes of autoantibodies against thyroglobulin in two rat models of autoimmune thyroiditis.

We have studied the isoelectric focusing (IEF) spectrotypes of autoantibodies against thyroglobulin in two rat models of Hashimoto's thyroiditis: (1) AUG strain rats immunized with autologous thyroglobulin in Freund's complete adjuvant; (2) PVG/c strain rats which have been thymectomized and sublethally irradiated. The IEF spectrotypes revealed differences between the two models. The anti-thyroglobulin response in immunized AUG rats is mainly oligoclonal or polyclonal, often with dominant clones in the spectrotype, similar to about 90% of Hashimoto's patients, whereas the response in the PVG/c rat is highly restricted. There were pronounced changes in spectrotype with time in the PVG/c, but not AUG, rats with considerable variation in the lifespan of individual clones. The maximum lifespan of an anti-self secreting B-cell clone in the PVG/c rat, determined by persistence of its clonotype, was at least 16 weeks