ABSTRACT
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.
ABSTRACT
Head and neck pathologies requiring surgical intervention are considered a high-risk subsite in the context of COVID-19 pandemic by virtue of its close proximity to the mucosa of the upper aerodigestive tract. Retrospective review of all head and neck surgical procedures is undertaken during the pandemic from 23rd April 2020 to 30th September 2020. One hundred procedures were performed on 98 patients. COVID-19 status determined by SARS-Cov-2 RT-PCR at baseline was negative for 81, positive in 8 and unknown in 11. The RT-PCR negative subgroup included 40 diagnostic procedures and 41 ablative and or reconstructive procedures for head and neck neoplasms. None of the patients or health-care workers converted to COVID-19-positive status during the duration of the hospital stay. There were no cases with 30-day mortality. Clavien-Dindo grading for postoperative complications was as follows: 1-4, 2-12, 3a-2, 3b-1. Eleven patients with unknown COVID-19 status at baseline underwent emergency tracheostomy in a COVID-19 designated operating room for upper airway obstruction secondary to head and neck cancer. Of the 8 procedures conducted on known cases of COVID-19, 6 were tracheostomies performed for COVID-19 ARDS. The rest were maxillectomy for acute invasive mucormycosis and incision and drainage for parotid abscess. A matched-pair analysis was performed with similarly staged historical cohort operated during January to December 2016 to compare peri-operative complication rates (Clavien-Dindo Score). Incidence of complication with higher Clavien Dindo Score (>/=3a) was found to be lower in those patients operated during the pandemic (p=0.007). By meticulous preoperative COVID-19 screening and isolation, head and neck surgical procedures can be continued to avoid delay in diagnosis and treatment without jeopardising the risk of transmission of COVID-19 to the patients or health-care workers.
ABSTRACT
SARS CoV-2 infection is associated with various hematological manifestations, including leucopenia, thrombocytopenia, thrombocytopenia. Severe thrombocytopenia is, however, rare and is associated with severe COVID-19. ITP remains an important differential among other causes. We report a case of HIV-TB-COVID-19 co-infection, without any feature of severe COVID, presenting with severe thrombocytopenia which resolved on its own; cause was attributed to immune-mediated effect of SARS CoV-2 virus.
ABSTRACT
BACKGROUND: Ongoing pandemic because of COVID-19 has spread across countries, with varied clinical features and severity. Awareness of clinical course among asymptomatic and symptomatology in symptomatic cases is essential for patients' management as well as optimal utilization of health services (in resource limited settings) based on clinical status and risk factors. This study aimed to describe the clinical characteristics and outcomes of patients admitted with COVID-19 illness in the initial phase of the pandemic in India. METHODS: It was an observational study. Patients aged 18 years or more, with confirmed SARS-CoV-2 infection, asymptomatic or mildly ill, were included. Patients with moderate-severe disease at admission or incomplete clinical symptomatology records were excluded. Data regarding demography, comorbidities, clinical features and course, treatment, results of SARS-CoV-2 RT-PCR, chest radiographs, and laboratory parameters were obtained retrospectively from hospital records. The outcome was noted in terms of course, patients discharged, still admitted (at the time of the study), or death. RESULTS: Out of 231 cases, most were males (78.3%) with a mean age of 39.8 years. Comorbidities were present in 21.2% of patients, diabetes mellitus and hypertension being the most common. The most common symptoms were dry cough (81, 35%), fever (64, 27.7%), sore throat (36, 15.6%); asymptomatic infection noted in 108 (46.8%) patients. The presence of comorbidities was an independent predictor of symptomatic disease (OR-2.66; 95%CI 1.08-6.53, P = 0·03). None of the patients progressed to moderate-severe COVID-19, and there were no deaths. CONCLUSIONS: A large proportion of patients remained asymptomatic whereas those with comorbidities were more likely to be symptomatic. Most with mild disease had a stable disease course, barring few complication in those with comorbidities. The pandemic continues to grow as large number of asymptomatic cases may go undiagnosed.
ABSTRACT
This study endeavours to comprehensively the study the spectrum of ENT manifestations in mild and asymptomatic COVID-19 and observe the natural course of anosmia and dysgeusia consequent to SARS-Cov-2 infection. A prospective cohort study was undertaken at a tertiary care centre in India on admitted patients with RT-PCR proven COVID-19. Patients were included provided the baseline National Early Warning Score (NEWS) was less than 4. Patients were screened for ENT manifestations using a questionnaire at baseline, 7 days, 14 days and 28 days. 225 patients were included in the study. Of these complete data at 4 weeks was available for 210. Out of 145 patients with mild COVID-19 (asymptomatic = 80), ENT manifestations accounted for 66.2% of all symptoms. Smell and taste disturbance had an overall incidence of 20% and 45% of ENT manifestations. Temporal trends of the recovery rate of anosmia and dysgeusia were 53.6% and 66.7% at 2 weeks, respectively. Corresponding rates at 3 weeks were 89.29% and 86.7%, respectively. By the end of 4 weeks 96% of the patients had recovered completely. The incidence of anosmia and dysgeusia in this study parallels the rates reported from other Asian countries, albeit, lower than the rates quoted from the Western Hemisphere. We recommend structured reporting of all ENT manifestations especially smell and taste disturbances to accurately identify individuals infected with SARS-Cov-2.
ABSTRACT
BACKGROUND: despite being in the 5th month of pandemic, knowledge with respect to viral dynamics, infectivity and RT-PCR positivity continues to evolve. AIM: to analyse the SARS CoV-2 nucleic acid RT-PCR profiles in COVID-19 patients. DESIGN: it was a retrospective, observational study conducted at COVID facilities under AIIMS, New Delhi. METHODS: patients admitted with laboratory confirmed COVID-19 were eligible for enrolment. Patients with incomplete details, or only single PCR tests were excluded. Data regarding demographic details, comorbidities, treatment received and results of SARS-CoV-2 RT-PCR performed on nasopharyngeal and oropharyngeal swabs, collected at different time points, was retrieved from the hospital records. RESULTS: a total of 298 patients were included, majority were males (75·8%) with mean age of 39·07 years (0·6-88 years). The mean duration from symptom onset to first positive RT-PCR was 4·7 days (SD 3·67), while that of symptom onset to last positive test was 17·83 days (SD 6·22). Proportions of positive RT-PCR tests were 100%, 49%, 24%, 8·7% and 20·6% in the 1st, 2nd, 3rd, 4th and >4 weeks of illness. A total of 12 symptomatic patients had prolonged positive test results even after 3 weeks of symptom onset. Age > = 60 years was associated with prolonged RT-PCR positivity (statistically significant). CONCLUSION: this study showed that the average period of PCR positivity is more than 2 weeks in COVID-19 patients; elderly patients have prolonged duration of RT-PCR positivity and requires further follow up.