Reflux Symptom Index (RSI), Videolaryngostroboscopy and Voice Analysis: A Triad of Non-Invasive Tools to Study Treatment Outcomes of Laryngopharyngeal Reflux Disease (LPRD).

Study post treatment improvement of Laryngopharyngeal Reflux Disease (LPRD) using non-invasive tools of Reflux symptom index (RSI), Reflux finding score (RFS) grading of videolaryngostroboscopy (VLS) and voice analysis. This study from December 2020 to April 2022 enrolled 100 adults with complaints suggestive of reflux symptoms and having Reflux Symptom Index (RSI) more than 13. All patients underwent VLS along with voice analysis. VLS findings were graded using Reflux Finding Score (RFS). Patients were advised for lifestyle modifications and proton pump inhibitors for 8 weeks when post treatment RSI, VLS and voice analyses were again documented. The age range was from 18 to 75 years. Males predominated. Lifestyle modification compliance was seen in 85% of the patients. We found a significant association (P = 0.001) for difference in pretreatment and posttreatment for both Reflux Symptom Index (RSI) parameters & Reflux Finding Score Index (RFS) parameters. Voice analysis pre and post treatment showed a significant association (P = 0.001) for fundamental frequency, jitter, shimmer, harmonic-to-noise ratio and maximum phonation time. The gold standard of diagnosis of LPRD is 24 h pH monitoring but has many false negatives and false positives due to intermittent reflux and inaccurate probe placement. This costly, time consuming and invasive procedure is not widely available amongst our speciality. Excellent visualisation of VLS allowed accurate RFS calculation. Voice analysis permitted early diagnosis of LPRD induced hoarseness before it became clinically significant. It also documented the treatment outcome. We conclude that an 8-weeks proton pump inhibitor treatment combined with lifestyle modification resulted in a significant improvement in the parameters of the non-invasive tools of RSI and RFS and voice analysis.

Donor Heart Recovery and Preservation Modalities in 2023.

Historically, heart transplantation (HT) has relied on the use of traditional cold storage for donor heart preservation. This organ preservation modality has several limitations, including the risk for ischemic and cold-induced graft injuries that may contribute to primary graft dysfunction and poor post-HT outcomes. In recent years, several novel donor heart preservation modalities have entered clinical practice, including the SherpaPak Cardiac Transport System of controlled hypothermic preservation, and the Transmedics Organ Care System of ex vivo perfusion. Such technologies are altering the landscape of HT by expanding the geographic reach of procurement teams and enabling both donation after cardiac death and the use of expanded criteria donor hearts. This paper will review the emerging evidence on the association of these modalities with improved post-HT outcomes, and will also suggest best practices for selecting between donor heart preservation techniques.

Aspirin and Hemocompatibility Events With a Left Ventricular Assist Device in Advanced Heart Failure: The ARIES-HM3 Randomized Clinical Trial.

IMPORTANCE: Left ventricular assist devices (LVADs) enhance quality and duration of life in advanced heart failure. The burden of nonsurgical bleeding events is a leading morbidity. Aspirin as an antiplatelet agent is mandated along with vitamin K antagonists (VKAs) with continuous-flow LVADs without conclusive evidence of efficacy and safety. OBJECTIVE: To determine whether excluding aspirin as part of the antithrombotic regimen with a fully magnetically levitated LVAD is safe and decreases bleeding. DESIGN, SETTING, and PARTICIPANTS: This international, randomized, double-blind, placebo-controlled study of aspirin (100 mg/d) vs placebo with VKA therapy in patients with advanced heart failure with an LVAD was conducted across 51 centers with expertise in treating patients with advanced heart failure across 9 countries. The randomized population included 628 patients with advanced heart failure implanted with a fully magnetically levitated LVAD (314 in the placebo group and 314 in the aspirin group), of whom 296 patients in the placebo group and 293 in the aspirin group were in the primary analysis population, which informed the primary end point analysis. The study enrolled patients from July 2020 to September 2022; median follow-up was 14 months. Intervention: Patients were randomized in a 1:1 ratio to receive aspirin (100 mg/d) or placebo in addition to an antithrombotic regimen. MAIN OUTCOMES AND MEASURES: The composite primary end point, assessed for noninferiority (-10% margin) of placebo, was survival free of a major nonsurgical (>14 days after implant) hemocompatibility-related adverse events (including stroke, pump thrombosis, major bleeding, or arterial peripheral thromboembolism) at 12 months. The principal secondary end point was nonsurgical bleeding events. RESULTS: Of the 589 analyzed patients, 77% were men; one-third were Black and 61% were White. More patients were alive and free of hemocompatibility events at 12 months in the placebo group (74%) vs those taking aspirin (68%). Noninferiority of placebo was demonstrated (absolute between-group difference, 6.0% improvement in event-free survival with placebo [lower 1-sided 97.5% CI, -1.6%]; P < .001). Aspirin avoidance was associated with reduced nonsurgical bleeding events (relative risk, 0.66 [95% confidence limit, 0.51-0.85]; P = .002) with no increase in stroke or other thromboembolic events, a finding consistent among diverse subgroups of patient characteristics. CONCLUSIONS AND RELEVANCE: In patients with advanced heart failure treated with a fully magnetically levitated LVAD, avoidance of aspirin as part of an antithrombotic regimen, which includes VKA, is not inferior to a regimen containing aspirin, does not increase thromboembolism risk, and is associated with a reduction in bleeding events. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT04069156.

Maternal and Pregnancy Outcomes Following Heart Transplantation in the United States.

BACKGROUND: Improved survival following heart transplantation (HT) has led to more recipients contemplating pregnancy, but data on outcomes are limited. OBJECTIVES: The authors used a national data set to investigate and describe outcomes of pregnancies and deliveries in the United States in HT recipients. METHODS: Diagnosis and procedure codes from the 2010-2020 Nationwide Readmissions Database identified delivery hospitalizations, history of HT, comorbid conditions, and outcomes. The authors compared rates of severe maternal morbidity (SMM), nontransfusion SMM, cardiovascular SMM (cSMM), and preterm birth from delivery hospitalization between HT recipients and no-HT recipients. The authors evaluated readmission to 330 days postpartum. Logistic and proportional hazard regressions were performed, adjusting for age, socioeconomic and facility characteristics, and clinical comorbidities. RESULTS: Among 19,399,521 deliveries, 105 were HT recipients. Compared with no-HT, HT recipients were at higher risk for all SMM (24.8% vs 1.7%), nontransfusion SMM (20.8% vs 0.7%), cSMM (7.3% vs 0.12%), and preterm birth (43.3% vs 8.2%), all P < 0.001. In adjusted analyses, HT recipients had 16-fold greater odds of SMM, 28-fold greater odds of nontransfusion SMM, 38-fold greater odds of cSMM, and 7-fold greater odds of preterm birth. HT recipients had higher morbidity rates during delivery hospitalization and higher readmission rates within 1 year following delivery (26.9% vs 3.8%; adjusted HR: 6.03 [95% CI: 3.73-9.75]). CONCLUSIONS: Delivery with history of HT is associated with significantly increased rates of SMM, preterm birth, and hospital readmission. These results provide data regarding pregnancy outcomes for use when counseling patients with HT history who are considering pregnancy or who are pregnant.

Real-world Experiences in the Transplantation of Hepatitis C-NAAT-positive Organs.

Background: Hepatitis C virus (HCV) nucleic acid amplification test (NAAT)-positive donors have increased the organ pool. Direct-acting antivirals (DAAs) have led to high rates of treatment success and sustained virologic response (SVR) in recipients with donor-derived HCV infection without significant adverse effects, although variability remains in the timing and duration of antivirals. Methods: This retrospective study analyzed all adult HCV-NAAT-negative transplant recipients who received an organ from HCV-NAAT-positive donors from November 24, 2018, to March 31, 2022, at Duke University Medical Center with protocolized delay of DAA initiation until after hospital discharge, with at least 180-d follow-up on all patients. Transplant and HCV-related outcomes were analyzed. Results: Two hundred eleven transplants (111 kidneys, 41 livers, 34 hearts, and 25 lungs) were performed from HCV-NAAT-positive donors to HCV-NAAT-negative recipients. Ninety percent of recipients became viremic within 7 d posttransplant. Ninety-nine percent of recipients were initiated on pangenotypic DAAs in the outpatient setting a median of 52 d posttransplant, most commonly with 12-wk courses of sofosbuvir-velpatasvir (lungs) and glecaprevir-pibrentasvir (heart, kidney, and liver). Ninety-seven percent of recipients had SVR after a first-line DAA; all ultimately achieved SVR at 12 wk after subsequent treatment courses. The median peak HCV RNA for all organ systems was 2 436 512 IU/mL; the median time from antiviral to undetectable RNA was 48 d, although differences were noted between organ groups. No patient deaths or graft losses were directly attributable to HCV infection. Conclusions: One hundred percent of transplant recipients of HCV-NAAT-positive organs ultimately developed SVR without significant adverse effects when HCV antivirals were initiated in the outpatient setting after transplant hospitalization, suggesting that this real-world treatment pathway is a viable option.

Seroprevalence and incidence of primary dengue infection and its correlation with fetomaternal prognosis in Western Rajasthan.

Background: Viral hemorrhagic fevers are becoming increasingly common in the tropics and subtropics. Dengue fever is currently the most important arthropod-borne viral disease because of its widespread distribution in more than 100 countries and its potential for extensive outbreaks of life-threatening disease. Material and Methods: This study was a hospital-based cross-sectional study conducted in the Microbiology Laboratory of Maternal and Child Tertiary Care Hospital in Western Rajasthan, India, between January 2021 and December 2021. Institutional Ethical Committee permission was obtained. All patients with clinical suspicion of dengue-like illness (DLI), attending outpatient department (OPD) or inpatient department (IPD), were included in the study after obtaining their written consent. A blood sample was collected, and the Dengue Duo rapid card test was conducted for the detection of nonstructural protein 1 (NS1) antigen and immunoglobulin (Ig) M or IgG antibody estimation. All positive samples were tested for IgM enzyme-linked immunosorbent assay (ELISA) test using MAC-ELISA. Results: Of 250 positive sample, the distribution of cases as per clinical features was as follows: all cases presented with fever (100%) followed by myalgia (24.5%), headache (16.06%), hemorrhagic manifestation (13.25%), rash (8.84%), and bleeding gums (2.01%). Thrombocytopenia was seen in 30.40% (76/250) of dengue fever cases. NS1 antigen was detected in 157 cases (62.80%) followed by IgG in 84 cases (33.60%), IgM in 77 cases (30.80%), NS1+IgG in 27 cases (10.80%), NS1 + IgM in 16 cases (6.40%), and NS1 + IgM + IgG in five cases (2%). Of 250 samples, 77 cases were IgM positive and 173 were IgM negative by the Dengue Duo card test. Among the 173 Dengue Duo IgM card negative, 131 cases (79.39%) were also detected negative by IgM ELISA and 42 cases (49.41%) were detected positive by IgM ELISA. The sensitivity was 50.59%, the specificity was 79.39%, the positive predictive value (PPV) was 55.84%, the negative predictive value (NPV) was 75.72%, and the diagnostic accuracy was 69.90%. The case fatality of the cases was 2.35%. Conclusion: Early diagnosis and treatment can prevent mortality in pediatric and pregnant females suffering from dengue and dengue-like illness. Facility and availability of ELISA kits should be adequate for early confirmation of suspected dengue patients by ELISA test.

A comparative study of vision-related quality of life (VRQoL) among glaucoma and nonglaucoma patients at a tertiary care center of North India.

Purpose: Glaucoma influences vision-related quality of life (VRQoL) of an individual in various ways. There are very limited studies on the Indian population and the northern part of India. Therefore, we conducted the present study to evaluate VRQoL in glaucoma patients as well as the association between the severity of glaucoma and VRQoL. Methods: An institution-based, cross-sectional, analytical study was conducted from August 2022 to October 2022 involving 190 participants (95 glaucoma patients and 95 controls). The glaucoma quality of life-15 (GQL-15) questionnaire was explained in their vernacular language by the interviewer. The scoring was given according to the validated scoring algorithm for the questionnaire. Results: Mean GQL score in the control group was 19.66 ± 5.5 and in glaucoma cases was 32.8 ± 10.2, whereas the mean score of mild glaucoma cases was 22.3 ± 4.83, moderate glaucoma cases was 36.3 ± 4.09, and severe glaucoma cases was 47.24 ± 3.03. Therefore, as the severity of glaucoma increases, the GQL score also increases, indicating poorer quality of life. Visual field loss was strongly positively correlated (correlation coefficient = 0.759, P < 0.01) with the GQL-15 score, while a weaker positive correlation was found with best corrected visual acuity (BCVA), cup disc ratio, and duration of treatment. Conclusion: Besides controlling or reducing intraocular pressure (IOP) to the target level, the goal of glaucoma treatment should be to provide an individual with good functional vision to maintain an acceptable quality of life. It would also help in providing patients with the best possible treatment, not only in terms of good vision but also in maintaining or improving their overall quality of life.

Pattern of Food Allergen Sensitivity Amongst Adult Allergic Rhinitis Patients: A Four Year Central Indian Study.

AIMS: Only limited Indian data is available regarding the food allergy pattern among allergic rhinitis patients in Indian population. This study aims to assess the pattern of food allergen sensitivity amongst allergic rhinitis patients in central India. MATERIAL AND METHODS: A total of 218 subjects with allergic rhinitis were enrolled in the study (from May 2018 to August 2022). Skin prick test was done in all subjects with proper technique and precautions by using 125 common food allergens and 75 aero allergens. The test readings were noted after 20 minutes by comparing the wheals so formed with the negative control of Saline and positive control of Histamine. Any reaction with a wheal diameter of 3mm or greater was considered as positive. RESULTS: While test results of both food and inhalant allergens were issued to individual patients, this study was restricted to detection and analysis of pattern of food allergens. Our study observed male preponderance with predominantly a third decade affliction. The most common food allergen in the study population was beetle nut (29.3%) followed by chilli powder and spinach (28.8% each). CONCLUSION: Along with aeroallergens, food allergens are also important provocateurs of allergic rhinitis. Diagnosing the offending food allergens and its avoidance reduces patient morbidity, need for pharmaceutical agents and subsequently drug dependence and its side effects. Offering subjects a replacement diet with food items of similar taste and nutritive character helps in sustainable avoidance therapy.

Metastatic Renal Cell Carcinoma: An Enigmatic Nasal Mass.

Metastatic Renal Cell Carcinoma rarely presents in head and neck and is even rarer in the sinonasal region. However, a sinonasal metastatic mass is usually of RCC origin. These metastases may present prior to the renal symptoms or may appear after primary treatment. Report a 60-year lady with epistaxis due to metastatic RCC. Calculate total published cases of sino-nasal metastasis of RCC. Classify according to sequence of primary and metastatic presentation. A computer aided search of PubMed and Google scholar databases was done using pertinent combinations of the keywords "renal cell carcinoma", "nose and paranasal sinus", "metastasis", "delayed metastasis" and "unusual presentation", revealing 1350 articles. 38 relevant articles were included in the review. Our case presented with epistaxis 3 years after primary RCC. She had a vascular left sided nasal mass which was excised enblock. Immunohistochemistry confirmed metastatic RCC. She is on oral chemotherapy and asymptomatic 1 year post excision. Literature search revealed 116 such cases. 19 patients presented within 10 years of RCC while 7 more were delayed metastasis. 17 cases presented primarily with nasal symptoms with subsequent incidental renal mass. Chronology of presentation was unavailable in the rest 73 cases. We recommend to consider the diagnosis of sinonasal metastatic RCC in a patient presenting with epistaxis or nasal mass, particularly with a past history of RCC. Also, any person with known diagnosis of RCC should undergo regular ENT examination for early diagnosis of sinonasal metastasis.

The association with organ procurement techniques and early cardiac transplant outcomes using cardiac MRI.

BACKGROUND: Heart transplantation (HT) has historically been limited by organ availability. Use of donation after circulatory death (DCD) donors addresses this limitation by utilizing previously unused hearts through use of the Organ Care System (OCS). OBJECTIVES: This study aimed to determine the impact of procurement and transportation method on allograft structure and function using early post-transplant cardiac magnetic resonance imaging (MRI). METHODS: Patients who underwent HT at our institution from February 1, 2020, through April 30, 2021 who underwent cardiac MRI imaging <60 days from transplant were included. Recipient and donor characteristics, clinical outcomes, and MRI findings were compared between those who underwent DCD transplantation using the OCS device (DCD-OCS), brain dead donation (DBD) using the OCS device (DBD-OCS), and DBD transported via cold storage (DBD-cold storage) using one-way analysis of variance. RESULTS: A total of 85 patients underwent HT with a cardiac MRI during the study period. Thirty-one (36%) patients received a DCD organ, 16 (19%) received a DBD-OCS organ and 38 (45%) received a DBD-cold storage organ. Rates of primary graft dysfunction (PGD) were significantly higher in DCD transplants (19.5% DCD vs. .0% DBD-OCS and 5.3% DBD-cold storage; p < .050 across three groups), but with no differences in mortality or rejection. There were no differences in cardiac MRI findings between the three transplant types, including presence of gadolinium hyperenhancement after transplant (all p > .050). CONCLUSIONS: We observed no differences in early cardiac MRI findings between patients that received DCD and DBD-OCS heart transplants compared with those receiving DBD-cold storage transplants.

Dexmedetomidine versus clonidine as an adjuvant to local anaesthetic in brachial plexus blocks: a meta-analysis of randomised controlled trials.

OBJECTIVE: This meta-analysis aimed to compare the efficacy and safety of dexmedetomidine and clonidine as an adjuvant to local anesthetics in BPBs. METHODS: Two investigators independently searched databases to identify all RCTs comparing the efficacy and/or safety of dexmedetomidine and clonidine as an adjuvant to local anesthetics in BPBs. All outcomes were pooled using the inverse variance method with a random-effect model. An I2 test was used to assess heterogeneity. The source of heterogeneity was explored through meta-regression. The quality of the evidence was assessed using the GRADE approach. RESULTS: Out of 123 full texts assessed, 24 studies (1448 patients) were included in the analysis. As compared to clonidine, dexmedetomidine groups showed significantly longer sensory block duration (MD = 173.31; 95% CI 138.02‒208.59; I2 = 99%; GRADE approach evidence: high); motor block duration (MD = 158.35; 95% CI 131.55‒185.16; I2 = 98%; GRADE approach evidence: high), duration of analgesia (MD = 203.92; 95% CI 169.25‒238.58; I2 = 99%; GRADE approach evidence- high), and provided higher grade quality of block (RR = 1.97; 95% CI 1.60‒2.41; I2 = 0%; GRADE approach evidence: moderate). The block positioning technique (regression coefficient: 51.45, p = 0.005) was observed as a significant predictor of the heterogeneity in the case of sensory block duration. No significant difference was observed for the risk of hypotension (RR = 2.59; 95% CI 0.63‒10.66; I2 = %). CONCLUSION: Moderate to high-quality evidence suggests dexmedetomidine is a more efficacious adjuvant to local anesthetic in BPBs than clonidine.

Incidence and clinical correlates of de-novo aortic regurgitation with a fully magnetically levitated left ventricular assist device: a MOMENTUM 3 trial portfolio analysis.

AIMS: We assessed the incidence, predictors and clinical correlates of de-novo aortic regurgitation (AR), which physiologically reduces left ventricular assist device (LVAD) effectiveness due to recirculation syndrome, in the MOMENTUM 3 trial portfolio of the fully magnetically levitated HeartMate 3 (HM3) pump using the randomized pivotal trial (PT) and post-trial continued access protocol (CAP). METHODS AND RESULTS: De-novo aortic regurgitation incidence at 2 years was analysed in the randomized PT and validated in the first 1000 implanted patients of the CAP. Patients with concomitant/prior aortic valve surgery or without baseline or post-implant echocardiograms were excluded from this analysis. AR severity was assessed qualitatively by site-adjudicated echocardiograms (significant AR was defined as moderate or severe grade on echocardiogram). Of 1028 patients enrolled in the PT, 918 were eligible for inclusion in this analysis (HM3, n = 465; HMII, n = 453). At 2 years of LVAD support, freedom from significant AR was greater in the HM3 (92%) than HMII (82%) (hazard ratio 0.45, 95% confidence interval 0.27-0.75, p < 0.01). Of 907 HM3 patients analysed from the first 1000 implanted CAP patients, the rate of freedom from significant AR was 90%, consistent with the PT (p = 0.3). In the combined HM3 group (n = 1372), multivariable Cox modelling identified increasing age and female sex as significant predictors. Survival free of urgent transplant or AR corrective procedure was similar between HM3 patients with and without significant de-novo AR. CONCLUSIONS: The development of moderate or severe grade de-novo AR is reduced with the fully magnetically levitated HM3 LVAD compared to the axial-flow HMII pump. The occurrence of significant de-novo AR with the HM3 pump is not associated with a worse outcome at 2 years of follow-up.

Dexmedetomidine versus clonidine as an adjuvant to local anaesthetic in brachial plexus blocks: a metaanalysis of randomised controlled trials

Abstract Objective: This meta-analysis aimed to compare the efficacy and safety of dexmedetomidine and Clonidine as an adjuvant to local anesthetics in BPBs. Methods: Two investigators independently searched databases to identify all RCTs comparing the efficacy and/or safety of dexmedetomidine and Clonidine as an adjuvant to local anesthetics in BPBs. All outcomes were pooled using the inverse variance method with a random-effect model. An I2 test was used to assess heterogeneity. The source of heterogeneity was explored through meta-regression. The quality of the evidence was assessed using the GRADE approach. Results: Out of 123 full texts assessed, 24 studies (1448 patients) were included in the analysis. As compared to Clonidine, dexmedetomidine groups showed significantly longer sensory block duration (MD = 173.31; 95% CI 138.02-208.59; I2 = 99%; GRADE approach evidence: high); motor block duration (MD = 158.35; 95% CI 131.55-185.16; I2 = 98%; GRADE approach evidence: high), duration of analgesia (MD = 203.92; 95% CI 169.25-238.58; I2 = 99%; GRADE approach evidence-high), and provided higher grade quality of block (RR = 1.97; 95% CI 1.60-2.41 ; I2 = 0%; GRADE approach evidence: moderate). The block positioning technique (regression coefficient: 51.45, p = 0.005) was observed as a significant predictor of the heterogeneity in the case of sensory block duration. No significant difference was observed for the risk of hypotension (RR = 2.59; 95% CI 0.63-10.66; I2 = %). Conclusion: Moderate to high-quality evidence suggests dexmedetomidine is a more efficacious adjuvant to local anesthetic in BPBs than Clonidine.

Role of cardiac magnetic resonance (CMR) in planning ventricular septal myomectomy in patients with hypertrophic obstructive cardiomyopathy (HOCM).

Septal myectomy is currently the gold standard treatment for symptomatic patients with hypertrophic obstructive cardiomyopathy (HOCM). The procedure needs to be tailored and performed in a personalized fashion, taking into consideration the anatomic and physiologic heterogeneity of this disease. The extent and location of surgical myectomy will depend on the location of the hypertrophy, with the goal of widening the outflow tract and improve the function of the mitral valve. CMR helps to identify hypertrophy not well visualized by TTE, providing more accurate wall thickness measurements and differentiating HOCM from other causes of LV hypertrophy. CMR also helps identify an abnormal attachment of papillary muscle to the MV or to the septal myocardium and mitral valve pathology. A collaborative approach with cardiac surgeons, radiologists and cardiologists will optimize preoperative planning to improve the success for surgical myectomy.

Effect of increased screen time on eyes during COVID-19 pandemic.

Purpose: The objective of the study was to assess the effect of increased screen time on ocular health during the coronavirus disease (COVID-19) crisis. Materials and Method: An online pretested, self-reported questionnaire with relevant details was generated through Google form and sent to participants. Chi-square or Fisher's exact test was used to investigate the associations between the qualitative variables. The associated risk factors of number and frequency of ocular health problems were analysed by univariate and multivariate logistic regression. Result: A total of 435 responses were considered where 48.5% (N = 211) were female participants and 51.5% (N = 224) were male. Average age of the participants was 35 years. 89% of the participants reported an increase in the screen time during the during the lockdown period. Younger age group reported to have greater screen time than the older participants (p = 0.001) and hence experienced more symptoms of digital eye strain (DES) (p = 0.003). The most common symptoms associated with digital eye strain in our study were eyestrain 52.8% (N = 230) and headache 31.3% (N = 136). In total, 81.37% (354/435) of participants had experienced at least one symptom related to digital screen usage. Conclusion: DES is non-vision-threatening but discomfort caused due to it can have implications on overall physical, mental, and social well-being. The study highlights the increase in digital screen time during the pandemic and the resultant eye strain. There is need of spreading awareness regarding the adverse effects of digital device use and the preventive measures to safeguard our ocular health.

Selection of Ideal Reference Genes for Gene Expression Analysis in COVID-19 and Mucormycosis.

Selection of reference genes during real-time quantitative PCR (qRT-PCR) is critical to determine accurate and reliable mRNA expression. Nonetheless, not a single study has investigated the expression stability of candidate reference genes to determine their suitability as internal controls in SARS-CoV-2 infection or COVID-19-associated mucormycosis (CAM). Using qRT-PCR, we determined expression stability of the nine most commonly used housekeeping genes, namely, TATA-box binding protein (TBP), cyclophilin (CypA), ß-2-microglobulin (B2M), 18S rRNA (18S), peroxisome proliferator-activated receptor gamma (PPARG) coactivator 1 alpha (PGC-1α), glucuronidase beta (GUSB), hypoxanthine phosphoribosyltransferase 1 (HPRT-1), ß-ACTIN, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in patients with COVID-19 of various severities (asymptomatic, mild, moderate, and severe) and those with CAM. We used statistical algorithms (delta-CT [threshold cycle], NormFinder, BestKeeper, GeNorm, and RefFinder) to select the most appropriate reference gene and observed that clinical severity profoundly influences expression stability of reference genes. CypA demonstrated the most consistent expression irrespective of disease severity and emerged as the most suitable reference gene in COVID-19 and CAM. Incidentally, GAPDH, the most commonly used reference gene, showed the maximum variations in expression and emerged as the least suitable. Next, we determined expression of nuclear factor erythroid 2-related factor 2 (NRF2), interleukin-6 (IL-6), and IL-15 using CypA and GAPDH as internal controls and show that CypA-normalized expression matches well with the RNA sequencing-based expression of these genes. Further, IL-6 expression correlated well with the plasma levels of IL-6 and C-reactive protein, a marker of inflammation. In conclusion, GAPDH emerged as the least suitable and CypA as the most suitable reference gene in COVID-19 and CAM. The results highlight the expression variability of housekeeping genes due to disease severity and provide a strong rationale for identification of appropriate reference genes in other chronic conditions as well. IMPORTANCE Gene expression studies are critical to develop new diagnostics, therapeutics, and prognostic modalities. However, accurate determination of expression requires data normalization with a reference gene, whose expression does not vary across different disease stages. Misidentification of a reference gene can produce inaccurate results. Unfortunately, despite the global impact of COVID-19 and an urgent unmet need for better treatment, not a single study has investigated the expression stability of housekeeping genes across the disease spectrum to determine their suitability as internal controls. Our study identifies CypA and then TBP as the two most suitable reference genes for COVID-19 and CAM. Further, GAPDH, the most commonly used reference gene in COVID-19 studies, turned out to be the least suitable. This work fills an important gap in the field and promises to facilitate determination of an accurate expression of genes to catalyze development of novel molecular diagnostics and therapeutics for improved patient care.