Objectives

This study aimed to assess whether the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Epsilon variant (B.1.429/427) is more virulent, leading to more hospitalization and more severe disease requiring intensive care unit (ICU) admission.

Methods

SARS-CoV-2 genomic surveillance was performed on respiratory samples from 231 unique patients, collected at a single large health system in Southern California between November 2020 and March 2021 during the winter surge.

Results

The frequencies of the Epsilon variant among outpatients, hospitalized patients, and ICU patients were indifferent.

Conclusions

Our study suggests that the Epsilon variant is not associated with increased hospitalization and ICU admission.

The application of next-generation sequencing extends from microbial identification to epidemiologic insight and antimicrobial resistance prediction. Despite this potential, the roadblock for clinical laboratories lies in implementation and validation of such complex technology and data analysis. Herein, a validation study used whole-genome sequencing (WGS) for pan-bacterial identification (ID) in a clinical laboratory, and assessed its clinical relevance. A diverse set of 125 bacterial isolates, including a subset without genus (25) and/or species (10) ID, were analyzed by de novo assembly and reference genome mapping. The 16S rRNA, rpoB, and groEL genes were used for ID. Using WGS, 100% (89 of 89) and 89% (79 of 89) of isolates were identified at the genus and species-levels, respectively. WGS also provided improved results for 71% (25/35) isolates originally reported with genus-only or descriptive IDs. Chart review identified cases in which improved genus and/or species-level ID by WGS may have had a positive impact on patient care. Reasons included the use of an ineffective antibiotic owing to unclear ID, use of antibiotics when not clinically indicated, and help with an outbreak investigation. The implementation of next-generation sequencing in a clinical microbiology setting is a challenging but necessary task. This study provides a model for the validation and implementation of bacterial ID by WGS in such a setting.

Purpose

To describe a unique case of LASIK flap fungal keratitis confirmed by next generation sequencing.

Observations

A 56-year-old female presented with refractory keratitis involving her LASIK flap 21 years after surgery. Confocal was positive for filamentous structures. The patient underwent immediate flap amputation followed by topical antifungal treatment. Corneal culture was positive for Acremonium sp. Metagenomic deep sequencing confirmed Acremonium as the primary source of infection and also identified Fusarium as a likely contributor of a mixed fungal infection. Sequencing also identified hay as the likely source of the infection. Treatment resulted in eradication of the infection. The patient's final best corrected visual acuity was 20/30 with rigid contact lens overrefraction.

Conclusions

Metagenomic deep sequencing is a novel diagnostic tool that is increasingly being utilized for diagnosis of refractory keratitis. This case demonstrates the diagnostic potential of deep sequencing for identifying post-LASIK keratitis and reinforces the utility of LASIK flap amputation in the setting of tectonic flap instability due to keratolysis.

Importance

This case highlights several important clinical points for treating LASIK flap keratitis and highlights the emerging role metagenomic sequencing has in the diagnosis of infectious keratitis. This is first known case using next generation sequencing to diagnose a post-LASIK infectious keratitis.

The current pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in the approval of numerous molecular diagnostic assays with various performance and technical capacities. There are limited data comparing performance among assays. We conducted a retrospective analysis of >10,000 test results among three widely used RT-PCR assays for coronavirus disease 2019 (CDC, Simplexa Direct, and TaqPath) to assess performance characteristics. We also retested remnant weakly positive specimens to assess analytical sensitivity. All assays had strong linear correlation and little bias among C_T values for PCR targets. In patients with first-test negative results (n = 811), most (795, 98.0%) remained negative for all subsequent testing. Retesting of weakly positive specimens (C_T > 30) showed sensitivities as follows: TaqPath (97.8%), CDC (91%), Simplexa (75.3%). Our analysis showed no performance difference among PCR targets within the same assay, suggesting a single target is sufficient for SARS-CoV-2 detection. Lower respiratory tract specimens had a higher negative predictive value (100%) than upper respiratory tract specimens (98%), highlighting the utility of testing lower respiratory tract specimens when clinically indicated. Negative predictive value did not increase on further repeated testing, providing strong evidence for discouraging unnecessary repeated testing for SARS-CoV-2.

Purpose

To describe a case of central corneal ulceration in a newborn secondary to congenital entropion.

Observations

Corneal ulcers during infancy are rare and may occur secondary to congenital structural anomalies, including congenital entropion. Correct anatomic eyelid position in newborns is challenging to determine with closed eyelids, and eyelid squeezing during crying and discomfort adds to this challenge.

Conclusions and importance

This report reinforces the importance of careful examination of the adnexa in infants with corneal ulcers while they are most comfortable, usually after topical anesthesia and prior to placement of eyelid speculum. Ophthalmologists caring for infants must be able to detect this condition because prompt entropion repair is necessary for corneal ulcer resolution and prevention of permanent vision loss.

Using next-generation sequencing (NGS), we developed and validated a whole-genome sequencing (WGS)-based clinical test for fungal species identification on clinical isolates. The identification is mainly based on the fungal ribosomal internal transcribed spacer (ITS) region as the primary marker, and additional marker and genomic analysis applied for species within the Mucorales family (using the 28S rRNA gene) and Aspergillus genus (using the beta-tubulin gene and k-mer tree-based phylogenetic clustering). The validation study involving 74 unique fungal isolates (22 yeasts, 51 molds, and 1 mushroom-forming fungus) showed high accuracy, with 100% (74/74) concordance on the genus-level identifications and 89.2% (66/74) concordance on the species level. The 8 discrepant results were due to either the limitation of conventional morphology-based methodology or taxonomic changes. After one year of implementation in our clinical laboratory, this fungal NGS test was utilized in 29 cases; the majority of them were transplant and cancer patients. We demonstrated the utility of this test by detailing five case studies, in which accurate fungal species identification led to correct diagnosis, treatment adjustment or was ruled out for hospital acquired infection. This study provides a model for validation and implementation of WGS for fungal identification in a complex health system that serves a large immunocompromised patient population.

Purpose

Acanthamoeba keratitis often is refractory to medical and surgical therapy, primarily because of the remarkable resilience of Acanthamoeba cysts. In this study, we directly compared the cysticidal activity and potency of several candidate medical therapies in vitro.

Design

Experimental study.

Participants

In vitro Acanthamoeba specimens obtained from 9 patients with keratitis seen at the Francis I. Proctor Foundation from 2008 through 2012.

Methods

The minimum cysticidal concentration (MCC) of povidone iodine, natamycin, and chlorhexidine was investigated using an established assay technique. The relative potency of each agent was estimated starting with concentrations commonly used in clinical practice and determining the number of two-fold dilutions required to reach the MCC. Statistical comparisons of relative potency were performed using bootstrap simulations and permutation tests.

Main outcome measures

Minimum cysticidal concentration and the number of two-fold dilutions required to reach the MCC.

Results

The MCC for chlorhexidine ranged from 3.1 to 25 μg/ml (median, 12.5 μg/ml; interquartile range [IQR], 6.25-12.5 μg/ml), the MCC for natamycin ranged from 390.6 to 3125 μg/ml (median, 390.6 μg/ml; IQR, 390.6-781.2 μg/ml), and the MCC for povidone iodine ranged from 0.3 to 78.1 μg/ml (median, 2.4 μg/ml; IQR, 0.6-9.8 μg/ml). Doses commonly used in clinical practice (povidone iodine 1%, natamycin 5%, and chlorhexidine 0.04%) were approximately 12, 7, and 5 two-fold dilutions higher than the drug's corresponding median MCC, respectively (P < 0.001, comparing 3 drugs). Povidone iodine 1% had the highest potency of the 3 medications tested, requiring more dilutions than natamycin 5% (P < 0.001) and chlorhexidine 0.04% (P < 0.001) to reach the MCC.

Conclusions

All 3 medications demonstrated in vitro cysticidal activity in each of the 9 isolates. The potency of 1% povidone iodine was greater than standard formulations of natamycin or chlorhexidine. Although its clinical efficacy is yet to be determined, povidone iodine may be considered as a potential adjuvant treatment in cases of recalcitrant Acanthamoeba keratitis.

Objectives

Cefiderocol is a novel siderophore cephalosporin with in vitro activity against multidrug-resistant (MDR), gram-negative bacteria and intrinsic structural stability to all classes of carbapenemases. We sought to identify gene variants that could affect the mechanism of action (MOA) of cefiderocol.

Methods

We report a case of bacteremia in a liver transplant candidate with a strain of carbapenem-resistant Escherichia coli that was found to be resistant to cefiderocol despite no prior treatment with this antimicrobial agent. Using whole-genome sequencing, we characterized the genomic content of this E coli isolate and assessed for genetic variants between related strains that were found to be cefiderocol susceptible.

Results

We identified several variants in genes with the potential to affect the mechanism of action of cefiderocol.

Conclusions

The cefiderocol resistance in the E coli isolate identified in this study is likely due to mutations in the cirA gene, an iron transporter gene.

Purpose

To study the feasibility and efficacy of a new remote wet lab for microsurgical education using a corneal suturing task.

Setting

Department of Ophthalmology, University of California San Francisco, San Francisco, California, USA.

Design

Prospective randomized controlled study.

Methods

Ten ophthalmology residents were stratified by postgraduate year and randomized to perform a corneal suturing task consisting of placing the 4 cardinal sutures for a penetrating keratoplasty in porcine eyes with or without remote ophthalmology attending feedback. Subsequently, both groups repeated the same task without remote feedback to test whether initial remote feedback affected subsequent performance. Finally, the group without feedback was crossed over to repeat the same corneal suturing task with remote feedback. The effectiveness of the remote wet lab was assessed subjectively by survey and objectively by grading each suture pass.

Results

Resident-reported comfort with corneal suturing improved significantly after the remote wet lab for all residents. Residents and attendings rated the remote wet lab as equally or more effective compared with previous in-person wet labs and overall effective in corneal suturing. Attendings rated the remote wet lab as effective in multiple domains of microsurgical education using a modified microsurgical global rating scale. Objective corneal suturing performance was similar for both groups.

Conclusions

The remote wet lab was feasible and effective for training ophthalmology residents in corneal suturing. This represents a new social distancing compliant platform for microsurgical education during the COVID-19 pandemic.

Background

Mycoplasma hominis can cause significant infections after solid organ transplantation (SOT). Treatment should be guided by susceptibility testing, but conventional lab methods are laborious with prolonged turnaround time (TAT). This case series compares the phenotypic and genotypic susceptibility profiles of M. hominis isolates identified from SOT patients.

Methods

This is a single-center retrospective study evaluating SOT recipients with confirmed M. hominis infections. Patients' demographic, clinical, microbiological, and radiographic data were collected. Culture of M. hominis isolates was performed according to current Clinical and Laboratory Standards Institute guidelines. Phenotypic susceptibility testing was performed by University of Alabama Diagnostic Mycoplasma Laboratory. Whole genome sequencing (WGS) was performed followed by bioinformatic analysis of known genetic determinants of resistance.

Results

Seven SOT recipients with M. hominis infections were identified. Two out of seven (28.5%) patients had resistance detected by phenotypic susceptibility testing (Case 5 to levofloxacin and Case 7 to tetracycline). Genomic analyses confirmed the presence of mutations in the parC and parE topoisomerase genes at positions conferring to fluoroquinolone resistance in the isolate from Case 5, while the tetracycline-resistant isolate from Case 7 harbored the tetM gene. The median TAT from the date of specimen collection was 24 days for phenotypic susceptibility testing and 14 days for genotypic susceptibility testing. All seven patients received antimicrobials directed toward M. hominis and recovered with complete resolution of infection.

Conclusions

WGS may offer a novel and more rapid methodology for M. hominis susceptibility testing to help optimize antimicrobial usage, but more data are needed.