Effect of Maturity And Birth Weight On Umbilical Cord Blood Lipid Profile: A Fetal Programming Phenomenon

Background:The recent research on "fetal programming hypothesis" has totally revived the mechanistic understanding of triggering factors responsible for development of cardiovascular diseases (CVDs). As deranged lipid profile is associated with increased predisposition to atherosclerotic CVDs, we planned to study effect of fetal maturity and birth weight on umbilical cord blood lipid profile. Methods: A hospital based cross sectional study including 200 neonates satisfying inclusion criteria was carried out. The umbilical cord blood samples of selected neonates were collected and analysed for lipid profile (Total Cholesterol (TC), Triglycerides (TG), High Density Lipoprotein Cholesterol (HDL-C), Low Density Lipoprotein Cholesterol (LDL-C) and Very Low Density Lipoprotein Cholesterol (VLDL-C)) in order to be compared among groups of neonates based on maturity and birth weight. Results: The preterm neonates had statistically significant higher levels of umbilical cord blood TC, TG, LDL-C and VLDL-C (with P values of 0.025, 0.001, 0.045, 0.007 and 0.045 respectively) while lower but statistically insignificant (p value of 0.131) levels of HDL-C in as compared to full term neonates. The low birth weight neonates also had statistically significant higher levels of TC, TG, LDL-C and VLDL-C (P values of 0.001, 0.032, 0.00001 and 0.032 respectively) but statistically significant (P value of 0.034) lower levels of HDL-C in umbilical cord blood as compared to normal birth weight neonates. Conclusion: The results suggest that prematurity as well as low birth weight have deleterious effect on umbilical cord blood lipid profile resulting in more atherogenic lipid levels. This could be regarded a triggering factor responsible for development of CVDs in later life. This observation not only conciliates with fetal programming hypothesis but also indicates that its effect is evident (in the form of altered lipid profile) even at the birth in such programmed neonates.

Multi-antigen and antibody assays (Seva TB Elisa) for the diagnosis of tuberculous pleural effusion.

Objective: Prospective evaluation of inhouse developed SEVA TB ELISA using cocktail of Mycobacterial antigens ES-31 and EST-6(containing ES-38 and ES-41) and their specific antibodies in the diagnosis of Tuberculous pleural effusion was done in a tertiary care hospital. Methods: Detection of circulating free and immune-complexed (IC) antigens and antibody by sandwich and indirect peroxidase ELISA respectively was done in pleural fluid and sera specimens. Total 33 patients with pleural effusion, including 24 patients diagnosed as tuberculous pleural effusion based on clinico-radiological, microbiological and biochemical profile (protein, LDH and ADA) of pleural effusion and nine patients with non-tuberculous pleural effusion, were studied. Results: Pleural fluid showing either antigen or immune-complexed antigen or antibody positive was considered as ELISA positive for tuberculous pleural effusion. Multi antigen and antibody assay (SEVATB ELISA) showed 100% specificity and 83% sensitivity in pleural fluid while 78% specificity and 92% sensitivity in serum of tuberculous pleuritis patients. Conclusion: This study showed usefulness of SEVATB ELISA, using cocktail of ES-31 and EST-6 antigens and their antibodies for antibody and antigen detection respectively in analysis of either sera or pleural fluid samples of suspected tuberculous pleuritis patients as an adjunct test to clinical diagnosis.

A sensitive and specific ES-31 antigen detection based fluorometric assay for confirmation of Mycobacterium tuberculosis in cell culture.

Confirmation of presence of M. tuberculosis bacilli on microscopic examination is very important in diagnosis of tuberculosis. The present study was undertaken to find the usefulness of mycobacterial ES-31 serine protease as a marker to detect tuberculosis bacilli using fluorescein isothiocyanate conjugated anti-ES-31 serine protease antibody. This immunofluorescence method was compared with Ziehl-Neelsen and auramine-O staining methods for detection of tuberculosis bacilli. Slides were prepared for each serially diluted tuberculosis H37Ra bacilli (1×107 bacilli/ml to 5 bacilli/ml). Slides for each dilution group were stained by ZN method, auramine-O and immunostaining methods using fluorescein isothiocyanate conjugated anti-ES-31 serine protease antibody. ZN staining method showed efficacy for detection of M. tuberculosis H37Ra upto 1×104 bacilli/ml while auramine-O method showed upto 1×102 bacilli/ml. The presence of bacilli was indicated by green fluorescence on immunostaining using anti-ES-31 antibody conjugate and this method was effective upto 10 bacilli/ml. The slides which were negative for ZN (1×103 cells/ml) and auramine-O (100 cells/ml) method showed positivity on restaining with immunofluorescent staining method. The results of this preliminary study showed that immunofluorescent staining method using specific anti-ES-31 antibody conjugate was more sensitive for detection of tuberculosis bacilli than ZN and auramine-O methods in samples of laboratory strain. The utility of this method will be studied further in clinical specimens.

Effect of HIV protease inhibitors and orlistat on mycobacterial ES-31 serine protease, a potential drug target in mycobacterium tuberculosis.

Background: Mycobacterial excretory secretory-31 (SEVA TB ES-31) antigen is shown to possess protease and lipase activities. Aim: To study the effect of commonly used HIV-protease inhibitors and lipase inhibitor Orlistat if any on mycobacterial ES-31 serine protease in vitro enzyme activity and on the growth of M.tb H37Ra bacilli in axenic culture. Methods: Effect of HIV-protease inhibitors namely Ritonavir, Lopinavir and Indinavir and Orlistat on protease activity of ES-31 was assessed using azocasein assay and on bacillary growth in axenic culture of Mycobacterium tuberculosis H37Ra. The concentration of ES-31 antigen in culture filtrate was determined by sandwich peroxidase ELISA using anti ES-31 antibody and the growth of bacilli by CFU count. Results: HIV-protease inhibitors such as Ritonavir, Lopinavir and Indinavir and lipase inhibitor Orlistat inhibited serine protease activity by 41.3 - 69.7% in vitro. These inhibitors also showed decreased bacterial growth in axenic culture and further confirmed by decreased concentration of ES-31 serine protease secretion in the culture fluid. Ritonavir showed maximum inhibition of 77% on the growth of the bacilli in axenic culture while anti obesity drug Orlistat showed 61% inhibition. Conclusion: SEVA TB ES-31 with serine protease and lipase activities may be a potential drug target in tuberculosis management.

Metabolic syndrome in adult population of rural Wardha, central India.

Background & Objectives: Prevalence of metabolic syndrome is high among Asians including Indians. Scarce information is available about the magnitude of metabolic syndrome in rural areas and hence present study in rural area of Wardha district, central India. Methods: In 300 randomly selected subjects, blood pressure and anthropometric measurements such as height, weight, waist circumference and hip circumference were noted. Blood sample was collected after overnight fasting and was subjected to biochemical quantification such as fasting blood sugar, total cholesterol, triglycerides, HDL-C, VLDL-C and LDL-C. Data were analyzed using ATP-III definition as well as by modifying the waist circumference cut-offs as per Asia-Pacific guidelines. Results: Overall metabolic syndrome as per ATP-III criteria was observed in 5.0 per cent adult rural population. When ATP-III criteria were modified using waist circumference cut-offs recommended by Asia-Pacific guidelines, metabolic syndrome was seen in 9.3 per cent. It was 10.7 per cent among females and 8.2 per cent among males. Receiver operating characteristic curve was plotted to find out the best cut-off of BMI to identify the individuals with metabolic syndrome. The best cut-off for BMI came out to be 23.32 kg/m2. Interpretation & conclusions: The magnitude of metabolic syndrome was low among rural adults of Wardha as compared to reported values in urban areas. BMI of 23.32 kg/m2 and higher was found to predict significant risk of metabolic syndrome in these study subjects. However, studies with larger sample need to be conducted to confirm these findings.

Detection of circulating free and immune-complexed antigen in pulmonary tuberculosis using cocktail of antibodies to mycobacterium tuberculosis excretory secretory antigens by peroxidase enzyme immunoassay.

Background: Decreased sensitivity has been a limiting factor of antigen assay for detection of tuberculosis. Assay of more than one antigen may improve sensitivity of an assay. Aim: To develop a simple, rapid and less-expensive serodiagnostic method compared to culture method for Pulmonary Tuberculosis. Method: A cocktail of affinity purified antibodies against Mycobacterium tuberculosis H37Ra antigens (SEVA TB ES-31, ES-43 and EST-6) was explored for detection of circulating free and Immune-Complexed (IC) cocktail antigen by microtitre plate Peroxidase sandwich ELISA. The assay was evaluated in 27 clinical sera of sputum acid fast bacilli (AFB) positive and 10 AFB negative but anti-tuberculosis therapy responded pulmonary tuberculosis patients and 20 normal sera as controls. Results: Assay of cocktail antigen showed marginal improvement in sensitivity compared to assay of ES-31 antigen alone. The assay for circulating free cocktail antigen showed a sensitivity of 77.7% for AFB positive cases and 70% for AFB negative cases compared to assay of ES-31antigen with sensitivity of 74% and 70% respectively. The assay for ICcocktail antigen showed sensitivity of 77.7% for AFB positive and 80% for AFB negative cases compared to assay of ICES- 31 antigen with sensitivity of 77% and 70% respectively. Specificity of antigen assay was found to be 90%. Detection of IC-antigen as adjunct assay improved the sensitivity of detection in AFB-ve but ATT responded cases. Peroxidase enzyme immunoassay of cocktail antigen showed a sensitivity of detection of 0.25 μg/ ml and levels of free and IC cocktail antigens were 1.70 ± 1.04 and 1.13 ± 0.047 μg/ ml in AFB positive patients’ sera. Conclusions: Peroxidase enzyme immunoassay for circulating antigen was found to be a useful serodiagnostic assay and in particular in AFB –ve cases responding to ATT.