Laboratory Research

Performance of the Qvella FAST Prep System in the Analysis of Positive Blood Cultures Compared to the Standard of Care at Corewell Health East

Brody Dams, B.S.¹, Carrie Maro, B.S.², Kristina Helm², Benjamin von Bredow, Ph.D.²

_{¹Oakland University William Beaumont School of Medicine, Rochester, MI
²Division of Microbiology, Department of Pathology, Corewell Health, Royal Oak, MI}

INTRODUCTION
Sepsis is a dysregulated systemic inflammatory response from infection. Blood culture results take up to 72 hours for complete identification of the pathogen and antibiotic susceptibility testing (AST). The Qvella FASTTM Prep (Qvella) creates a Liquid ColonyTM (LC) from a positive blood culture, which is then available for MALDI-TOF analysis and AST. Our goal was to determine if the Qvella was as accurate compared to the standard of care in identifying pathogens and their antibiotic resistance profiles from positive blood cultures.

METHODS
A retrospective study utilized positive blood cultures at Corewell Health Royal Oak. LC samples were derived from these cultures and processed on the Qvella. Samples were then inoculated on agar plates for AST and underwent MALDI-TOF analysis for comprehensive species identification. Blood cultures inoculated with known isolates from a microbial isolate bank were also utilized for greater variety of species and resistance profiles. 

RESULTS
A total of 90 samples were identified on MALDI-TOF from the LC. 47 samples were correctly identified at the species level and 15 at the genus level. 28 samples produced no ID. None of the samples were incorrect at the genus level. However, several attempts were often required to obtain any results from the LC. Essential and categorical agreements for AST were 96.1% and 95.8% for Gram+ species, and 97.9% and 94.9% for Gram- species, respectively.

CONCLUSIONS
The Qvella presents a promising technology in delivering more timely blood culture results. It did provide highly accurate results for both species identification and AST when results were available. However, the rate of failed identification and frequency of re-processing samples for results presents a significant hurdle in implementing this technology for clinical practice. AST results had some categorical disagreements and higher very major error rates for select antimicrobials which were likely due to small sample sizes of resistant organisms.

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Effects of Resolvin-D2 in lipopolysaccharide-induced retinal inflammation

Min Young Kim, B.S.¹, Mohamed Moustafa, Pharm. D.^2,3, Sonali Sharma, Ph.D.^2,3, Ahmed Elmarakby, Ph.D.^4,5, Mohamed Al-Shabrawey, M.D. / Ph.D.^6,7

_{¹Oakland University William Beaumont School of Medicine, Rochester, MI
²Eye Research Center, Oakland University William Beaumont School of Medicine (OUWB-SOM), Rochester, MI, USA
³Eye Research Institute, Oakland University, Rochester, MI, USA
⁴Department of Oral Biology and Diagnostic Sciences, Dental College of Georgia, Augusta University, Augusta, GA, USA
⁵Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta University, Augusta, GA, USA
⁶Oakland University William Beaumont School of Medicine, Rochester, MI
⁷Eye Research Center, Oakland University William Beaumont School of Medicine (OUWB-SOM), Rochester, MI, USA}

INTRODUCTION
Elevated lipopolysaccharide (LPS) contributes to diabetic retinopathy (DR). We have shown that diabetes and LPS modulates 12/15-lipoxygenase (12/15-LO). Here, we investigated the role of LPS in modulating retinal 12/15-LO-derived bioactive lipids and whether resolvin-D2 (RvD2) or docosahexaenoic acid (DHA) reduces LPS-induced inflammation.

METHODS
LPS level was assessed in blood samples from patients with type-2 diabetes compared to control using ELISA. Five groups of wild-type (WT) mice received treatments with intraperitoneal injections of saline (control), LPS (4 mg/kg), LPS+RvD2 (100ng/ mouse), LPS+DHA (50mg/kg), LPS+baicalein as an 12/15-LO inhibitor (20mg/kg). 12/15-LO knockout mice received saline (control) or LPS (4mg/kg). Treatments were administered 3 days before and after LPS, and retinal tissues and blood were collected on day 7 for RT-PCR and LC/MS analysis. Human retinal endothelial cells (HRECs) were treated with TNFα, RvD2, or both, and analyzed for cytokine and VCAM-1 expression using Bioplex and western blot. Data were analyzed using ANOVA.

RESULTS
Levels of LPS were significantly increased in serum of diabetic patients compared to non-diabetic. LPS significantly reduced blood levels of RvD and increased 12/15-LO metabolites and increased retinal 12/15-LO, TNFα, and ICAM-1. Baicalein, DHA, and RvD2 ameliorated the levels of TNFα, and ICAM-1 while increasing the levels of IL-10, an anti-inflammatory cytokine. DHA supplementation restored plasma RvD2 in LPS-treated mice. Interestingly, deletion of 12/15-LO did not reduce LPS-induced retinal inflammation. In HRECs, RvD2 did not significantly alter TNFα-induced IL-6 cytokine release or VCAM-1 expression.

CONCLUSIONS
Increased LPS in diabetic patients is consistent with its role in DR. RvD2 and DHA are potential therapies for retinal inflammation.  Lack of protection in HREC suggests that retinal cells other than HREC may be responsible for initiating retinal inflammatory responses in diseases such as in DR.

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In-House Gene Sequencing Reveals Rare Variants in FEVR, Norrie Disease and Persistent Fetal Vascular Syndrome

Vincent Le, B.A.¹, Savyo Krikor, B.A.², Wendy Dailey, M.D. / Ph.D.², Kimberly A. Drenser, M.D. / Ph.D.³, Kenneth P. Mitton, Ph.D.²

_{¹Oakland University William Beaumont School of Medicine, Rochester, MI
²Pediatric Retinal Research Laboratory, Eye Research Institute, Oakland University, Rochester, MI
³Associated Retinal Consultants PC, Royal Oak, MI, United States}

INTRODUCTION
Familial Exudative Vitreoretinopathy (FEVR), Norrie Disease (ND), and Persistent Fetal Vascular Syndrome (PFVS) belong to a family of rare retinopathies. Current studies suggest that FEVR specifically is a multigenic condition with incomplete penetrance.

METHODS
Blood samples were acquired from 20 pediatric patients and family members with FEVR/ND/PFVS, seen at Associated Retinal Consultants, PC of Royal Oak, Michigan, USA. Genomic DNA was extracted followed by Ampliseq gene library creation and sequencing with the Illumina iSeq-100. A custom targeted 7-gene panel was used, covering seven FEVR related genes: NDP, FZD4, LRP5, TSPAN12, KIF11, CTNNB1, and ZNF408. Full coverage of 83 exons with an additional 25 bp of adjacent intron sequences was acquired. VCF files with fully passing variants (Q30 quality reads and minimum read depth >10) were analyzed via the Ensembl Variant Effect Predictor Database and consequences were assessed via ClinVar.

RESULTS
16 of 20 patients presented with variants in coding regions, with a total of 35 gene-altering variants identified. A missense variant (ZNF408: Ser225Phe, AF = 0.0000006912) was identified in a patient diagnosed with FEVR. The same variant was observed in the patient’s asymptomatic father. In addition, three novel variants in NDP were discovered in three separate patients with ND/PFVS; a premature stop codon (NDP: Arg37Ter), a deletion (NDP: Leu13_Met19del), and a frameshift that induced a premature stop codon (NDP: Met32TrpfsTer9). 

CONCLUSIONS
The missense variant (Ser225Phe) in ZNF408 is reported to be of uncertain significance by ClinVar but is extremely rare. The father with the same variant in the absence of FEVR implies that the variant cannot be the sole factor for the patient’s condition. The novel premature stop codons in NDP (Arg37Ter, Met32TrpfsTer9) render the Norrin protein nonfunctional, and the novel deletion in NDP (Leu13_Met19del) is hypothesized to alter the trafficking signal of Norrin, contained within the protein’s N-terminal segment. 

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Anatomic Landmarks for Safe Subcutaneous Defibrillator Implantation

Mohammad Hossein Mohammadzadeh, B.S.¹, Malli Barremkala, M.D.², Brian D. Williamson, Ph.D.³, James Grogan, Ph.D.², Peter Khalil, M.D.³

_{¹Oakland University William Beaumont School of Medicine, Rochester, MI
²Department of Foundational Medical Studies, Oakland University William Beaumont School of Medicine, Rochester, MI
³Corewell Health William Beaumont University Hospital, Royal Oak, MI}

INTRODUCTION
Subcutaneous implantable cardioverter-defibrillators (S-ICDs) provide life-saving therapy for malignant arrhythmias without transvenous leads. Recommended generator placement is between the latissimus dorsi muscle (LDM) and serratus anterior muscle (SAM) along the mid-axillary line; however, limited anatomical data exist to guide safe implantation. Variability in the anterior LDM border and proximity of the long thoracic nerve (LTN) may increase complication risk. This study aimed to define these relationships to improve surgical safety.

METHODS
Eighteen cadavers (12 female, 6 male; mean age 79.6 years) were dissected. Measurements included the distance from the back to the anterior LDM border (A) at the 5th and 7th ribs and the anterior-posterior chest diameter (B). The A/B ratio was calculated. The LTN location relative to the LDM and its entry into the SAM were recorded from the 4th to 6th ribs.

RESULTS
The mean distance to the anterior LDM border was 7.6 cm (range 5.5–10.8 cm). The mean chest diameter was 21.5 cm (18.6–24.8 cm), yielding an A/B ratio of 0.35 ± 0.1 (0.27–0.45), with no sex differences. The LTN was anterior to the LDM at the 4th rib and posterior at the 5th and 6th ribs. It entered the SAM at the 4th rib in 6.6% and at the 5th and 6th ribs in 46.6% each. No LTN extended superficial to the LDM below the 6th rib.

CONCLUSIONS
The anterior LDM border is variable and consistently posterior to the mid-axillary line, suggesting standard incision landmarks may be suboptimal. The proximity of the LTN highlights the need for careful dissection to avoid nerve injury. These findings provide anatomical guidance to improve S-ICD implantation safety.

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One Carbon Metabolism and Aging Eye Disease: Exploring Preventive Therapeutic Targets

Neelesh Peddireddy , B.S.¹, Rana Abdelkarim, B.S.², Omar Elzayat, B.S.², Charlene Hsiung, B.S.², Amany Tawfik, M.D.²

_{¹Oakland University William Beaumont School of Medicine, Rochester, MI
²Oakland University Eye Research Institute (ERI), Oakland University William Beaumont School of Medicine Eye Research Center (ERC), Rochester, MI}

INTRODUCTION
Homocysteine (Hcy) is a key intermediate in one-carbon metabolism and requires many cofactors for metabolism including B vitamins. B vitamin deficiency can contribute to hyperhomocysteinemia (HHcy) which has been associated with diabetic retinopathy (DR) and age-related macular degeneration (AMD). Early diagnosis and management of aging eye disease remains time and money intensive. We tested the hypothesis that B vitamin supplementation (B6, B9, B12) will accelerate Hcy metabolism through the remethylation pathway, clearing the high Hcy levels in blood, and decreasing retinal dysfunction using an experimental mouse model.

METHODS
48 male and female CBS mice (mice with HHcy) and wild type (WT) mice were placed on a commercialized diet (regular, deficient, vitamin rich) for six-nine months. CBS mice were divided into three groups (8 mice/group), put on normal, vitamin B deficient, and supplementation diet. The WT mice were divided into two groups, one group of 8 mice were placed on a normal diet, and 16 were put on a vitamin B deficient diet. Once vitamin B levels were low, 8 WT mice were placed on a vitamin supplemented diet while the remaining 8 remained on deficient diet. Mice were evaluated every 8 weeks with Optical Coherence Tomography (OCT) imaging to evaluate retinal structures and layers in 3 test groups. Images were taken with Phoenix Micron IV retinal imaging microscope and analyzed with Insight 2D software.

RESULTS
Analysis from OCT images showed noticeable morphological changes in retina between CBS and WT mice on regular, deficient, and B Vitamin rich supplemented diet. Mice with vitamin B supplementation had improved retinal structure and thickness compared to deficient.

CONCLUSIONS
B vitamin supplementation improved metabolism of Hcy and decreased retinal dysfunction prompting discussion that B vitamins can be a potential therapeutic target and preventive method for aging diseases with HHcy such as diabetic retinopathy and age-related macular degeneration.

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T3: A Potential Therapy for Alzheimer’s Disease

Diana Ramo, B.S.¹, Luca Cucullo, Ph.D.², Michael Powers, M.S.³, Minelly Gonzalez, M.S.³

_{¹Oakland University William Beaumont School of Medicine, Rochester, MI
²Department of Foundational Medical Studies, Oakland University William Beaumont School of Medicine, Rochester, MI
³Oakland University, Rochester, MI}

INTRODUCTION
Alzheimer’s disease (AD) is a neurodegenerative condition primarily characterized by abnormal β-amyloid (Aβ) plaques and Tau neurofibrillary tangles in the brain. Unfortunately, current management strategies targeting Aβ plaques are predominately preventative instead of curative. Notably, triiodothyronine (T3), the active form of Thyroxine (T4), has been found to increase insulin sensitivity as well as the expression of both GLUT1 and GLUT3. Prior studies have demonstrated a correlation between hypothyroidism, and abnormal levels of TSH, T4, and T3, in AD patients. Proper thyroid hormone homeostasis is essential for healthy glucose metabolism in the brain. T3 also suppresses harmful amyloid precursor protein (APP) cleavage leading to Aβ plaque formation. Finally, T3 has been established as a pivotal hormone for neurodevelopment and neurogenesis in both developing and diseased brains. These encouraging findings strongly support the hypothesis that T3 could play an important therapeutic role in AD.

METHODS
Serum samples were analyzed from 10 healthy subjects/patients (male and female; age ≥ 65 years with no signs suggestive of dementia, hypothyroidism, or other neurodegenerative diseases) and 10 AD patients (male and female; ≥ 65 years with clinical signs of dementia). We will measure TSH, T4, T3, and rT3 by ELISA. To further corroborate the ELISA data, western blot analysis was carried out to support initial findings. These data were correlated to AD disease severity through analysis using an unpaired two-tailed t-test with Welch’s correction. Statistical significance was defined as p < 0.05.

RESULTS
Despite limited power (n=10 per group), Alzheimer’s disease patients exhibited trends toward elevated rT3 levels and reduced fT4/fT3 ratios compared with healthy controls, suggesting altered peripheral thyroid hormone metabolism.

CONCLUSIONS
These findings suggest altered thyroid hormone metabolism in Alzheimer’s disease and support a role of T3 dysregulation in AD progression, warranting further studies to investigate therapeutic modalities targeting this pathway.

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Identification of Tripeptide Modulators of ACE2 Activity Using a High Throughput Screen

David Walker, B.S.¹, Vardan Karamyan, Pharm. D., Ph.D.²

_{¹Oakland University William Beaumont School of Medicine, Rochester, MI
²Department of Foundational Medical Studies, Oakland University William Beaumont School of Medicine, Rochester, MI}

INTRODUCTION
Angiotensin converting enzyme 2 (ACE2) works in the renin angiotensin aldosterone system to decrease circulating angiotensin II by converting it to angiotensin (1-7) by removing a C-terminus phenylalanine.  ACE2 has received increased interest in research due to its role in COVID-19 pathogenesis, as a key binding site for COVID-19 cellular attachment and a suspected mechanism of symptom expression. While ACE inhibitors have been a common pharmacological option for medical management in vivo angiotensin II levels, no current ACE2 inducer has been identified in literature.

METHODS
We screened 8000 peptide combinations through microarray to identify ACE2 binding activity from the screen. From the 8000 peptides, 22 were selected that expressed high binding activity. The selected peptide combinations were tested for effect on ACE2 enzymatic activity through a cleaved fluorescent peptide protocol, and thermal shift protocols.

RESULTS
Of the peptides analyzed four peptide combinations have been shown to decrease ACE2 enzymatic activity, with no identified inducer. Thermal shift analysis of the peptides and ACE 2 revealed no change in the denaturation of ACE when compared with controls.  

CONCLUSIONS
Continued research into the field of ACE2 modulators continues to be an open and active opportunity for drug discovery through biopharmaceutical models. We hope that or research serves as a groundwork for future drug discovery.

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