Genome-sequencing of ocular fluid in a patient with syphilitic uveitis: Strain identification and antimicrobial resistance detection

Hypertensive and HLA-B27 negative uveitis: Disease course and complications.

BackgroundThe aims of this study are to investigate the clinical characteristics of patients with anterior hypertensive uveitis and to compare the characteristics between patients in cytomegalovirus (CMV)-positive and CMV-negative groups in their aqueous humor samples.Immunocompetent patients (n = 42) with a history of chronic and/or recurrent hypertensive anterior uveitis underwent ophthalmic examination and serological tests. Among the 42 patients with hypertensive anterior uveitis, aqueous humor sampling was performed in 21, and they were analyzed for viral deoxyribonucleic acids using the polymerase chain reaction (PCR).ResultsThe average age of the 42 patients with hypertensive anterior uveitis was 57.6 years, and 29 (69.0 %) of the subjects were males. Of the patients, 22 (52.4 %) underwent glaucoma surgery, and the average corneal endothelial cell counts were 1908 cells/mm2. Among the 21 patients who underwent an aqueous sampling, 6 were positive for CMV-DNA, while 15 were negative. The frequency of glaucoma surgery was similar between groups (CMV positive vs. CMV negative, 66.0 vs. 66.0 %, P = 0.701). However, 66.7 % of the CMV-positive group underwent glaucoma tube shunt surgery, whereas 80 % of the CMV-negative group underwent trabeculectomy or received an ExPRESS glaucoma filtration device (Alcon, Fort Worth, TX) for glaucoma surgery (P = 0.095). The corneal endothelial cell counts were significantly lower in the CMV-positive group (CMV positive vs. CMV negative, 1245 ± 560 vs. 1981 ± 387 cells/mm2; P = 0.009).ConclusionsCMV was found to be an etiological factor in patients with hypertensive anterior uveitis in Korea. Special caution is needed for patients with CMV-induced hypertensive anterior uveitis, considering its adverse effect on the corneal endothelium.Electronic supplementary materialThe online version of this article (doi:10.1186/s12348-016-0100-5) contains supplementary material, which is available to authorized users.

The aim of this study was to report a novel case of hypertensive uveitis with intravitreal faricimab. This is a case report. A 69-year-old woman undergoing treatment of bilateral diabetic macular edema with intravitreal faricimab presented for routine review. Ophthalmic examination was performed including VA, intraocular pressure, gonioscopy, and slitlamp examination. Findings consistent with hypertensive uveitis prompted further infectious/inflammatory/infiltrative uveitis screen. The patient developed hypertensive uveitis in the left eye (four weeks after the third injection) with an intraocular pressure of 42 mmHg. Slitlamp examination revealed fine keratic precipitates and mild anterior uveitis. Anterior chamber angle was open on gonioscopy, and there was no vitritis or vasculitis. At the review a week later, the patient had developed hypertensive uveitis in the right eye (six weeks after the fourth injection) with intraocular pressure of 35 mmHg. Slitlamp examination revealed fine keratic precipitates, open angles, and mild vitritis. There was no vasculitis. At both presentations, the patient had preserved VA with no significant visual symptoms. The hypertensive uveitis resolved in both eyes with a course of steroid and antihypertensive eye drops. The uveitis screen was negative apart from elevated urine protein (negative beta-2 microglobulin), which could be explained by known diabetes and hypertension. Hypertensive uveitis is a potential adverse reaction to intravitreal faricimab. This case highlights the importance of monitoring intraocular pressure in patients undergoing treatment with faricimab and emphasizes the need for reporting other cases in the community.

The seroprevalence of Cytomegalovirus (CMV) ranges from 40 to 100 % being highest in less developed areas and is a significant cause of ocular disease both in immunocompromised and immunocompetent persons. CMV retinitis has long been recognized as a major cause of blindness in patients with human immunodeficiency virus infection and perinatally acquired infections. It is increasingly also being found as a potential cause of blindness in immunocompetent persons where it manifests mainly as a hypertensive anterior uveitis that has a good response to ganciclovir/valganciclovir but also a high relapse rate. The main reasons for visual loss in CMV anterior uveitis are glaucomatous optic neuropathy from refractory or repeated elevations in intraocular pressure and or corneal endothelial damage as the CMV infection also results in endothelial cell loss. Corticosteroids may aggravate the inflammation and should be avoided in hypertensive uveitis unless a viral infection can be excluded via aqueous sampling for viral nucleic acid and/or evidence of intraocular antibody production.

Resident Perspectives 56-5

Purpose. To review the clinical outcome of patients with hypertensive uveitis. Methods. Retrospective review of uveitis patients with elevated intraocular pressure (IOP) > 25 mmHg and >1-year follow-up. Data are uveitis type, etiology, viral (VU) and nonviral uveitis (NVU), IOP, and medical and/or surgical treatment. Results. In 61 patients, IOP values are first 32.9 mmHg (SD: 9.0), highest 36.6 mmHg (SD: 9.9), 3 months after the first episode 19.54 mmHg (SD: 9.16), and end of follow-up 15.5 mmHg (SD: 6.24). Patients with VU (n = 25) were older (50.6 y/35.7 y, p = 0.014) and had more unilateral disease (100%/72.22% p = 0.004) than those with NVU (n = 36). Thirty patients (49.2%) had an elevated IOP before topical corticosteroid treatment. Patients with viral uveitis might have higher first elevated IOP (36.0/27.5 mmHg, p = 0,008) and maximal IOP (40.28/34.06 mmHg, p = 0.0148) but this was not significant when limited to the measurements before the use of topical corticosteroids (p = 0.260 and 0.160). Glaucoma occurred in 15 patients (24.59%) and was suspected in 11 (18.03%) without difference in viral and nonviral groups (p = 0.774). Conclusion. Patients with VU were older and had more unilateral hypertensive uveitis. Glaucoma frequently complicates hypertensive uveitis. Half of the patients had an elevated IOP before topical corticosteroid treatment.

Antimicrobial resistance (AMR) is a significant and growing public health threat. Sequencing of bacterial isolates is becoming more common, and therefore automatic identification of resistant bacterial strains is of pivotal importance for efficient, wide-spread AMR detection. To support this approach, several AMR databases and gene identification algorithms have been recently developed. A key problem in AMR detection, however, is the need for computational approaches detecting potential novel AMR genes or variants, which are not included in the reference databases. Toward this direction, here we study the relation between AMR and relative solvent accessibility (RSA) of protein variants from an in silico perspective. We show how known AMR protein variants tend to correspond to exposed residues, while on the contrary their susceptible counterparts tend to be buried. Based on these findings, we develop RSA-AMR, a novel relative solvent accessibility-based AMR scoring system. This scoring system can be applied to any protein variant to estimate its propensity of altering the relative solvent accessibility, and potentially conferring (or hindering) AMR. We show how RSA-AMR score can be integrated with existing AMR detection algorithms to expand their range of applicability into detecting potential novel AMR variants, and provide a ten-fold increase in Specificity. The two main limitations of RSA-AMR score is that it is designed on single point changes, and a limited number of variants was available for model learning.

Concerns regarding antimicrobial resistance (AMR) have resulted in the World Health Organization (WHO) designating so-called global priority pathogens (GPPs). However, little discussion has focused on the diagnosis of GPPs. To enable the simultaneous identification of pathogens and AMR, we developed a modular real-time nucleic acid amplification test (MRT-NAAT). Sequence-specific primers for each modular unit for MRT-NAAT pathogen identification and AMR sets were designed. The composition of the reaction mixture and the real-time PCR program were unified irrespective of primer type so to give MRT-NAAT modularity. Standard strains and clinical isolates were used to evaluate the performance of MRT-NAAT by real-time PCR and melting curve analysis. Probit analysis for the MRT-NAAT pathogen identification set was used to assess the limit of detection (LoD). The MRT-NAAT pathogen identification set was made up of 15 modular units 109-199bp in product size and with a Tms of 75.5-87.5°C. The LoD was < 15.548fg/μL, and nine modular units successfully detected the target pathogens. The MRT-NAAT AMR set included 24 modular units 65-785bp in product size with a Tms of 75.5-87.5°C; it showed high performance for detecting GPP target genes and variants. MRT-NAAT enables pathogen identification and AMR gene detection and is time-effective. By unifying the reaction settings of each modular unit, the modularity where combinations of primers can be used according to need could be achieved. This would greatly help in reflecting the researcher's need and the AMR status of a certain region while successfully detecting pathogens and AMR genes.

The diagnostic workup of patients suspected of having vitreoretinal lymphoma (VRL) is primarily based on vitreous fluid analysis, including the recently emerging myeloid differentiation primary response gene 88 (MYD88) mutation analysis. Aqueous humor paracentesis is a relatively less invasive and safer procedure than taking vitreous fluid specimens, and aqueous humor-based MYD88 mutation analysis would provide an additional liquid biopsy tool to diagnose and monitor patients with VRL. To investigate whether the detection of MYD88 L265P by highly sensitive droplet digital polymerase chain reaction (ddPCR) is feasible in the vitreous fluid and aqueous humor of patients with VRL. This cohort study includes aqueous humor and vitreous fluid samples from patients with VRL who were treated at the University Medical Center Utrecht, in Utrecht, the Netherlands, from August 2005 to August 2017. Ocular fluids were randomized and masked before MYD88 L265P analysis, which was performed using an in-house validated ddPCR platform. Patients with uveitis were included as a comparison group. The presence of MYD88 L265P mutation detected by ddPCR in AH and VF. The study included 96 samples from 63 individuals, including 23 patients with VRL (of whom 10 were female and 13 male, with a mean [SD] age of 72 [7.3] years) and 40 individuals with uveitis (of whom 23 were female and 17 male, with a mean [SD] age of 58 [20.9] years). In 17 of 23 patients with VRL (74%), MYD88 L265P was detected; it was not detected in any of the patients with uveitis. It was detectable in both vitreous fluid and aqueous humor samples. In the paired samples, the mutation was detected in 8 of 9 aqueous humor samples (89%) of the MYD88 L265P-positive vitreous fluid samples. In vitreous fluid, the MYD88 ddPCR test showed a sensitivity of 75% (95% CI, 50%-92%) and a positive predictive value of 100%; in aqueous humor, sensitivity was 67% (95% CI, 42%-92%), and positive predictive value was 100%. Specificity was 100% in both fluids. After treatment, the mutation was no longer detectable in any ocular fluids. The high concordance between aqueous humor and vitreous fluid samples suggests that use of the easily accessible aqueous humor is nearly as informative as vitreous fluid in the identification of key somatic mutations in patients with VRL. This approach may provide an additional minimally invasive tool for accurate diagnosis, detection of recurrence, and monitoring of treatment.

Introduction: Retinoblastoma (Rb) is the most common childhood intraocular cancer. Tissue biopsy of Rb can cause tumor spread, so it is contraindicated. We demonstrated that aqueous humor (AH), an ocular fluid, is a high-yield liquid biopsy enabling in vivo detection of tumor-derived cell-free DNA (cfDNA) thus overcoming the contraindication of biopsy. Somatic genomic alterations, including both somatic copy number alterations (SCNAs) and single nucleotide variations (SNVs) on RB1 gene, have been able to be identified in the same clinical samples but with two separate sequencing runs. In this study, we first developed a single targeted sequencing method to identify both SCNAs and RB1 SNVs. With this method, we further investigated the degree of genomic concordance between paired tumor and AH samples from the same Rb eye. Materials and Methods: 11 paired AH and Rb tumor samples were included in the study. cfDNA of AH and tumor DNA of enucleated Rb eyes were isolated with QIAgen commercial kits. DNAs were constructed into whole genome libraries followed by hybridization target enrichment (Agilent SureSelect). The enrichment assay covers the whole length of RB1 and MycN genes, the all-exon regions of BCOR and CREBBP genes, and a whole genome CNV backbone. Libraries were submitted to Illumina HiSeq 4000 platform for fastq data generation with 2 × 150bp mode. The bioinformatics pipeline was optimized to generate SCNA profiles from targeted sequencing reads, along with SNV calling. Results: For SCNA profiles, 11/11 AH samples (100%) and 8/11 tumor samples (72.72%) have positive RB-SCNA signatures. Strong concordances were observed between AH and tumor SCNA profiles (median = 90.1%) with targeted sequencing reads. In total, 9 disease-driving RB1 SNVs were identified in 6 patients (54.5%). 7/9 (77.8%) of the variants were shared between AH and tumor samples, while the AH and tumor each contained one unique SNV. In all SNVs, the AH displayed a higher allele frequency. Notably, 4/11 samples have focal RB1 gene deletion detected with SCNA profiling, which may explain the difficulties of RB1 SNV detection in some samples. Further, one case was driven by a MYCN gene amplification with no RB1 alterations. Conclusions: This study presented highlights the utility of a single method to call both SVNs and SCNAs in a single clinical sample, with enriched tumor information detected in AH compared with tumor. This study further strengthens the utility of AH as a liquid biopsy platform for Rb eyes. Citation Format: Liya Xu, Mike J. Schmidt, Rishvanth K. Prakabar, Peter Kuhn, James Hicks, Jesse Berry. Simultaneous somatic copy number alterations and single nucleotide variants detection in paired aqueous humor and tumors from retinoblastoma eyes. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3553.

Accurate and sensitive taxonomic profiling is essential for any metagenomic analysis to reveal microbial community structure and for potential functional prediction. Antimicrobial resistance (AMR) detection is also a critical task in the clinical diagnosis of infection and antimicrobial therapy. By incorporating Oxford Nanopore Technologies (ONT) sequencing, users benefit from the high-confidence alignment of long reads for taxonomic classification, even among bacteria with similar genomes. Portable ONT devices, such as VolTRAX with MinION, allow short turnaround time for detection and can be used in a lightweight laboratory setting. However, error-prone ONT sequencing reads are still challenging for existing software for accurate taxonomic classification of microbes and detection of AMR down to the drug level. In this paper, we present MegaPath-Nano, the successor to NGS-based MegaPath. It is a high-precision compositional analysis software with drug-level AMR detection for ONT metagenomic sequencing data. MegaPath-Nano performs 1) thorough multi-level filtering against decoy and human reads while removing noisy alignments, 2) alignment-based taxonomic classification with RefSeq down to strain-level, with an alignment-reassignment algorithm to tackle the challenge of non-unique alignments, based on global alignment distribution, and 3) comprehensive downstream drug-level AMR detection, integrating five AMR databases. In our benchmarks using the Zymo metagenomic dataset, MegaPath-Nano performed better than other existing software for taxonomic classification. We also sequenced five real patient isolates using MinION to benchmark its performance of AMR detection. MegaPath-Nano was the most accurate and provided the most comprehensive output at both the drug and class level of AMR prediction against other state-of-the-art software. MegaPath-Nano is open-source and available at https://github.com/HKU-BAL/MegaPath-Nano.

Molecular basis of antimicrobial resistance in Mycoplasma genitalium

Bacteriology testing and antimicrobial resistance detection capacity of national tiered laboratory networks in sub-Saharan Africa: an analysis from 14 countries

Preliminary assessment of nanopore-based metagenomic sequencing for the diagnosis of prosthetic joint infection

Though several studies have shown that the biochemical function of nitric oxide (NO) in the eye might play an important role in the regulation of intraocular pressure (IOP), local control of ocular blood flow and loss of retinal ganglion cells by apoptosis, it is unclear whether the role of NO is similar in the pathogenesis of different kinds of glaucoma: primary open-angle glaucoma (POAG), chronic closed-angle glaucoma (CCAG) and neovascular glaucoma (NVG). To further explore this issue, we measured the concentrations of NO in aqueous humor and plasma samples from patients with POAG (n = 31), CCAG (n = 76), NVG (n = 8) and cataract (n = 30). All of the NVG patients suffered from severe proliferative diabetic retinopathy, while other patients were free of any other systemic disease. The NO levels in both aqueous humor and plasma samples were assessed by chemiluminescence assay. We found that the NO levels in aqueous humor samples were greatly varied in patients with POAG (36.2 ± 3.3 µM), CCAG (47.7 ± 3.4 µM) and NVG (65.8 ± 5.4 µM), and all of them were significantly higher than in cataract patients (27.0 ± 2.9 µM; p < 0.05). Except NVG patients whose NO levels in plasma samples were highest (24.1 ± 3.5 µM) among all groups, the plasma NO levels were not significantly different between the other glaucoma patients and the cataract patients. We therefore concluded that significant variation of the elevated NO levels in aqueous humor samples from the patients with different types of glaucoma may reflect their differences in the pathogenesis.