Diagnostic accuracy of Dengue NS1 lateral flow immunoassay in comparison to reverse transcriptase polymerase chain reaction and enzyme linked Immunosorbent Assay.

The most widely used invitro diagnostic qualitative screening method for dengue virus infection is the lateral flow immunoassay technique. Testing of dengue non-structural antigen NS1 offers specificity in determining the active infection while testing of IgM and IgG helps in differentiating the primary and secondary dengue infections. The ELISA functions as the golden standard for dengue testing and PCR credits for the most accurate determination tool at the genetic level. The RT-PCR endorsed NS1 gene and in ELISA or LFIA NS1 antigen is used as the marker owing to the specificity and lesser chances of mutation effects. This study evaluated the performance of AG-Q Dengue NS1 LFIA kit in comparison with RT-PCR quantification cycle (Cq) Values and ELISA NS1 quantitation. The study also focused on differentiating the samples among dengue serotypes using the RealStar Dengue Type RT-PCR Kit 1.0. Dengue serotype 2 is the prominent viral strain in Kerala region succeeded by serotype 3 and 1 with a prevalence rate of 64 %, 20 % and 6 % respectively. Dengue serotype 4 was not reported during this study period. 10 % co-infection with DENV 1 & DENV 2 was also reported. The AG-Q Dengue NS1 kit stood as efficient in screening by providing positive results with samples having RT-PCR Cq values up to 43 and ELISA NS1 quantification minimum of 14 Panbio units.

Impact of Covishield Vaccination in Terms of SARS CoV-2 Neutralizing Antibody Expression.

The vaccination efficacy can indirectly be assessed through the quantification of neutralizing antibodies. Very few data are available on Covishield efficacy in terms of neutralizing antibody expression upon vaccination. This study is focused on profiling of neutralizing antibody expression during and after the Covishield two shot vaccination and observing COVID-19 infection in vaccinated participants during the period. SARS CoV-2 neutralizing antibody concentrations in samples were estimated using electrochemiluminescence immunoassay kit for Lifotronics eCL8000. The sampling had been done sequentially at 45th, 85th day after 1st dose and 15th day after 2nd dose Covishield vaccination. Parallelly, in order to confirm the total SARS CoV-2 IgG response in COVID-19 infection, measured the IgG using SARS CoV-2 IgG lateral flow immunoassay test kit. The subjects previously infected with COVID-19 before 1st dose vaccination demonstrated high neutralizing antibody (> 10AU/ml). In COVID-19 uninfected subjects, there was a sudden incline in neutralizing antibody after the 2nd dose. Infection with SARS CoV-2 between 1st and 2nd dose of Covishield vaccination implicate that the level of neutralizing antibody in serum after 1st dose was not adequate to combat the virus and prevent infection. We observed COVID-19 infection in participants even after 2nd dose of vaccination. Interestingly, there was no protection against SARS CoV-2 even with a high neutralizing antibody expression of 188.5 AU/mL after the 2nd dose. Findings of Covishield efficacy in different cohort samples before and after 2 doses of Covishield vaccination provide impetus for improvement or development of next generation vaccines.

Diagnostic Accuracy of SARS-CoV-2 Nucleocapsid Antigen Self-Test in Comparison to Reverse Transcriptase-Polymerase Chain Reaction.

BACKGROUND: Currently, the rapid antigen test (RAT) and reverse transcriptase-polymerase chain reaction (RT-PCR) are considered the main stakeholders in COVID-19 diagnosis. In RT-PCR, any of at least 2 evolutionary conserved genes (RdRP, E-, N-, ORF1ab gene) and S-gene of SARS-CoV-2 are endorsed, and in RAT, the nucleocapsid antigen (N-Ag) of SARS-CoV-2 is considered due to its stability and fewer chances of mutation effects. In the present work, we evaluated the performance of the AG-Q COVID-19 N-Ag self-test kit and conducted a validation study in comparison with the RT-PCR. METHODS: AG-Q COVID-19 N-Ag rapid test kit is an Indian Council of Medical Research (ICMR) approved product developed and marketed by Agappe Diagnostics Limited. The RT-PCR assay was performed with a COVIPATH COVID-19 RT-PCR kit from Thermo Fisher Scientific. RESULTS: We observed 19 false-negative results in antigen self-tests, including samples of threshold cycle (Ct) values 22/22 (N-gene/ORF1ab-gene) in RT-PCR, indicating inadequate sampling by the patients in self-tests, leading to false-negative results and increased chances of the disease spreading. Based on the RT-PCR Ct value vs antigen self-test comparison, it is evident that proper sampling is crucial in performing antigen self-tests. Also, there were weak positive results in antigen self-tests with a Ct value of 18/19 in RT-PCR. CONCLUSIONS: Although the sensitivity and diagnostic accuracy offered by the AG-Q COVID-19 N-Antigen self-test in comparison with RT-PCR fulfills the ICMR tenets for RAT, this study recommends the laboratory/hospital-based RAT execution would be appropriate, rather than the self-test.

Sequential Profiling of Anti-SARS CoV-2 IgG Antibody in Post COVID-19 Patients.

Upon SARS CoV-2 infection, humoral immune system triggers production of anti-SARS CoV-2 IgM and IgG antibodies. Currently, antibodies against SARS CoV-2 spike protein receptor binding domain play a central role in disease protection, making them potential target for in vitro diagnostics applications. This study determines the expression level and sustainability of anti-receptor binding domain (RBD) SARS CoV-2 IgG in post COVID-19 patients. Anti-RBD SARS CoV-2 IgG antibodies in patient serum were analysed by standardised indirect ELISA using SARS CoV-2 spike receptor binding domain protein and HRP conjugated anti-human IgG antibody (anti-h IgG). The study was conducted using 35 adult patient samples with confirmed SARS CoV-2 infection. Additionally, correlation between antibody response after each stage and disease symptoms in post COVID-19 patients were studied. Maximum antibody titre was seen at Day 40 and decreased relatively to Day 180 in antibody positive samples when compared with controls. Overall, more IgG antibody expression is observed in patients who suffered from loss of smell and taste at Day 40. 71% of the positive subjects in this study showed high SARS CoV-2 IgG antibody concentration of above 10 ng/mL and 37% showed strong antibody concentration above 20 ng/mL at the peak of seroconversion.

Development and Troubleshooting in Lateral Flow Immunochromatography Assays.

The development of Lateral Flow Immunochromatography Assay can be divided into two levels; standardizing membrane characteristics and optimizing molecular level immunoassay reaction between analyte and detector molecules. In the preliminary phase the reaction specificity of capture and detector antibodies with the analyte has to be checked with other techniques like ELISA. Molarity and pH of conjugation buffer have prime importance in the immunoreaction among analyte and antibodies. Epitope mapping of the capture and detector antibodies is also recommended to confirm the specificity of the assay. Standardization of membrane characteristics directly relates to the sensitivity of the assay through its porosity, hydrophobicity, protein holding/releasing capacity and wicking rate. Under optimised condition a perfect Lateral Flow Immunochromatography Assay should have high on-rate (target binding efficiency), low off-rate (target releasing efficiency) and low Cross-reactivity. In this manuscript, we share our experience, especially on developmental strategies and troubleshooting, that we have experienced during Lateral Flow Immunochromatography Assay kit development.

COVID-19: Current Trends in Invitro Diagnostics.

The novel coronavirus SARS-CoV-2 is the seventh known species of coronavirus, infectious to human beings. The pandemic COVID-19 spread all over the world with an unprecedented spreading rate after its first appearance in Wuhan, China. As a novel viral disease there in no antiviral treatment or vaccine for the COVID-19. At present, the early detection and the quarantine of infected patients are the ways to stop the spreading of the disease. This review will discuss about the current invitro diagnostic methods used worldwide for the early and accurate diagnosis of COVID-19. Currently the nucleic acid based polymerase chain reaction is used as the reliable diagnostic platform and antigen/antibody detection immunoassays are playing the role of screening tests for early detection and prognosis in COVID-19 treatment.

Platelet parameters in children with chromosome 22q11 deletion and conotruncal heart defects.

BACKGROUND AND OBJECTIVES: The 22q11 deletion syndrome is associated with a wide spectrum of phenotypic features, hence clinical diagnosis is difficult. Individuals with this syndrome are found to have a risk of developing transfusion associated graft-versus-host reaction, if they are given nonirradiated blood. Our aim was to elucidate whether chromosome 22q11 deletion in children with syndromic conotruncal heart defects is associated with platelet abnormality. MATERIALS AND METHODS: The genetic analysis was performed by standard cytogenetic and Fluorescence in situ hybridization technique. The platelet parameters in 39 patients with chromosome 22q11 deletion were compared with 154 cases without deletion. RESULTS: In deletion versus no deletion group, the mean of mean platelet volume (MPV) was 10.5 ± 2.5 vs 7.6 ± 1.5 fL, platelet count was 225 ± 80.7 and 339 ± 127.3 × 10 9 /L and frequency of high MPV was 49% vs 7% (P < .0001). The MPV was associated with a sensitivity of 90.9% and a specificity of 79.6% at a cutoff value of 8.32 fL, (area under the ROC curve 91%). A nonsignificant negative correlation was found between MPV and platelet count (r = -0.152; P = .361) in children with deletion. CONCLUSION: A cutoff value of 8.32 fL for MPV can be an indicator of high risk of chromosome 22q11 deletion in individuals with syndromic conotruncal defects. Individuals with chromosome 22q11 deletion should be given irradiated blood especially during cardiac surgery. Further investigation should clarify the etiology behind variation in frequency of high MPV in different conotruncal lesions.

Dietary Fats and Oxidative Stress: A Cross-Sectional Study Among Coronary Artery Disease Subjects Consuming Coconut Oil/Sunflower Oil.

Coconut oil has been used by the people of Kerala as a cooking medium for several decades. Due to its alleged hypercholesterolemic activity, general population in recent times is shifting to cooking oils rich in polyunsaturated fats, the most popular being sunflower oil. The effect of long-term consumption of sunflower oil on oxidative stress in humans is not well investigated. We studied oxidative stress among coronary artery disease (CAD) patients who were consuming coconut oil or sunflower oil as a part of their routine diet. Men, aged 35-70 years, with established CAD, who presented to the hospital for routine cardiac evaluations, were enrolled in this observational study. Group 1 and 2 consisted of 73 and 80 subjects consuming coconut oil and sunflower oil respectively for over a period of 2 years. Lipid profile and parameters for oxidative stress were evaluated among them. Conventional lipid parameters did not differ significantly between the two groups. Mean vitamin C concentration was significantly reduced for subjects on sunflower oil compared to those consuming coconut oil (P = 0.044). Malondialdehyde was higher for sunflower oil consumers compared to coconut oil consumers (P < 0.0001). Other parameters such as oxidized LDL, GSH, GPx and SOD were not found to be significantly different between the two groups. The results of the present study show that coconut oil did not induce hypercholesterolemia compared to sunflower oil. On the other hand, sunflower oil group had elevated oxidative stress compared to coconut oil group.

A randomized study of coconut oil versus sunflower oil on cardiovascular risk factors in patients with stable coronary heart disease.

BACKGROUND AND RATIONALE: Coronary artery disease (CAD) and its pathological atherosclerotic process are closely related to lipids. Lipids levels are in turn influenced by dietary oils and fats. Saturated fatty acids increase the risk for atherosclerosis by increasing the cholesterol level. This study was conducted to investigate the impact of cooking oil media (coconut oil and sunflower oil) on lipid profile, antioxidant mechanism, and endothelial function in patients with established CAD. DESIGN AND METHODS: In a single center randomized study in India, patients with stable CAD on standard medical care were assigned to receive coconut oil (Group I) or sunflower oil (Group II) as cooking media for 2 years. Anthropometric measurements, serum, lipids, Lipoprotein a, apo B/A-1 ratio, antioxidants, flow-mediated vasodilation, and cardiovascular events were assessed at 3 months, 6 months, 1 year, and 2 years. RESULTS: Hundred patients in each arm completed 2 years with 98% follow-up. There was no statistically significant difference in the anthropometric, biochemical, vascular function, and in cardiovascular events after 2 years. CONCLUSION: Coconut oil even though rich in saturated fatty acids in comparison to sunflower oil when used as cooking oil media over a period of 2 years did not change the lipid-related cardiovascular risk factors and events in those receiving standard medical care.

Protective role of extracts of grape skin and grape flesh on ethanol-induced oxidative stress, inflammation and histological alterations in rat brain.

Ethanol consumption has deleterious effects on all organs and especially on brain. In our study, the radical scavenging properties of grape skin and grape flesh (contains natural antioxidants like polyphenols) was determined in vitro and for in vivo studies, male Wistar rats (16-18 week-old) and 100-120 gm weight were divided in five groups of six animals each. One group were fed with ethanol (1.6 gm/kg body weight), while second group were fed with ethanol (1.6 g/kg body weight) and grape skin extract (2.5 g/kg body weight), the third group were fed with ethanol (1.6 g/kg body weight) and grape flesh extract, daily once for 12 weeks, while the fourth group were fed with ethanol (1.6 g/kg body weight) and tocopheryl acetate (80 mg/kg/day) daily once for 12 weeks. Results of in vitro studies indicate that grape skin extracts showed significant radical scavenging properties (ROS). There was also significantly alteration of serum cytokines in our study.

Analysis of gene mutations among South Indian patients with maple syrup urine disease: identification of four novel mutations.

Maple syrup urine disease (MSUD) is predominantly caused by mutations in the BCKDHA, BCKDHB and DBT genes, which encode for the E1alpha, E1beta and E2 subunits of the branched-chain alpha-keto acid dehydrogenase complex, respectively. Because disease causing mutations play a major role in the development of the disease, prenatal diagnosis at gestational level may have significance in making decisions by parents. Thus, this study was aimed to screen South Indian MSUD patients for mutations and assess the genotype-phenotype correlation. Thirteen patients diagnosed with MSUD by conventional biochemical screening such as urine analysis by DNPH test, thin layer chromatography for amino acids and blood amino acid quantification by HPLC were selected for mutation analysis. The entire coding regions of the BCKDHA, BCKDHB and DBT genes were analyzed for mutations by PCR-based direct DNA sequencing. BCKDHA and BCKDHB mutations were seen in 43% of the total ten patients, while disease-causing DBT gene mutation was observed only in 14%. Three patients displayed no mutations. Novel mutations were c.130C>T in BCKDHA gene, c. 599C>T and c.121_122delAC in BCKDHB gene and c.190G>A in DBT gene. Notably, patients harbouring these mutations were non-responsive to thiamine supplementation and other treatment regimens and might have a worse prognosis as compared to the patients not having such mutations. Thus, identification of these mutations may have a crucial role in the treatment as well as understanding the molecular mechanisms in MSUD.

Dietary grapes (Vitis vinifera) feeding attenuates ethanol-induced oxidative stress in blood and modulates immune functions in mice.

Ethanol metabolism is known to induce overwhelming production of reactive oxygen species (ROS) and also to cause associated immune dysfunction. Several interventional agents of plant origin, in particular fruits and vegetables have been used to counteract these alterations induced by ethanol. In this study, we investigated the efficacy of dietary feeding of skin and flesh of grapes (Vitis vinifera L.) on the alterations in immune and vascular functions in mice with liver abnormalities induced by chronic ethanol consumption. Results revealed that feeding of both grape skin and flesh (2.5 g/kg body wt/day) effectively attenuated the oxidative stress and alterations in immune function and angiogenesis induced by chronic ethanol consumption (1.6 g/kg body wt/day for 12 weeks) in mice. The antioxidant actions of the grape skin and flesh as observed in this study might be attributed to the polyphenols present in the grapes.

Effects of long term ethanol consumption mediated oxidative stress on neovessel generation in liver.

Angiogenesis, the growth of new blood vessels, is essential during tissue repair. Though most molecular mechanisms of angiogenesis are common to the liver and other organs, there was no report available whether alcoholic liver disease also causes angiogenesis. In this study, we examined the effects of long term ethanol (1.6 g/kg body weight/day) consumption on angiogenic responses in the liver of male Wistar strain albino rats (16-18 weeks old, weighing 200-220 g) up to 36 weeks. Chronic ethanol consumption was associated with not only elevated oxidative stress, and altered cytokines expression, but also developed large von Willebrand factor, fibrosis and activation of matrix metalloproteinases. Moreover, vascular endothelial growth factor-receptor 2 (VEGF-R2, fetal liver kinase 1: Flk-1/KDR) expression and neovessel generation in the rat liver were noted after 36 weeks of ethanol consumption. Thus our study provides novel evidence that long-term ethanol consumption is associated with angiogenesis through delicate and coordinated action of a variety of mediators.

Composition of plasma and atheromatous plaque among coronary artery disease subjects consuming coconut oil or sunflower oil as the cooking medium.

OBJECTIVES: Coconut oil, which is rich in medium-chain saturated fatty acids, is the principal cooking medium of the people of Kerala, India. Replacement of saturated fat with polyunsaturated fat is effective in reducing serum cholesterol levels. However, the effect of substituting coconut oil with sunflower oil on the fatty acid composition of plaque has not been thoroughly investigated. We therefore evaluated and compared the fatty acid composition of plasma and plaque among subjects consuming coconut oil or sunflower oil as the cooking medium. METHODS: Endarterectomy samples and plasma samples were obtained from subjects who underwent coronary artery bypass grafts (n = 71). The subjects were grouped based on the type of oil they were using as their cooking medium (coconut oil or sunflower oil). The fatty acid composition in the plaques and the plasma was determined by HPLC and the data were analyzed statistically. RESULTS: Sunflower oil consumers had elevated concentrations of linoleic acid (p = 0.001) in plasma, while coconut oil users had higher myristic acid levels (p = 0.011) in plasma. Medium-chain fatty acids did not differ significantly between the two groups in the plasma. Medium-chain fatty acids were detected in the plaques in both groups of subjects. In contrast to previous reports, long-chain saturated fatty acids dominated the lipid content of plaque in this population, and the fatty acid composition of plaque was not significantly different between the two groups. No correlation between fatty acids of plasma and plaque was observed in either group. CONCLUSION: A change in cooking medium, although it altered the plasma fatty acid composition, was not reflected in the plaque composition.

Organ Specific Tumor Markers: What's New?

Tumor markers are molecules produced in the body in response to cancer. An ideal tumor marker should have high sensitivity and specificity, should be cheap, and should be easily detected in body fluids. Identification of novel markers is important and it is expected that with the advent of newer technologies, more reliable markers will be discovered. This review discusses the currently available tumor markers for different malignancies.

Comparison of haematological parameters in patients with non-alcoholic fatty liver disease and alcoholic liver disease.

INTRODUCTION: Readily available laboratory tests are extremely useful in achieving a better understanding of diseases, and thereby, allow thoughtful management decisions to be made. The examination of peripheral blood smears usually provides excellent clues to the cause of the disease. The main objective of this study was to compare the haematological parameters of patients with non-alcoholic fatty liver disease (NAFLD) and alcoholic liver disease (ALD), and assess whether these tests have any discriminant value between the two conditions. METHODS: The haematological parameters were investigated in 105 NAFLD patients, 40 ALD patients, 32 alcoholics and 77 normal participants. RESULTS: The haemoglobin, red blood cell, haematocrit, lymphocyte count and platelet count were significantly reduced, while the mean corpuscular volume, mean corpuscular haemoglobin and prothrombin time expressed as an international normalised ratio (PT/INR) were significantly elevated in ALD patients compared to the other groups. The platelet count was significantly reduced, while the PT/INR and erythrocyte sedimentation rate (ESR) were significantly elevated in alcoholics compared to normal participants. ESR was also significantly elevated in ALD patients compared to normal participants and NAFLD patients. Compared to the control group, the NAFLD patients have significantly higher haematocrit and significantly lower platelet count. CONCLUSION: Chronic alcoholism is associated with inflammation and haematotoxic effects, while NAFLD has limited effect on haematological parameters.

Variation in prevalence of chromosome 22q11 deletion in subtypes of conotruncal defect in 254 children.

AIM: To determine the frequency of chromosomal aberrations particularly 22q11 deletion in Indian children ≤2 years with different types of conotruncal malformations and their association with abnormal aortic arch. Additionally, extracardiac features were also studied. METHODS: Conventional cytogenetic and fluorescence in situ hybridization analyses were performed in 254 patients with conotruncal defects. Multivariable logistic regression analysis was performed to ascertain extracardiac features helpful in identifying high-risk patients with deletion. RESULTS: Chromosomal abnormalities were identified in 52 (21%) children, of whom 49 (94%) showed 22q11 deletion and 3 (6%) had abnormalities of chromosome 6, 2 and X. None of the 11/254 children with tetralogy of Fallot with absent pulmonary valve showed deletion. The association of 22q11 deletion with right sidedness of the aortic arch varied with the type of conotruncal defect. The eight extracardiac features in combination showed 93.5% agreement with the presence of deletion. CONCLUSION: The extracardiac features along with specific type of conotruncal defect and associated cardiovascular anomaly should alert the clinician for 22q11 deletion testing. However, if deletion analysis is not possible, specific extracardiac features (six dysmorphic facial features, thin long fingers and hypocalcemia) can help to identify an increased risk of 22q11 deletion in patients with conotruncal defect.

Organic acidurias: an updated review.

Organic acidurias are an important class of inherited metabolic disorders arising due to defect in intermediary metabolic pathways of carbohydrate, amino acids and fatty acid oxidation. This review summarizes the current knowledge about the important organic acidurias in the Indian population. Specifically, diagnosis and principles of treatment of organic acidurias are covered. The salient features of common organic acidurias as well as their prevalence in various parts of the world are reviewed in some detail.

Diagnosis of major organic acidurias in children: two years experience at a tertiary care centre.

Organic acid disorders are inherited metabolic disorders in which organic acids accumulate in tissues and biological fluids of affected individuals. Classical organic acidurias include methylmalonic aciduria, propionic aciduria, isovaleric aciduria and maple syrup urine disease (MSUD). They are considered the most frequent metabolic disorders among severely ill children. Patients frequently present with acute symptoms early in life. 420 cases clinically suspected to have organic aciduria, with upper age limit of 12 years for a 2-year period (January 2007-December 2008) were enrolled into this study. Metabolic acidosis and neurological symptoms were the most common signs. Screening tests and thin layer chromatography were done for detection of organic acidurias. Identification and quantitation of organic acids in urine and quantification of amino acids in blood were done by high performance liquid chromatography. Out of 420 patients, 45 patients (10.7%) were found to have organic acidurias. 15 cases of methylmalonic aciduria, 16 cases of propionic aciduria, 13 cases of MSUD, and one case of isovaleric aciduria were diagnosed. Results demonstrate the importance of testing for organic acidurias. Since organic aciduria may cause irreversible brain damage if not treated, we recommend selective screening amongst severely ill children despite implied extra costs.