Development of RNA-Based Assay for Rapid Detection of SARS-CoV-2 in Clinical Samples.

INTRODUCTION: The ongoing spread of pandemic coronavirus disease-19 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is of growing concern. Rapid diagnosis and management of SARS-CoV-2 are crucial for controlling the outbreak in the community. Here, we report the development of a first rapid-colorimetric assay capable of detecting SARS-CoV-2 in the human nasopharyngeal RNA sample in less than 30 min. METHOD: We utilized a nanomaterial-based optical sensing platform to detect RNA-dependent RNA polymerase gene of SARS-CoV-2, where the formation of oligo probe-target hybrid led to salt-induced aggregation and change in gold-colloid color from pink to blue visibility range. Accordingly, we found a change in colloid color from pink to blue in assay containing nasopharyngeal RNA sample from the subject with clinically diagnosed COVID-19. The colloid retained pink color when the test includes samples from COVID-19 negative subjects or human papillomavirus-infected women. RESULTS: The results were validated using nasopharyngeal RNA samples from positive COVID-19 subjects (n = 136). Using real-time polymerase chain reaction as gold standard, the assay was found to have 85.29% sensitivity and 94.12% specificity. The optimized method has detection limit as little as 0.5 ng of SARS-CoV-2 RNA. CONCLUSION: We found that the developed assay rapidly detects SARS-CoV-2 RNA in clinical samples in a cost-effective manner and would be useful in pandemic management by facilitating mass screening.

Influence of genetic polymorphism in renin-angiotensin system-candidate genes on urinary trefoil family factor 3 levels in children with congenital anomalies of kidney and urinary tract.

BACKGROUND: This cross-sectional study aimed to determine the influence of genetic polymorphism in two renin-angiotensin system (RAS)-candidate genes on urinary trefoil family factor 3 (TFF3) levels in children with congenital anomalies of kidney and urinary tract (CAKUT). METHODS: The study included fifty children with CAKUT (PUV, VUR, and PUJO) and twenty age-matched controls. Urinary TFF3 levels were measured by enzyme-linked immunosorbent assay. Detection of genetic polymorphisms in two genes, i.e., I/D polymorphism (SNP at rs4340) in angiotensin-converting enzyme (ACE) and A/T polymorphism in the angiotensin II receptor type-2 (AT2R) due to point mutation at rs3736556 was performed by polymerase chain reaction. Progressive deterioration in kidney function was defined as fall in GFR to < 60 ml/min/1.73 m2 and/or progressive scarring. RESULTS: In our cohort, the genotypic distribution of patients and controls showed no difference. Progressive functional deterioration was significantly associated with the presence of D allele (p = 0.0004), A allele (p = 0.005), and both (p < 0.0001) in patients. Significantly raised TFF3 levels were detected in the urine of children having D allele (D/D > I/D > I/I; p < 0.0001) and A allele (A/A > A/T > TT; p < 0.0001). Also, children with both D/D and A/A allelic genotypes had significantly elevated urinary TFF3 compared to those having either of them. CONCLUSIONS: The presence of D allele and/or A allele is significantly associated with progressive functional deterioration and elevated urinary TFF3 levels. These findings support the role of angiotensin II-AT2R-NF-κB interaction in progressive deterioration of kidney function and subsequent TFF3 expression in CAKUT.

Association of kidney functions with a cascade of care for diabetes and hypertension in two geographically distinct Indian cohorts.

AIM: Type 2 diabetes (T2DM) and hypertension (HTN) are the main modifiable risk factors of chronic kidney disease (CKD), among the known traditional and non-traditional risk factors. METHODS: We determined the prevalence and care-cascade of these modifiable CKD-risk factors and their association with socioeconomic status in adjoining Lucknow and Puducherry cities of India. RESULTS: 439 participants reported no CKD were recruited. Serum analysis revealed an Estimated Glomerular Filtration Rate (eGFR) ≥ 90 ml/min/1.73 m2 in 60.36% of the population. Of them, 55.85% had HTN and/or T2DM as CKD-risk factors; however, less than half of this population was unaware of their CKD-risk status. Awareness and treatment were significantly higher in Puducherry and were associated with literacy, occupation, and residence place. Although the CKD-risk population was about two times higher in Puducherry than Lucknow, the populations with mild-low eGFR were comparable in the two regions. Moreover, in Lucknow, mild-low eGFR and low awareness were more prevalent among the younger participants (<30 years), relative to Puducherry. CONCLUSIONS: Despite a higher prevalence of CKD-risk factors in Puducherry, populations with mild-low eGFR were comparable to Lucknow. More heightened awareness and better care cascade for CKD-risk factors in Puducherry may prevent or delay eGFR reduction.

Ethylmalonic encephalopathy ETHE1 p. D165H mutation alters the mitochondrial function in human skeletal muscle proteome.

Ethylmalonic encephalopathy (EE) is a rare autosomal recessive inborn error of metabolism. To study the molecular effects of ETHE1 p. D165H mutation, we employed mass spectrometry-based mitochondrial proteome and phosphoproteome profiling in the human skeletal muscle. Eighty-six differentially altered proteins were identified, of which thirty-seven mitochondrial proteins were differentially expressed, and most of the proteins (37%) were down-regulated in the OXPHOS complex-IV. Also, nine phosphopeptides that correspond to eight mitochondrial proteins were significantly affected in EE patient. These altered proteins recognized are involved in several pathways and molecular functions, predominantly in oxidoreductase activity. This is the first study that has integrated proteome and phosphoproteome of skeletal muscle and identified multiple proteins associated in the pathogenesis of EE.

Urinary biomarkers as point-of-care tests for predicting progressive deterioration of kidney function in congenital anomalies of kidney and urinary tract: trefoil family factors (TFFs) as the emerging biomarkers.

BACKGROUND: Children with congenital anomalies of kidney and urinary tract (CAKUT) are at high risk of progressive deterioration of kidney function and further developing stage 5 chronic kidney disease (CKD 5), even after a successful surgery. This prospective study was designed to determine whether urinary biomarkers can predict progressive deterioration of kidney function in children with CAKUT. METHODS: The study included 50 consecutive children, aged < 14 years, who were diagnosed with congenital uropathies (PUV, VUR, and PUJO) and 20 age-matched controls. Examination of four urinary biomarkers, i.e., trefoil family factors (TFF) 1 and 3, neutrophil gelatinase-associated lipocalin (NGAL) and microalbuminuria (MALB) was done at the beginning of follow-up. Kidney function was assessed, at the beginning and after 12-months of follow-up, by technetium-99m diethylene triamine pentaacetic acid (DTPA) and technetium-99m dimercaptosuccinic acid (DMSA) scans. Progressive deterioration in the kidney function was defined as a fall in the GFR from ≥ 60 to < 60 ml/min/1.73 m2 on comparing the baseline and latest DTPA scans; and/or new-onset cortical scar/scars or increase in the size of previous scar/scars on serial DMSA scans. Group 1 and group 2 included children without and with progressive functional deterioration respectively. RESULTS: The median (IQR) age of children with CAKUT and controls was 3 (1.5-5) and 2.3 (1.2-3.6) years, respectively, and showed no significant difference (p = 0.29). Median concentrations of TFF1, TFF3, NGAL, and microalbumin in patients were 44.5, 176.5, 281.2, and 15.5 mcg/gCr, respectively, and were significantly elevated as compared to controls (p < 0.05). Children belonging to group 2 had significantly higher concentration of biomarkers as compared to those in group 1. TFF3 was found have the highest AUC (0.9198) on ROC curve for predicting progressive functional deterioration. CONCLUSION: Urinary TFFs, NGAL, and microalbumin significantly correlate with progressive deterioration of kidney function in children harboring CAKUT. TFF3, with the strongest prediction of functional deterioration, is an emerging peptide showing sufficient potential to be included in the biomarker panel. Graphical abstract.

Clinico-pathological and Molecular Spectrum of Mitochondrial Polymerase γ Mutations in a Cohort from India.

Polymerase γ catalytic subunit (POLG), a nuclear gene, encodes the enzyme responsible for mitochondrial DNA (mtDNA) replication. POLG mutations are a major cause of inherited mitochondrial diseases. They present with varied phenotypes, age of onset, and severity. Reports on POLG mutations from India are limited. Hence, this study aimed to describe the clinico-pathological and molecular observations of POLG mutations. A total of 446 patients with clinical diagnosis of mitochondrial disorders were sequenced for all exons and intron-exon boundaries of POLG. Of these, 19 (4.26%) patients (M:F: 10:9) had POLG mutations. The age of onset ranged from 5 to 55 years with an overlapping phenotypic spectrum. Ptosis, peripheral neuropathy, seizures, and ataxia were the common neurological features observed. The most common clinical phenotype was chronic progressive external ophthalmoplegia (CPEO) and CPEO plus (n = 14). Muscle biopsy showed characteristic features of mitochondrial myopathy in fourteen patients (14/19) and respiratory chain enzyme deficiency in eleven patients (11/19). Multiple mtDNA deletions were seen in 47.36% (9/19) patients. Eight pathogenic POLG variations including two novel variations (p.G132R and p.V1106A) were identified. The common pathogenic mutation identified was p.L304R, being present in eight patients (42.1%) predominantly in the younger age group followed by p.W748S in four patients (21%). To the best of our knowledge, this is the first extensive study from India, highlights the clinico-pathological and molecular spectrum of POLG mutations.

Prevalence of hearing loss in India.

Background Despite abundant literature, a clear and coherent understanding of hearing loss (HL) in India is limited by the wide disparity in studies. Methods We did a review of published peer-reviewed journal articles. Studies reporting the prevalence and degree of HL in India from 1980 to 2020 were included. Information was gathered on the population characteristics, methodology applied and the prevalence of hearing impairment. The data were analysed to identify trends and at-risk sections of population in various categories. Results Four hundred and forty studies were identified after a database search; 29 full-length articles were selected for final analysis. Using a 3-step screening protocol, hearing impairment (abnormal auditory brainstem response/auditory steady state response) in neonates ranged between 1.59 and 8.8 per 1000 births. Among 'at risk' neonates, it ranged from 7 to 49.18 per 1000 births. In children the prevalence of HL was 6.6% to 16.47%. Otitis media was the most common cause of HL in children. Community-based studies (all ages) reported prevalence of HL between 6% and 26.9% and prevalence of disabling HL between 4.5% and 18.3%. Rural areas and elderly showed a higher prevalence of hearing impairment. Conclusion Despite India's improving health indices, hearing disability remains persistently high. It is a major contributor to the loss of personal potential and a financial strain for the individual and the country. A large-scale multicentric study to identify the degree and type of HL, social awareness campaigns, widespread neonatal screening, strengthening treatment facilities and well-funded rehabilitation programmes can counter the rising prevalence of hearing impairment.

A Systematic Study of the Prevalence and Risk Factors of CKD in Uddanam, India.

INTRODUCTION: Despite reports of a high prevalence of chronic kidney disease (CKD) from the coastal Uddanam region of Andhra Pradesh, India, there are no accurate data on the distribution of kidney function abnormalities and CKD risk factors in this region. METHODS: A total of 2419 participants were recruited through multistage cluster random sampling from 67 villages. Serum creatinine and urine protein creatinine ratio were measured using validated methodologies. All abnormal estimated glomerular filtration rate (eGFR) and urine protein creatinine ratio values were reconfirmed after 3 months. A range of sociodemographic factors were evaluated for their association with CKD using Poisson regression. RESULTS: Of 2402 eligible subjects (mean ± SD age, 45.67 ± 13.29 years; 51% female), 506 (21.07%) had CKD (mean ± SD age, 51.79 ± 13.12 years; 41.3% female). A total of 246 (10.24%) had eGFR <60 ml/min/1.73 m2, whereas 371 (15.45%) had an elevated urine protein creatinine ratio (>0.15 g/g). The poststratified estimates, adjusted for age and sex distribution of the region for CKD prevalence, are 18.7% (range, 16.4%-21.0%) overall and 21.3% (range, 18.2%-24.4% ) and 16.2% (range, 13.7%-18.8%) in men and women, respectively. Older age, male sex, tobacco use, hypertension, and family history of CKD were independently associated with CKD. Compared with those with higher eGFR, those with eGFR <60 ml/min/1.73m2 were older, were more likely to be uneducated, manual laborers/farmers, or tobacco users, and were more likely to have hypertension, a family history of CKD, a diagnosis of heart disease, and a lower body mass index. Among those with low eGFR, there was no difference between those with urine protein creatinine ratio <0.15 or >0.15, except a lower frequency of males in the former. CONCLUSION: We confirmed the high prevalence of CKD in the adult population of Uddanam. The cause was not apparent in a majority. Subjects with a low eGFR with or without elevated proteinuria were phenotypically distinct from those with proteinuria and preserved eGFR. Our data suggest the need to apply a population-based approach to screening and prevention and studies to understand the causes of CKD in this region.

Options of Renal Replacement Therapy in CKDu.

Patients with advanced Chronic Kidney Disease of Unknown origin (CKDu) need to plan for renal replacement therapy. The patients usually affected are probably best served with living-related renal transplantation. Potential donors from the same area are possibly at risk for developing CKDu and need close monitoring post kidney donation. Peritoneal dialysis (PD) is probably a better option as hemodialysis (HD) centers are located in urban areas only and patients have the convenience of receiving therapy at home. The "PD first" pilot project of Sri Lanka is a unique initiative that trains community physicians to offer PD to patients with advanced CKDu. In Telengana and Andhra Pradesh, the Aarogyasri insurance scheme provides for poor patients to avail of free HD and transplantation in government and private hospitals. Much more needs to be done to care for all those who are affected. A public-private partnership model for providing comprehensive care to patients with advanced CKDu can be undertaken in all areas affected by CKDu that makes renal replacement therapy (RRT) available and accessible, irrespective of financial and social limitations.

Chronic Kidney Disease of Unknown Origin - What do we know?

The global burden of CKD is considerable and has risen dramatically over the past 20 years. Recently, CKD of unknown origin (CKDu), a form of CKD among rural agricultural communities has been reported worldwide. There is no strong evidence for a single cause of CKDu. Basically, it is a phenotypic environmentally acquired disease with a combination of occupational and social factors. Across all geographical regions, CKDu was most frequently associated with men, middle age, snake bite, infection, and exposure to agrochemicals, heavy metals, herbal drugs, heat stress, and dietary exposures. CKDu has emerged as a challenge in certain regions of the world as there is no acceptable global definition for CKDu. There is an urgent need for health promotion at individual and community levels for early screening of risk factors and timely management. It is also important to strengthen the health service networks for a better quality of life and patient safety as well as adequate financing. Further etiological and interventional research is needed to reduce preventable regional risk factors as well as to develop proactive and comprehensive approaches to prevent and treat the disease.

Study to Test and Operationalize Preventive Approaches for CKD of Undetermined Etiology in Andhra Pradesh, India.

INTRODUCTION: High prevalence of chronic kidney disease (CKD) not associated with known risk factors has been reported from coastal districts of Andhra Pradesh. The Study to Test and Operationalize Preventive Approaches for Chronic Kidney Disease of Undetermined Etiology in Andhra Pradesh (STOP CKDu AP) aims to ascertain the burden (prevalence and incidence) of CKD, the risk factor profile, and the community perceptions about the disease in the Uddanam area of Andhra Pradesh. METHODS: Study participants will be sampled from the Uddanam area using multistage cluster random sampling. Information will be collected on the demographic profile, occupational history, and presence of conventional as well as nonconventional risk factors. Glomerular filtration rate (GFR) will be estimated using the Chronic Kidney Disease Epidemiology Collaboration equation, and proteinuria will be measured. All abnormal values will be confirmed by repeat testing after 3 months. Cases of CKD not associated with identified etiologies will be identified. Biospecimens will be stored to explore future hypotheses. The entire cohort will be followed up every 6 months to determine the incidence of CKD and to identify risk factors for decline in kidney function. Qualitative studies will be performed to understand the community perceptions and expectations with respect to the interventions. IMPLICATIONS: CKD is an important public health challenge in low- and middle-income countries. This study will establish the prevalence and determine the incidence of CKD not associated with known risk factors in a reported high-burden region, and will provide insights to help design targeted health systems responses. The findings will contribute to the policy development to tackle CKD in the region and will permit international comparisons with other regions with similar high prevalence.