Extraction-Free Detection of SARS-CoV-2 Viral RNA Using LumiraDx's RNA Star Complete Assay from Clinical Nasal Swabs Stored in a Novel Collection and Transport Medium.

Background: The rapid detection of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is vital for patient care. The LumiraDx™ SARS-CoV-2 RNA Star Complete (RSC) is an Emergency Use Authorization-recognized molecular test using nasal/nasopharyngeal swabs immersed in a viral/universal transport medium (VTM/UTM). However, there is a critical need for an alternative medium for point-of-care testing (POCT). This study aimed to investigate Xtract-Free (XF), a novel collection medium for transport and direct (extraction-free) use with nucleic acid tests. Methods: Using serially diluted SARS-CoV-2 viral RNA (vRNA) in a routine UTM and XF, a limit of detection (LOD) was established via an RSC test and a quantitative reverse transcription PCR (RT-qPCR). Additionally, the results obtained from a panel of 108 clinical "car-side" nasal swabs collected in XF during the coronavirus pandemic and assessed using the "gold-standard" RT-qPCR assay were compared to Lumira's RSC assay. Results: The average replicate RT-qPCR cycle threshold (CT) values for vRNA in XF and UTM were observed to be equivalent. An LOD for which five out of five replicates were detected using XF or VTM was approximately 2000 copies/mL. The nasal swabs collected in XF exhibited 93.9% positive percent agreement (sensitivity) and 100% negative percent agreement (specificity) compared to the RT-qPCR. Three specimens tested positive via an RT-qPCR were negative when tested via RSC; however, all three samples had CT values ≥ 36.4. Conclusions: XF is equivalent to VTM/UTM and is compatible for use with the RSC test. Furthermore, XF can be used directly with RT-qPCRs and rapid antigen testing without the requirement for separate nucleic acid extraction (an extraction-free process), making it ideal for cost-effective high-throughput and decentralized respiratory testing. Impact Statement: This study is the first to evaluate LumiraDx's SARS-CoV-2 RNA Star Complete assay in concert with Xtract-Free (XF), a novel collection medium containing a proprietary RNase-inactivating technology for the rapid, "extraction-free" detection of SARS-CoV-2 RNA from clinical nasal swabs. Specimens collected in XF combined with rapid LumiraDx detection provide a safe and sensitive alternative to VTM/UTM, and Molecular Transport medium (MTM) for high throughput, "extraction-free" molecular detection.

Erratum to "Opsonic monoclonal antibodies enhance phagocytic killing activity and clearance of Mycobacterium tuberculosis from blood in a quantitative qPCR mouse model" [Heliyon 5 (9), (September 2019), e02260].

[This corrects the article DOI: 10.1016/j.heliyon.2019.e02260.].

An Alternative Psychophysical Diagnostic Indicator of the Aging Eye.

PURPOSE: Impaired adaptation to changes in lighting levels as well as mesopic visual function is a common complaint in those over the age of 65. The use of photostress is a well-established method to test the adaption rate and the response of the visual cycle. In this study, we test visual function recovery to mesopic luminance stimuli following a long duration photostress in young and elderly subjects. If successful in strongly differentiating aging macular function, these methods may also be useful in the study of pathologies such as age-related macular degeneration. METHODS: A group of 12 older normal subjects (mean age 75.1 ± 4.79) and a control group of 5 younger normal subjects (mean age 26.2 ± 4.19) were subjected to macular photostress using the OraLux photostress system. The OraLux system provides a diffuse light source bleaching 84% of cone photopigment while maintaining an exposure safety factor of 200 times less than the maximum safe exposure. After each photostressing session, macular recovery was tracked using a foveal, variable contrast, flickering stimulus of mean luminance in the high mesopic range. Recovery was tracked for 300 seconds. The endpoint was time to recovery to each individual's baseline sensitivity as determined by two static sensitivity trials prior to photostress. RESULTS: Proportional hazards analysis of recovery time yielded a statistically significant difference between the older group and the young group (HR = 0.181; p=0.0289). The estimated hazard ratio of 0.181 indicates that older subjects return to baseline at less than one-fifth the rate of younger subjects. The hazards ratio remained statistically significant after adjusting for visual acuity (HR = 0.093; p=0.0424). CONCLUSION: Photostress recovery of flicker sensitivity under mesopic conditions is a strong differentiator of aging macular function. This agrees with subject-reported complaints in reduced luminance conditions after exposure to bright lights such as night driving. The qualitative similarity between the aging retina and changes in early AMD suggests that flicker recovery following photostress may be useful as a surrogate endpoint in AMD clinical trials.

Opsonic monoclonal antibodies enhance phagocytic killing activity and clearance of Mycobacterium tuberculosis from blood in a quantitative qPCR mouse model.

BACKGROUND: Patients with impaired immunity often have rapid progression of tuberculosis (TB) which can lead to highly lethal Mycobacterium tuberculosis (MTB) sepsis. Opsonic monoclonal antibodies (MABs) directed against MTB that enhance phagocytic killing activity and clearance of MTB from blood may be useful to enhance TB immunity. METHODS: BALB/c mice were immunized with ethanol-killed MTB (EK-MTB) and MABs were produced and screened by ELISA for binding to killed and live Mycobacterium smegmatis (SMEG) and MTB. MAB opsonophagocytic killing activity (OPKA) was examined using SMEG with HL60 and U-937 cells and MTB with U-937 cells. Clearance of MTB from blood was evaluated in Institute of Cancer Research (ICR) mice given opsonic anti-MTB MABs or saline (control) 24 h prior to intravenous infusion with 108 CFUs gamma-irradiated MTB (HN878). MTB levels in murine blood collected 0.25, 4 and 24 h post-challenge were assessed by qPCR. MAB binding to peptidoglycan (PGN) was examined by ELISA using PGN cell wall mixture and ultra-pure PGN. RESULTS: Two MABs (GG9 and JG7) bound to killed and live SMEG and MTB (susceptible and resistant), and promoted OPKA with live MTB. MAB JG7 significantly enhanced OPKA of MTB. Both MABs significantly enhanced clearance of killed MTB from murine blood at 4 and 24 h as measured by qPCR. These opsonic MABs bound to PGN, a major cell wall constituent. CONCLUSIONS: Anti-MTB MABs that promote bactericidal phagocytic activity of MTB and enhance clearance of killed MTB from the blood, may offer an immunotherapeutic approach for treatment of MTB bacteremia or sepsis, and augment treatment of multi-drug resistant (MDR) or extensively drug resistant (XDR) TB.

Cone photoreceptor macular function and recovery after photostress in early non-exudative age-related macular degeneration.

PURPOSE: To identify parameters from cone function and recovery after photostress that detect functional deficits in early non-exudative age-related macular degeneration (AMD) and to determine the repeatability of these parameters. METHODS: Cone-mediated visual function recovery after photostress was examined in three groups of subjects: young normal subjects (ages 20-29; N=8), older normal subjects (ages 50-90; N=9), and early non-exudative AMD subjects (ages 50-90; N=12). Eight AMD and four normal subjects were retested 1 year after the initial evaluation. Early Treatment Diabetic Retinopathy Study (ETDRS) visual acuity (VA) and parameters of cone function (baseline cone sensitivity and cone recovery half-life following photobleach) were measured and compared between AMD and normal subjects. Short-term repeatability was assessed for each subject's initial evaluation. Long-term repeatability was assessed by comparing outcomes from the initial evaluation and 1-year follow-up. RESULTS: The mean baseline cone threshold was significantly worse in subjects with early AMD compared to older normal subjects (-1.80±0.04 vs -1.57±0.06 log cd/m2p=0.0027). Moreover, the baseline cone threshold parameter exhibited good short-term (intraclass correlation coefficient [ICC]=0.88) and long-term (ICC=0.85) repeatability in all subjects. The cone intercept parameter and ETDRS VA were not significantly different between AMD and older normal subject groups. Cone recovery half-life was significantly different between older normal and AMD subject groups (p=0.041). Neither ETDRS VA nor cone function parameters were significantly different for any group at the 1-year follow-up. CONCLUSION: The baseline cone threshold shows potential as a novel parameter to assess visual dysfunction in early AMD. This outcome consistently detected deficits in AMD subjects, and differentiated them from age-matched controls with high test-retest repeatability.

Multi- and Extensively Drug Resistant Mycobacterium tuberculosis in South Africa: a Molecular Analysis of Historical Isolates.

Modern advances in genomics provide an opportunity to reinterpret historical bacterial culture collections. In this study, genotypic antibiotic resistance profiles of Mycobacterium tuberculosis isolates from a historical 20-year-old multidrug-resistant tuberculosis (MDR-TB) culture collection in South Africa are described. DNA samples extracted from the phenotypically MDR-TB isolates (n = 240) were assayed by Hain line probe assay (LPA) for the confirmation of MDR-TB and by Illumina Miseq whole-genome sequencing (WGS) for the characterization of mutations in eight genes (rpoB, katG, inhA, rpsL, pncA, embB, gyrA, and rrs) that are known to code for resistance to commonly used anti-TB agents. LPA identified 71.3% of the TB isolates as MDR-TB, 18.3% as rifampin (RIF) monoresistant, 2% as isoniazid (INH) monoresistant, and 8.3% as susceptible to both RIF and INH (RIF+INH). In a subset of 42 randomly selected isolates designated as RIF+INH resistant by Löwenstein-Jensen (LJ) culture in 1993, LPA and WGS results confirmed MDR-TB. In all five INH-monoresistant isolates by LPA and in all but one (the wild type) of the 34 successfully sequenced RIF-monoresistant isolates, WGS revealed matching mutations. Only 26% of isolates designated as susceptible by LPA, however, were found to be wild type by WGS. Novel mutations were found in the rpoB (Thr480Ala, Gln253Arg, Val249Met, Val251Tyr, Val251Phe), katG (Trp477STOP, Gln88STOP, Trp198STOP, Trp412STOP), embB (Thr11Xaa, Gln59Pro), and pncA (Thr100Ile, Thr159Ala, Ala134Arg, Val163Ala, Thr153Ile, DelGpos7, Phe106Ser) genes. Three MDR-TB isolates showed mutations in both the gyrA and rrs genes, suggesting that extensively drug-resistant tuberculosis existed in South Africa well before its formal recognition in 2006.

Blink: Characteristics, Controls, and Relation to Dry Eyes.

Blink is a complex phenomenon that is profoundly affected by diverse endogenous and exogenous stimuli. It has been studied in the context of cognition, emotional, and psychological states, as an indicator of fatigue and sleepiness, particularly in the automobile and transportation industry, in visual tasking, and finally, as it relates to tear film stability and ocular surface health. The fact that it is highly variable and has input from so many sources makes it very difficult to study. In the present review, the behavior of blink in many of these systems is discussed, ultimately returning in each instance to a discussion of how these factors affect blink in the context of dry eyes. Blink is important to ocular surface health and to an individual's optimal functioning and quality of life. Disturbances in blink, as cause or effect, result in a breakdown of tear film stability, optical clarity, and visual function.

Diurnal Tracking of Blink and Relationship to Signs and Symptoms of Dry Eye.

PURPOSE: To assess diurnal changes in the signs and symptoms of dry eyes and their relationship to diurnal interblink interval (IBI) in normal subjects and in subjects with dry eye. METHODS: Blink data were collected from 9:00 AM to 8:00 PM during 2 days of normal activity using an electrocardiogram monitoring device. All subjects recorded ocular discomfort (0-5 scale) and primary activity hourly each day in a diary. Inferior and central fluorescein staining was graded by slit lamp (0-4) at the start and end of each day. Blink activity was detected using an algorithm based on recognition of the waveform corresponding to the kinematic properties of the blink signal. RESULTS: Normal subjects (N = 12) reported negligible symptoms, and results did not show a diurnal change in group hourly IBI. Mean daily IBI for the group with dry eye (N = 15) (4.63 ± 1.63 s) was shorter than that for the normal group (5.28 ± 1.48 s) (P = 0.0483). Correlation of diurnal symptoms and mean hourly IBI was relatively weak (r = -0.248). A repeated-measures model found IBI to be significantly associated with the time of day (P = 0.0028). Inferior corneal staining showed a small but significant diurnal increase for both normal group and group with dry eyes. CONCLUSIONS: Diurnal blink tracking reveals significant trending with symptoms. Diurnal change in IBI may be an appropriate surrogate for symptoms in the study of dry eye.

Improved Detection by Next-Generation Sequencing of Pyrazinamide Resistance in Mycobacterium tuberculosis Isolates.

The technical limitations of common tests used for detecting pyrazinamide (PZA) resistance in Mycobacterium tuberculosis isolates pose challenges for comprehensive and accurate descriptions of drug resistance in patients with multidrug-resistant tuberculosis (MDR-TB). In this study, a 606-bp fragment (comprising the pncA coding region plus the promoter) was sequenced using Ion Torrent next-generation sequencing (NGS) to detect associated PZA resistance mutations in 88 recultured MDR-TB isolates from an archived series collected in 2001. These 88 isolates were previously Sanger sequenced, with 55 (61%) designated as carrying the wild-type pncA gene and 33 (37%) showing mutations. PZA susceptibility of the isolates was also determined using the Bactec 460 TB system and the Wayne test. In this study, isolates were recultured and susceptibility testing was performed in Bactec 960 MGIT. Concordance between NGS and MGIT results was 93% (n = 88), and concordance values between the Bactec 460, the Wayne test, or pncA gene Sanger sequencing and NGS results were 82% (n = 88), 83% (n = 88), and 89% (n = 88), respectively. NGS confirmed the majority of pncA mutations detected by Sanger sequencing but revealed several new and mixed-strain mutations that resolved discordancy in other phenotypic results. Importantly, in 53% (18/34) of these isolates, pncA mutations were located in the 151 to 360 region and warrant further exploration. In these isolates, with their known resistance to rifampin, NGS of pncA improved PZA resistance detection sensitivity to 97% and specificity to 94% using NGS as the gold standard and helped to resolve discordant results from conventional methodologies.

Automated Grading System for Evaluation of Superficial Punctate Keratitis Associated With Dry Eye.

PURPOSE: To develop an automated method of grading fluorescein staining that accurately reproduces the clinical grading system currently in use. METHODS: From the slit lamp photograph of the fluorescein-stained cornea, the region of interest was selected and punctate dot number calculated using software developed with the OpenCV computer vision library. Images (n = 229) were then divided into six incremental severity categories based on computed scores. The final selection of 54 photographs represented the full range of scores: nine images from each of six categories. These were then evaluated by three investigators using a clinical 0 to 4 corneal staining scale. Pearson correlations were calculated to compare investigator scores, and mean investigator and automated scores. Lin's Concordance Correlation Coefficients (CCC) and Bland-Altman plots were used to assess agreement between methods and between investigators. RESULTS: Pearson's correlation between investigators was 0.914; mean CCC between investigators was 0.882. Bland-Altman analysis indicated that scores assessed by investigator 3 were significantly higher than those of investigators 1 and 2 (paired t-test). The predicted grade was calculated to be: Gpred = 1.48log(Ndots) - 0.206. The two-point Pearson's correlation coefficient between the methods was 0.927 (P < 0.0001). The CCC between predicted automated score Gpred and mean investigator score was 0.929, 95% confidence interval (0.884-0.957). Bland-Altman analysis did not indicate bias. The difference in SD between clinical and automated methods was 0.398. CONCLUSIONS: An objective, automated analysis of corneal staining provides a quality assurance tool to be used to substantiate clinical grading of key corneal staining endpoints in multicentered clinical trials of dry eye.

Optimizing Reading Tests for Dry Eye Disease.

PURPOSE: The aim of this study was to evaluate the visual function information obtained from multiple reading tests in a dry eye population. METHODS: In this case-control, single-center clinical research center-based study, 15 subjects with dry eye (mean age 65 years) and 10 normal subjects (mean age 40 years) were included. The Standardized Mini-Mental Examination was given to assure that subjects had normal cognitive function. Reading tests were both sentence based (Radner reading acuity test, reading contrast sensitivity test at a fixed print size, and menu-reading test) and paragraph based (Wilkins test and International Reading Speed Texts [IReST]). Wilkins and IReST tests were slightly modified to increase difficulty and visual stress. The main outcome measures were cognitive function, fatigue, dry eye symptoms, reading acuity, reading rate, and blink rate. RESULTS: Results showed significantly lower rates in all reading tests in subjects with dry eye than in normal subjects; in the age-matched subgroup, only the menu and contrast sensitivity tests lost significance. Fatigue was significantly related to the IReST test, both at normal and critical print sizes. Reflex tearing was monitored and shown to significantly decrease the reading rate. IReST scores were considered the baseline, and the percent change from one test to IReST was also used to differentiate dry eye and normal subjects. The blink rate, symptoms, and demographics were not significantly correlated with reading tests. CONCLUSIONS: Reading is a relevant end point that differentiates dry eye and normal subjects. This study evaluated a variety of reading tests for relevance as a measurable assessment of visual function in dry eye disease.

Whole-Genome Sequence for Methicillin-Resistant Staphylococcus aureus Strain ATCC BAA-1680.

We report here the whole-genome sequence of the USA300 strain of methicillin-resistant Staphylococcus aureus (MRSA), designated ATCC BAA-1680, and commonly referred to as community-associated MRSA (CA-MRSA). This clinical MRSA isolate is commercially available from the American Type Culture Collection (ATCC) and is widely utilized as a control strain for research applications and clinical diagnosis. The isolate was propagated in ATCC medium 18, tryptic soy agar, and has been utilized as a model S. aureus strain in several studies, including MRSA genetic analysis after irradiation with 470-nm blue light.

Automated grading system for evaluation of ocular redness associated with dry eye.

BACKGROUND: We have observed that dry eye redness is characterized by a prominence of fine horizontal conjunctival vessels in the exposed ocular surface of the interpalpebral fissure, and have incorporated this feature into the grading of redness in clinical studies of dry eye. AIM: To develop an automated method of grading dry eye-associated ocular redness in order to expand on the clinical grading system currently used. METHODS: Ninety nine images from 26 dry eye subjects were evaluated by five graders using a 0-4 (in 0.5 increments) dry eye redness (Ora Calibra™ Dry Eye Redness Scale [OCDER]) scale. For the automated method, the Opencv computer vision library was used to develop software for calculating redness and horizontal conjunctival vessels (noted as "horizontality"). From original photograph, the region of interest (ROI) was selected manually using the open source ImageJ software. Total average redness intensity (Com-Red) was calculated as a single channel 8-bit image as R - 0.83G - 0.17B, where R, G and B were the respective intensities of the red, green and blue channels. The location of vessels was detected by normalizing the blue channel and selecting pixels with an intensity of less than 97% of the mean. The horizontal component (Com-Hor) was calculated by the first order Sobel derivative in the vertical direction and the score was calculated as the average blue channel image intensity of this vertical derivative. Pearson correlation coefficients, accuracy and concordance correlation coefficients (CCC) were calculated after regression and standardized regression of the dataset. RESULTS: The agreement (both Pearson's and CCC) among investigators using the OCDER scale was 0.67, while the agreement of investigator to computer was 0.76. A multiple regression using both redness and horizontality improved the agreement CCC from 0.66 and 0.69 to 0.76, demonstrating the contribution of vessel geometry to the overall grade. Computer analysis of a given image has 100% repeatability and zero variability from session to session. CONCLUSION: This objective means of grading ocular redness in a unified fashion has potential significance as a new clinical endpoint. In comparisons between computer and investigator, computer grading proved to be more reliable than another investigator using the OCDER scale. The best fitting model based on the present sample, and usable for future studies, was [Formula: see text] is the predicted investigator grade, and [Formula: see text] and [Formula: see text] are logarithmic transformations of the computer calculated parameters COM-Hor and COM-Red. Considering the superior repeatability, computer automated grading might be preferable to investigator grading in multicentered dry eye studies in which the subtle differences in redness incurred by treatment have been historically difficult to define.

Investigation of extended blinks and interblink intervals in subjects with and without dry eye.

BACKGROUND: The purpose of this study was to investigate the occurrence and duration of extended blinks ≥ 70 msec and their associated interblink intervals in normal subjects and in subjects with mild to moderate dry eye. METHODS: This single-center, prospective, double-blind study included 11 subjects with dry eye and eight subjects with normal eyes. Extended blinks were defined as lid closure in at least two successive video frames (≥70 msec). Digital video imaging of each subject's eyes was recorded while the subject viewed a 10-minute documentary. The subjects did not know that blink was the outcome being measured. Following capture, the videos were manually analyzed in a masked fashion for the occurrence of extended blinks. The length of the interblink interval (ie, time between blinks) before and after these extended blinks (the interblink interval ratio) was calculated, as well as differences in lid contact times. RESULTS: The dry eye group had a median extended blink duration which was 2.53 times longer than that of the normal group. For subjects with dry eye, interblink intervals post-extended blink were significantly longer than interblink intervals pre-extended blink (P < 0.001). Interblink intervals did not lengthen significantly in normal subjects. In both groups, the duration of the extended blink was significantly (P = 0.001) and positively correlated with interblink interval ratio (post-extended to pre-extended blink interblink interval), such that for each doubling of extended blink duration, the interblink interval ratio increased by 10%. Blinks longer than one second in duration occurred almost exclusively in subjects with dry eye. CONCLUSION: THIS STUDY REPORTS THREE CENTRAL FINDINGS: blink duration tended to be longer in subjects with dry eye; a lengthening of the interblink interval after an extended blink occurred in subjects with dry eye but not in those without dry eye; and a longer blink duration was associated with a significantly increased interblink interval ratio in all subjects.

Next-generation ion torrent sequencing of drug resistance mutations in Mycobacterium tuberculosis strains.

A novel protocol for full-length Mycobacterium tuberculosis gene analysis of first- and second-line drug resistance was developed using the Ion Torrent Personal Genome Machine (PGM). Five genes-rpoB (rifampin), katG (isoniazid), pncA (pyrazinamide), gyrA (ofloxacin/fluoroquinolone), and rrs (aminoglycosides)-were amplified and sequenced, and results were compared to those obtained by genotypic Hain line probe assay (LPA) and phenotypic Bactec MGIT 960 analysis using 26 geographically diverse South African clinical isolates collected between July and November 2011. Ion Torrent sequencing exhibited 100% (26/26) concordance to phenotypic resistance obtained by MGIT 960 culture and genotypic rpoB and katG results by LPA. In several rifampin-resistant isolates, Ion Torrent sequencing revealed uncommon substitutions (H526R and D516G) that did not have a defined mutation by LPA. Importantly, previously uncharacterized mutations in rpoB (V194I), rrs (G878A), and pncA (Q122Stop) genes were observed. Ion Torrent sequencing may facilitate tracking and monitoring geographically diverse multidrug-resistant and extensively drug-resistant strains and could potentially be integrated into selected regional and reference settings throughout Africa, India, and China.

Amplification of Herpes simplex type 1 and Human Herpes type 5 viral DNA from formalin-fixed Alzheimer brain tissue.

It is known that nucleic acids from formalin-fixed tissues are not nearly as good templates for DNA amplification as those extracted from fresh tissues. However, specimens stored in most pathologic archives are initially fixed in formalin. The possibility of an infectious etiology of several diseases including Alzheimer's underscores the usefulness of archived tissue in assessing the association of infectious agents with specific pathology. In this report, we describe in detail a method resulting in robust amplification of HSV1 and Human Herpes type (HHV) 5 viral DNA targets using formalin-fixed Alzheimer brain frontal and temporal tissue as source of amplification template. Herpes simplex type 2 viral DNA was not detected in the limited samples examined in this study. Amplicons were verified by sequence analysis. Brain tissue stored in formalin longer than 1 year prior to post-formalin-fixation analysis gave rise to significantly shorter amplicons consistent with the observation that template DNA integrity decreases significantly with increasing time of storage in formalin. Thus, this report should be useful in PCR-based investigations assessing the regional presence of viral DNAs in formalin-fixed brain tissue.