Study of Association of Serum Prolactin Levels with Insulin Resistance in Type 2 Diabetes Mellitus Patients.

Objective: Prolactin (PRL) regulates glucose metabolism and insulin sensitivity. The study aimed to evaluate the role of PRL in glucose homeostasis and its association with insulin resistance in patients with diabetes mellitus (DM). Methods and Materials: This cross-sectional, observational study included 100 patients (25-60 years) with T2DM. Primary information including demographics, anthropometric measurements, and biochemical measures (complete blood count, glucose parameters, liver and kidney function test, lipid profile, thyroid function test, serum fasting insulin levels, serum PRL levels) was collected. Results: A total of 100 patients, 50 men and 50 women (25 premenopausal and 25 postmenopausal), were enrolled in this study. The correlation between serum cholesterol and PRL was found to be statistically non-significant (P = 0.129) in men and significant (P = 0.041) in women. There was an inverse relationship between fasting plasma glucose and serum PRL levels in both men (r = -0.88; P < 0.0001) and women patients (r = -0.768; P < 0.0001). Negative correlation between postprandial plasma glucose and PRL was found to be statistically significant (r = -0.398; P = 0.048) in postmenopausal women. The comparison in both men and women indicated an inverse correlation between serum PRL and glycated haemoglobin levels. There was a significant negative correlation between homeostasis model assessment-estimated insulin resistance (HOMA-IR) and PRL levels in both men (r = -0.362; P = 0.039) and women patients (r = -0.362; P = 0.003). Homeostasis model assessment of ß cell function (HOMA-ß), which directly correlates with residual pancreatic beta cell function, was positively correlated with prolactin levels, irrespective of gender and menopausal status of female subjects. Conclusion: Serum PRL levels correlate with improved glycaemic control.

Rapid diagnosis of TB using Aptamer-based assays for Mycobacterium tuberculosis antigens in children and adolescents.

Background: Despite advances establishing microbiological evidence of tuberculosis (TB) is still a concern in children due to the limitation of availability of sample and predominance of extrapulmonary TB, there is unmet need for diagnostic tests which are low cost, rapid and sensitive and specific. Methods: This study evaluated the utility of aptamer-based assay for detecting mycobacterium tuberculosis antigens HspX and MPT 64 in rapid diagnosis of TB in children up to 18 years of age in a tertiary medical college. A total of 100 children were sequentially enrolled with presumptive pulmonary (n = 52 and extrapulmonary n = 48) TB based on clinico-radiological characteristics. The samples were evaluated with ALISA technique for TB antigens and compared with the results of ZN microscopy, GeneXpert and mycobacterial culture MGIT. Results: The enrolled children had mean age (11.7 + 4.4 years) with both pulmonary (n = 52) and extrapulmonary TB (n = 48). Our study results concluded poor results of smears (11% positivity, sensitivity: 17.7%, NPV: 42.7%) and better of GeneXpert (positivity: 42%, sensitivity of 67.4%, NPV: 65.5%) and culture (positivity 57%, sensitivity 91.9%, NPV 88.3%). HspX antigen by ALISA had comparable results (positivity: 49%, sensitivity: 62.9%; NPV: 54.9%). MPT 64 antigen by ALISA also had similar results (positivity: 45%, sensitivity: 58% and NPV 52, 3%). Sensitivity and specificity were higher in pulmonary TB compared to EPTB for both antigens. HspX antigen assay by ALISA and MPT 64 ALISA over existing microbiological diagnostic methods had additional of 13%. Conclusion: ALISA technique for mycobacterium antigens HspX and MPT 64 was rapid, low-cost test (1-3$/test) high sensitivity and specificity and comparable to currently available methods.

Nanodot zirconium trisulfide modified conducting thread: A smart substrate for fabrication of next generation biosensor.

In present work, we report an eco-friendly, flexible and highly conducting cotton thread (CT) as a smart substrate for the development of biosensing platform towards ultrasensitive detection of swine flu serum amyloid A (SAA) biomarker. The biosensor was fabricated by optimized coating of CT with poly(3,4-ethylenedioxythiophene): poly(styrenesulfonate) (PEDOT:PSS) conductive ink followed by incorporation of nanodot zirconium trisulfide (nZrS3) which helped in enhancing the electrochemical properties and improving stability of PEDOT:PSS polymeric film. The fabricated nZrS3/PEDOT:PSS/CT electrode was then used for sequential immobilization of monoclonal antibodies of SAA (anti-SAA) and bovine serum albumin (BSA). The synthesized nanomaterials and fabricated electrodes were characterized through X-ray diffraction, Fourier-transform infrared spectroscopy, transmission electron microscopy, scanning electron microscopy and contact angle analyser techniques. The electrochemical response of the fabricated smart thread based biosensor (BSA/anti-SAA/ZrS3/PEDOT:PSS/CT) was recorded against SAA using chronoamperometry technique which revealed superior sensitivity {30.2 µA [log (µg mL-1)]-1 cm-2}, excellent lower detection limit (0.72 ng mL-1) and prolonged shelf life up to 48 days. The response of the biosensor was also validated by analysing the electrochemical response of SAA spiked serum samples and the obtained results showed good correlation with that of standard samples.

MPT51 and MPT64-based antigen detection assay for the diagnosis of extrapulmonary tuberculosis from urine samples.

In view of WHO's "End-TB" strategy, we developed a non-invasive, urine-based ELISA, targeting 2 Mycobacterium tuberculosis antigens namely MPT51 and MPT64 for extrapulmonary TB (EPTB) diagnosis. Suspected EPTB patients (n = 137) [Pleural TB, Abdominal TB and Tuberculous meningitis] were categorized in "Definite" EPTB (n = 10) [Xpert-MTB/RIF and/or culture-positive], "Probable" EPTB (n = 77) and "Non-EPTB" (n = 50) groups using defined composite reference standards. ROC-curves were generated using ELISA results of "Definite" EPTB and "Non-EPTB" groups for both antigens independently and cut-off values were selected to provide 86.3% (95%CI:73.3-94.2) specificity for MPT51 and 92% (95%CI:80.8-97.8) for MPT64. The sensitivity of MPT51-ELISA and MPT64-ELISA was 70% (95%CI:34.7-93.3) and 90% (95%CI:55.5-99.7) for "Definite" EPTB group and 32.5% (95%CI:22.2-44.1) and 30.8% (95%CI:20.8-42.2) for "Probable" EPTB group, respectively. Combining the results of both ELISAs showed a 100% (95%CI:69.1-100) sensitivity in "Definite" EPTB group and 41.6% (95%CI:30.4-53.4) in "Probable" EPTB group, with an 80% (95%CI:66.3-89.9) specificity. The results demonstrated the potential of urine-based ELISAs as screening tests for EPTB diagnosis.

Biochemical interaction of human hemoglobin with ionic liquids of noscapinoids: Spectroscopic and computational approach.

In this work, we have developed noscapine based ionic liquids i.e., Noscapine (MeNOS) and 9-Bromonoscapine (MeBrNOS) as cation supported with bis(trifluoromethylsulfonyl)amide (NTf2-) as anion. We have reported the mechanism of binding interaction between noscapine based ILs and human hemoglobin (Hb) using various spectroscopic and computational techniques. The corresponding thermodynamics studies showed that the binding is exothermic in nature and major forces responsible for binding are Van der waals and hydrogen bonding interaction. The fluorescence spectra showed that the intensity of Hb decreases in the presence of [MeNOS]NTf2 and [MeBrNOS]NTf2 both shows static quenching. The secondary structural changes in Hb were observed and calculated by using CD spectroscopy. Molecular docking studies revealed that both the ILs show strong binding in ß1 fragment of the tetrameric structure of Hb, but the binding of [MeNOS]NTf2 is relatively stronger than [MeBrNOS]NTf2 and the results are supported by MD simulations.

Utility of cell-free transrenal DNA for the diagnosis of Tuberculous Meningitis: A proof-of-concept study.

Tuberculous Meningitis (TBM) diagnosis remains a grave challenge. We evaluated the utility of extracellular vesicles (EVs) as a source of cell-free transrenal-mycobacterial DNA (cf-Tr-MTB DNA) for TBM diagnosis from urine samples. We developed a qPCR-assay targeting a highly repetitive 36-bp sequence specific to Mycobacterium tuberculosis complex. EVs were isolated from urine samples of suspected TBM groups (n = 44) [categorized using composite reference standard as 'Definite' TBM (n = 8), 'Probable' TBM (n = 15), 'Possible' TBM (n = 21)] and 'Non-TBM' group (n = 26). cf-Tr-MTB DNA-based qPCR assay was applied to DNA isolated from EVs (EV-DNA) and EV-free-fraction (EV-free DNA). ROC-curves were generated using qPCR results of 'Definite' TBM and 'Non-TBM' category in both EV-DNA and EV-free DNA samples and cut-off values were selected to provide 100% (95%CI:69.1-100) specificity. The cf-Tr-MTB DNA assay gave a sensitivity of 54.5% (95%CI:38.8-69.6) for EV-DNA and 77.3% (95%CI:62.1-88.5) for EV-free DNA in the TBM group (n = 44). The combination of EV-DNA and EV-free DNA results (corresponding to performance cf-Tr MTB DNA assay in urine), gave an overall sensitivity of 81.8% (95%CI:67.2-91.8) in the TBM group. Our results confirmed EVs as one of the sources of cf-Tr-MTB DNA and we believe the cf-Tr-MTB DNA-based qPCR assay has a potential application for TBM diagnosis.

Assessment of DNA aptamers targeting GlcB and HspX antigens for application in the diagnosis of abdominal tuberculosis.

The diagnosis of abdominal tuberculosis (aTB) is challenging and there is an urgent need for an accurate diagnostic test. We have developed a high affinity DNA aptamer against GlcB antigen of Mycobacterium tuberculosis (Mtb). We further compared the diagnostic utility of in-house-generated high affinity DNA aptamers and polyclonal antibodies against two Mtb antigens, namely GlcB and HspX, in ascitic fluid samples. These diagnostic reagents were assessed in patients (n = 94) who were categorized as 'Definite TB', 'Probable TB', 'Possible TB' (taken together as aTB) and 'Non-TB' disease. Receiver operating characteristic curves were used to derive cut-off values to provide ≥93% specificity. Aptamer Linked Immobilized Sorbent Assay (ALISA) for HspX and GlcB exhibited a sensitivity of ∼84% and 50%, respectively (p-value <0.01). In contrast, antibody-based ELISA exhibited a lower sensitivity of ∼18% and ∼28% for HspX and GlcB, respectively (p-value <0.0001 and p = 0.05 for HspX and GlcB ELISA vs. ALISA, respectively). HspX ALISA detected 32/38 aTB cases, while Xpert detected only 9 samples. In conclusion, HspX aptamer-based test was found to be superior to the other tests for diagnosing aTB and it nearly fulfils the sensitivity criteria of WHO's 'Target Product Profile' for extrapulmonary tuberculosis (sensitivity ≥80%, specificity 98%).

PCOS patients differ in meal timings rather than total caloric or macronutrient intake in comparison to weight matched controls.

BACKGROUND: There is paucity of data related to dietary patterns in women with PCOS with heterogenous phenotypes compared to weight matched healthy women. In the present study, we studied the influence of diet and dietary patterns in relation to BMI and phenotype of PCOS women. DESIGN: Case control study in a tertiary care hospital. PATIENTS: 168 PCOS women and 102 age matched healthy women were recruited. All were subjected to clinical examination, biochemical, hormonal and dietary assessment based on 2-day dietary recall and direct interview by dieticians. RESULTS: There was no difference in the total energy and macronutrient distribution among the lean and obese PCOS women compared to weight matched controls. There was a higher junk intake and lower fibre intake among PCOS patients compared to controls. There was no difference in the total energy or macronutrient distribution or meal timings among different phenotypes of PCOS. Around 40% PCOS women had a late/missed breakfast pattern compared to 15% of healthy controls (p-0.00). In multivariable logistic regression analysis, we observed that late breakfast and late lunch patterns were associated with PCOS independent of BMI (OR-3.3[CI- 1.7-6.8] and 3.0 [CI- 1.2-6.9]). Junk intake was correlated with BMI, glucose and cholesterol levels and dairy intake was correlated with hirsuitism score. CONCLUSION: We demonstrate for the first time that PCOS women differ significantly from weight matched controls in the timings of their breakfast and lunch intake rather than the total caloric intake and macronutrient distribution. Whether correction of meal timings and food choices can improve the phenotypic manifestations of PCOS remains to be seen.

Antitussive noscapine and antiviral drug conjugates as arsenal against COVID-19: a comprehensive chemoinformatics analysis.

Coronavirus pandemic has caused a vast number of deaths worldwide. Thus creating an urgent need to develop effective counteragents against novel coronavirus disease (COVID-19). Many antiviral drugs have been repurposed for treatment but implicated minimal recovery, which further advanced the need for clearer insights and innovation to derive effective therapeutics. Strategically, Noscapine, an approved antitussive drug with positive effects on lung linings may show favorable outcomes synergistically with antiviral drugs in trials. Hence, we have theoretically examined the combinatorial drug therapy by culminating the existing experimental results with in silico analyses. We employed the antitussive noscapine in conjugation with antiviral drugs (Chloroquine, Umifenovir, Hydroxychloroquine, Favlplravir and Galidesivir). We found that Noscapine-Hydroxychloroquine (Nos-Hcq) conjugate has strong binding affinity for the main protease (Mpro) of SARS-CoV-2, which performs key biological function in virus infection and progression. Nos-Hcq was analyzed through molecular dynamics simulation. The MD simulation for 100 ns affirmed the stable binding of conjugation unprecedentedly through RMSD and radius of gyration plots along with critical reaction coordinate binding free energy profile. Also, dynamical residue cross-correlation map with principal component analysis depicted the stable binding of Nos-Hcq conjugate to Mpro domains with optimal secondary structure statistics of complex dynamics. Also, we reveal the drugs with stable binding to major domains of Mpro can significantly improve the work profile of reaction coordinates, drug accession and inhibitory regulation of Mpro. The designed combinatorial therapy paves way for further prioritized in vitro and in vivo investigations for drug with robust binding against Mpro of SARS-CoV-2.

Longitudinal Study to Find the Association of Serum Phosphorus Level with FGF23 in Three Different Hyperphosphatemia Management Groups of Stage 3 and 4 Chronic Kidney Disease (CKD) Patients.

Background: There is paucity of clinical evidence on target serum phosphorus levels in early chronic kidney disease (CKD). Present longitudinal study was done to find target phosphorus level and its association with fibroblast growth factor (FGF23) in three different hyperphosphatemia management groups. Methods: This 1-year, prospective, randomized controlled, open-labelled study was conducted among three equally allocated treatment groups that consisted of 120 screened early CKD patients totally. Group 1 patients were given dietary phosphorus modification (n = 40), group 2 patients were administered calcium-based phosphate binders (n = 40), and group 3 patients were given non-calcium-based phosphate binders (n = 40). Three-monthly dietary assessment, MDRD estimated glomerular filtration rate (eGFR), phosphorus, calcium, iPTH, alkaline phosphatase, and six-monthly FGF23, 2D echocardiography, and X-ray of chest and abdomen were performed. Association of three categories of phosphorus level up to 3.9, 4-5, and >5mg/dl, rate of progression of all parameters, and correlation with FGF23 were studied among all three groups. Results: At baseline, all clinical and biochemical parameters were equally distributed with a controlled nutritional phosphate among all groups. There was no significant difference of FGF23 levels from all the three categories of phosphorus level among all groups. Serum phosphorus at the level of 5 mg/dl was associated with iPTH and eGFR at 1 year. Over 1 year, there was a significant decline in serum phosphorus levels in group 1 (P 0.02), group 2 (P 0.00), and group 3 (P 0.05). FGF23 declined significantly only in group 3 (P 0.00). There was no correlation of FGF23 with serum phosphorus levels (P 0.13). However, FGF23 correlated positively with iPTH (P 0.03, r = 0.19). Conclusion: Serum phosphorus levels upto 5mg/dl had no effect on FGF23 at early CKD stages. Although different treatment groups showed significant phosphorus reduction, non-calcium phosphate binder had a major impact on FGF23 reduction.

Prevalence of Vascular Calcification in Chronic Kidney Disease Stage 4 and 5 Patients and its Correlation with Inflammatory Markers of Atherosclerosis.

Vascular calcification is one of the independent risk factors associated with cardiovascular disease (CVD) in chronic kidney disease (CKD) patients. This study evaluated the prevalence of vascular calcification in Indian patients with CKD stages 4 and 5. This was a prospective study conducted between January 2011 and June 2012. CKD stage 4 and 5 patients of either sex aged >18 years were screened for aortic vascular calcification using digital X-ray lumbar spine and multislice computed tomography (CT) scan. In addition, details of inflammatory markers [high-sensitivity C-reactive protein (hs-CRP) and interleukin (IL-6)] were also collected. A total of 150 patients (stage 4, n = 98; stage 5, n = 56) were screened for vascular calcification, and the mean age was 56.56 years. Patients with CKD stage 5 had significantly higher (P ≤0.05) serum creatinine and lower estimated glomerular filtration rate, total cholesterol, and low-density lipoprotein than CKD stage 4. Significantly, more patients with CKD stage 5 had a history of CVD. A total of 113 (75.3%) patients had vascular calcification [aortic calcification index (ACI) >0] with significantly higher prevalence in CKD stage 5 (85.72%) as compared to CKD stage 4 (69.15%). Patients having high aortic calcification (ACI >20%) were older (P = 0.0013); had a higher frequency of diabetes, and CVD; and had significantly (P <0.05) higher blood urea, serum creatinine, phosphorus, Ca × PO4 product, intact parathyroid hormone, hs-CRP, and IL-6. The higher CKD stage was associated with a higher prevalence of vascular calcification and higher aortic calcification index (ACI). CT techniques (electron beam CT and multislice CT) are the gold standards for detection and quantification of progression of vascular calcification.

Sigma Metric Evaluation of Drugs in a Clinical Laboratory: Importance of Choosing Appropriate Total Allowable Error and a Troubleshooting Roadmap.

Objectives Stringent quality control is an essential requisite of diagnostic laboratories to deliver consistent results. Measures used to assess the performance of a clinical chemistry laboratory are internal quality control and external quality assurance scheme (EQAS). However, the number of errors cannot be measured by the above but can be quantified by sigma metrics. The sigma scale varies from 0 to 6 with "6" being the ideal goal, which is calculated by using total allowable error (TEa), bias, and precision. However, there is no proper consensus for setting a TEa goal, and influence of this limiting factor during routine laboratory practice and sigma calculation has not been adequately determined. The study evaluates the impact of the choice of TEa value on sigma score derivation and also describes a detailed structured approach (followed by the study laboratory) to determine the potential causes of errors causing poor sigma score. Materials and Methods The study was conducted at a clinical biochemistry laboratory of a central government tertiary care hospital. Internal and external quality control data were evaluated for a period of 5 months from October 2019 to February 2020. Three drugs (carbamazepine, phenytoin, and valproate) were evaluated on the sigma scale using two different TEa values to determine significant difference, if any. Statistical Analysis Bias was calculated using the following formula: Bias% = (laboratory EQAS result - peer group mean) × 100 / peer group mean Peer group mean sigma metric was calculated using the standard equation: Sigma value = TEa - bias / coefficient of variation (CV)%. Results Impressive sigma scores (> 3 sigma) for two out of three drugs were obtained with TEa value 25, while with TEa value 15, sigma score was distinctly dissimilar and warranted root cause analysis and corrective action plans to be implemented for both valproate and carbamazepine. Conclusions The current study evidently recognizes that distinctly different sigma values can be obtained, depending on the TEa values selected, and using the same bias and precision values in the sigma equation. The laboratories should thereby choose appropriate TEa goals and make judicious use of sigma metric as a quality improvement tool.

Prevalence of Macroprolactinemia in People Detected to Have Hyperprolactinemia.

Background Macroprolactinemia is an analytic laboma encountered as a part of prolactin assay. No data are available on the burden of macroprolactinemia in Indians. This study aimed to determine the prevalence and predictors of macroprolactinemia among people with hyperprolactinemia. Methods Consecutive patients detected to have serum prolactin > 18 ng/mL as per the upper reference limit were further screened for macroprolactin by post-polyethylene-glycol (PEG)-precipitation test. Macroprolactinemia was defined as post-PEG recovery of prolactin < 40%. Results The four most common underlying etiologies for the testing of hyperprolactinemia were polycystic ovary syndrome ( n = 402; 32.71%), pituitary adenomas ( n = 318; 25.87%), drug-induced hyperprolactinemia ( n = 224; 18.23%), and infertility ( n = 126; 10.25%). A total of 1,229 patients (male:female = 191:1038) having mean age 30.46 ± 10.14 years had hyperprolactinemia, of which 168 (13.7%) were diagnosed to have macroprolactinemia. Macroprolactinemia was significantly higher in females than males (15.03 vs. 6.28%; p < 0.001). Age quartile-based analysis revealed no difference in occurrence of macroprolactinemia. Only 34 patients (2.76%) with macroprolactinemia (< 40% recovery of prolactin post-PEG precipitation) had raised prolactin levels after recovery. These patients primarily had underlying pituitary pathology. Conclusion Macroprolactinemia is not uncommon in people being tested for hyperprolactinemia. We should not hesitate to screen for macroprolactinemia in patients who have incidentally been detected to have hyperprolactinemia.

Carbohydrate-rich Meals Have no Impact on Post-prandial Lipid Parameters in Indians with Subclinical and Overt Primary Hypothyroidism.

BACKGROUND AND AIMS: The impact of altered cholesterol metabolism on post-prandial lipids in Indians with hypothyroidism is not known. This study evaluated the impact of overt primary hypothyroidism (OPH) and subclinical hypothyroidism (ScH) on post-prandial lipids after a standardised, carbohydrate-rich, mixed meal. METHODS: Endocrinology outpatients were screened for possible inclusion into the study. Patients >18 years of age with hypothyroidism who were not taking levothyroxine and who did not present with any comorbidities underwent biochemical evaluation following a carbohydrate-rich, mixed meal. Assessments included total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein-cholesterol (HDL-C), triglycerides, lipoprotein-A (Lp-A), apolipoprotein-A1 (apo-A1), apolipoprotein-B (apo-B), insulin and fasting glucose. Assessments were carried out 1 hour, 2 hours and 4 hours after the meal. Patients were compared against healthy matched controls recruited from healthcare professionals in the hospital (asymptomatic and apparently healthy nursing staff, reception staff and ward staff). RESULTS: Data from 194 patients (161 with ScH and 33 with OPH) and 40 euthyroid controls were analysed. Anthropometry, body mass index, glycaemia and insulin resistance were comparable among patients with OPH and ScH, and controls. LDL-C and Lp-A were significantly higher in those with OPH, compared with ScH and controls, at baseline, 1 hour, 2 hours and 4 hours after mixed meal consumption (all p<0.05). There was progressive and similar decline in post-prandial TC, LDL-C and Lp-A in all three groups. Triglycerides were similar among the OPH, ScH and control groups, both in fasting and post-prandial state, with a progressive and similar increase in post-prandial triglycerides in all three groups. CONCLUSION: This study demonstrated that severity of hypothyroidism had no impact on post-prandial TC, LDL-C and Lp-A. In addition, hypothyroidism had no impact on post-prandial triglycerides. Therefore, we conclude that lipid profile can be reliably estimated in a non-fasting state in individuals with ScH and OPH.

Utility of circulating cell-free Mycobacterium tuberculosis DNA for the improved diagnosis of abdominal tuberculosis.

Abdominal tuberculosis (ATB) continues to pose a major diagnostic challenge for clinicians due to its nonspecific clinical presentation, variable anatomical location and lack of sensitive diagnostic tools. In spite of the development of several assays till date; no single test has proved to be adequate for ATB diagnosis. In this study, we for the first time report the detection of circulating cell-free Mycobacterium tuberculosis (M. tuberculosis) DNA (cfMTB-DNA) in ascitic fluid (AF) samples and its utility in ATB diagnosis. Sixty-five AF samples were included in the study and processed for liquid culture, cytological, biochemical and molecular assays. A composite reference standard (CRS) was formulated to categorize the patients into 'Definite ATB' (M. tuberculosis culture positive, n = 2), 'Probable ATB' (n = 16), 'Possible ATB' (n = 13) and 'Non-TB' category (n = 34). Two molecular assays were performed, namely, the novel cfMTB-DNA qPCR assay targeting M. tuberculosis devR gene and Xpert MTB/RIF assay (Xpert), and their diagnostic accuracy was assessed using CRS as reference standard. Clinical features such as fever, loss of weight, abdominal distension and positive Mantoux were found to be strongly associated with ATB disease (p<0.05). cfMTB-DNA qPCR had a sensitivity of 66.7% (95% CI:40.9,86.7) with 97.1% specificity (95% CI:84.7,99.9) in 'Definite ATB' and 'Probable ATB' group collectively. The sensitivity increased to 70.9% (95% CI:51.9,85.8) in the combined 'Definite', 'Probable' and 'Possible' ATB group with similar specificity. The cfMTB-DNA qPCR assay performed significantly better than the Xpert assay which demonstrated a poor sensitivity of ≤16.7% with 100% (95% CI:89.7,100) specificity (p<0.001). We conclude that cfMTB-DNA qPCR assay is an accurate molecular test that can provide direct evidence of M. tuberculosis etiology and has promise to pave the way for improving ATB diagnosis.

Neuroprotective Role of Oral Vitamin D Supplementation on Consciousness and Inflammatory Biomarkers in Determining Severity Outcome in Acute Traumatic Brain Injury Patients: A Double-Blind Randomized Clinical Trial.

BACKGROUND AND OBJECTIVE: Early management of traumatic brain injury (TBI) is essential. We aimed to evaluate the efficacy of vitamin D over early clinical outcome and serum cytokine levels in patients with moderate to severe brain injury. METHODS: Thirty-five patients with moderate to severe traumatic brain injury who were admitted to the ICU unit were recruited into the study. Subjects were randomly allocated to a treatment regimen comprising either a one-time oral dose of 120,000 IU (two tablets of 60,000 IU each) of vitamin D (n = 20) or 8 mg of saccharide (two tablets of 4 g each) as placebo (n = 15). The main parameters evaluated included duration of mechanical ventilation and ICU stay, Glasgow Coma Scale (GCS) and cytokine levels (interleukin (IL)-6, tumour necrosis factor (TNF)-α, interferon (IFN)-γ, IL-2). RESULTS: The results indicated an improvement in the level of consciousness after 7 days in the vitamin D-treated group compared with placebo. An elevation in GCS score by 3.86 units in the vitamin D-treated group with a 0.19-unit descent in the control group was recorded. Duration of mechanical ventilation was reduced in the vitamin D-treated group compared with the control group (4.7 days vs. 8.2 days, p value 0.0001). A noticeable reduction was recorded in inflammatory biomarkers (cytokines) in the vitamin D-treated group (IL-6 p = 0.08, TNF-α p = 0.02, IL-2 p = 0.36) with notable elevation in IFN-γ (p = 0.65) compared to the control group. CONCLUSION: In the acute phase of moderate to severe traumatic brain injury, vitamin D supplementation plays a vital role and has a favourable effect on the consciousness level of patients. Clinical trial Registry (CTRI) No. CTRI/2019/05/019259.

Multiepitope Subunit Vaccine to Evoke Immune Response against Acute Encephalitis.

Acute encephalitis syndrome outbreak has emerged as a major health concern on both national and international scales. Brain inflammation/infections caused by Japanese encephalitis virus (JEV) can lead to death. The cases are growing in numbers globally, and this emergent health concern requires an effective and viable vaccine to strengthen the body's immune system against this deadly virus. Proteomic analyses of JEV revealed the envelope protein as a potential target for vaccine development by patient samples analysis. Hence, in this study, we aimed to design a multiepitope subunit vaccine for acute encephalitis using the advanced structural biology and immunoinformatics approaches. We report the multiepitope subunit vaccine consisted of the putative T-cell epitope (MHC-1 and MHC-2 restricted) and B-cell epitope and with high antigenicity and immunogenicity. The TAP affinity epitopes along with adjuvants were engineered to the vaccine, to ensure the ease transportation inside the host and elicitation of a strong immune response. The specificity of vaccine construct was evaluated by molecular docking with major histocompatibility complex (MHC) receptors and host membrane receptor TLR2. High docking scores and a close interaction to the binding groove of receptors confirmed the potency and specificity of the vaccine. Also, molecular dynamics simulation studies confirmed the stable interaction of vaccine with TLR2 for a long run (100 ns), which showed the prolonged elicitation of the strong immune response. Peptide dynamics studies showed the flexible, strong, and stable binding of vaccine with minimal deviation in root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), and secondary structure estimation (SSE) plots till 100 ns simulation run. The in silico immune simulation approach based on the position-specific scoring matrix and machine learning methods resulted in the strong immune response reinforcement statistics of immune cells (T-cells, B-cells population, and memory cells) in response to vaccine candidate. The favorable results and well-correlated data of varied in silico techniques paved for a potent multiepitope vaccine and helped us to propose the mechanism of action of designed vaccine and generation of the immune response against acute encephalitis syndrome.

Theranostic Application of a Novel G-Quadruplex-Forming DNA Aptamer Targeting Malate Synthase of Mycobacterium tuberculosis.

The successful management of tuberculosis (TB) requires efficient diagnosis and treatment. Further, the increasing prevalence of drug-resistant TB highlights the urgent need to develop novel inhibitors against both drug-susceptible and drug-resistant forms of disease. Malate synthase (MS), an enzyme of the glyoxylate pathway, plays a vital role in mycobacterial persistence, and therefore it is considered as an attractive target for novel anti-TB drug development. Recent studies have also ascribed an adhesin function to MS and established it as a potent diagnostic biomarker. In this study, a panel of Mycobacterium tuberculosis (Mtb) MS-specific single-stranded DNA aptamers was identified by Systematic Evolution of Ligands by EXponential enrichment (SELEX). The best-performing G-quadruplex-forming 44-mer aptamer, MS10, was optimized post-SELEX to generate an 11-mer aptamer, MS10-Trunc. This aptamer was characterized by various biochemical, biophysical, and in silico techniques. Its theranostic activity toward Mtb was established using enzyme inhibition, host cell binding, and invasion assays. MS10-Trunc aptamer exhibited high affinity for MS (equilibrium dissociation constant [KD] ∼19 pM) and displayed robust inhibition of MS enzyme activity with IC50 of 251.1 nM and inhibitor constant (Ki) of 230 nM. This aptamer blocked mycobacterial entry into host cells by binding to surface-associated MS. In addition, we have also demonstrated its application in the detection of tuberculous meningitis (TBM) in patients with sensitivity and specificity each of >97%.

Correlation between Clinical Features, Biochemical Parameters, and Histopathological Findings in Women with Patterned Baldness: A Study from North India.

BACKGROUND: Androgenetic alopecia (AGA) is a non-scarring alopecia with a characteristic pattern in genetically predisposed men and women. Hormonal abnormalities namely hyperandrogenism, hair cycle defects, genetic predisposition, and follicular miniaturization have been implicated as the causative factors for AGA. AIM: To analyze women with patterned hair loss and correlate their clinical findings with the histopathology and biochemical parameters. MATERIALS AND METHODS: Female patients between 18 and 45 years of age with a history of hair loss on the crown, temporal area, and recession of hairline were clinically examined. These patients were then subjected to histopathological examination, and on confirmation of diagnosis of female pattern hair loss (FPHL), they were included in the study. Their morning blood sample was taken on 3rd-5th day of the menstrual cycle for hormonal analysis. The study was carried out on 30 patients and 30 age- and sex-matched controls. RESULTS: A statistically significant difference was observed between the mean values of dehydroepiandrosterone sulfate, prolactin, androstenedione, and free triiodothyronine of cases and controls. The most common histopathological finding in our study was an increase in the percentage of telogen hair. CONCLUSION: The biochemical findings in our study corroborate the role of hyperandrogenism as one of the major etiological factors in FPHL with the role of adrenal androgens being central, and therefore all female patients with FPHL should be evaluated for underlying hormonal imbalances. The role of histopathology in FPHL can be used as a prognostic marker.

Structural switching electrochemical DNA aptasensor for the rapid diagnosis of tuberculous meningitis.

BACKGROUND: Tuberculous meningitis (TBM) is the most devastating manifestation of extra-pulmonary tuberculosis. About 33% of TBM patients die due to very late diagnosis of the disease. Conventional diagnostic methods based on signs and symptoms, cerebrospinal fluid (CSF) smear microscopy or liquid culture suffer from either poor sensitivity or long turnaround time (up to 8 weeks). Therefore, in order to manage the disease efficiently, there is an urgent and unmet need for a rapid and reliable diagnostic test. METHODS: In the current study, to address the diagnostic challenge of TBM, a highly rapid and sensitive structural switching electrochemical aptasensor was developed by combining the electrochemical property of methylene blue (MB) with the molecular recognition ability of a ssDNA aptamer. To demonstrate the clinical diagnostic utility of the developed aptasensor, a blinded study was performed on 81 archived CSF specimens using differential pulse voltammetry. RESULTS: The electrochemical aptasensor developed in the current study can detect as low as 10 pg HspX in CSF background and yields a highly discriminatory response (P<0.0001) for TBM and not-TBM categories with ~95% sensitivity and ~97.5% specificity and has the ability to deliver sample-to-answer in ≤30 minutes. CONCLUSION: In summary, we demonstrate a new aptamer-based electrochemical biosensing strategy by exploiting the target-induced structural switching of H63 SL-2 M6 aptamer and electroactivity of aptamer-tagged MB for the detection of HspX in CSF samples for the diagnosis of TBM. Further, the clinical utility of this sensor could be extended for the diagnosis of other forms of tuberculosis in the near future.