Cardiopulmonary Exercise Test Using Arm Ergometry in Children With Spina Bifida: A Prediction Model for O2peak.

PURPOSE: This study aimed to develop a prediction model for peak oxygen uptake ((Equation is included in full-text article.)O2peak) in children with spina bifida (SB), considering peak workload (Wpeak), peak heart rate, age, sex, anthropometric measures, walking level, physical activity level, and level of the lesion. METHODS: Data of 26 participants with SB performing a graded arm crank test were used to develop the prediction model. An unrelated data set of participants with SB was used for validation. RESULTS: The following equation was developed to predict (Equation is included in full-text article.)O2peak of participants with SB: (Equation is included in full-text article.)O2peak (mL/min) = 194+18 × Wpeak - 110 × sex (adjusted R(2) = 0.933, SEE = 96 mL/min). Bland-Altman analysis showed a nonsignificant mean difference between the measured and predicted values of (Equation is included in full-text article.)O2peak (-0.09 L/min) and limits of agreement of -0.4036 and 0.2236 L/min. CONCLUSIONS: The prediction model shows promising results; however, further validation using the same protocol is warranted before implementation in clinical practice.

Comparison of a dipstick enzyme-linked immunosorbent assay with commercial assays for detection of Japanese encephalitis virus-specific IgM antibodies.

BACKGROUND: Japanese encephalitis (JE) is a major public health concern in Asia including India. OBJECTIVES: To evaluate an in-house developed dipstick enzyme-linked immunosorbent assay (ELISA) test vis-à-vis two commercial kits for detection of JE virus-specific IgM antibodies. SETTING AND DESIGN: Comparative study carried out in Research and Development centre. MATERIALS AND METHODS: A total of 136 specimens comprising 84 serum and 52 CSF samples were tested by in-house dipstick ELISA, Pan-Bio IgM capture ELISA (Pan-Bio, Australia) and JEV CheX IgM capture ELISA (XCyton, India). RESULTS: The overall agreement among all three tests was found to be 92% with both serum and cerebrospinal fluid (CSF) samples. The sensitivity of the dipstick ELISA was found to be 91% with serum and 89% with CSF samples respectively. The specificity of the dipstick ELISA with reference to both commercial assays was found to be 100% in serum and CSF samples in this study. CONCLUSIONS: The in-house dipstick ELISA with its comparable sensitivity and specificity can be used as a promising test in field conditions since it is simple, rapid and requires no specialized equipment.

Comparison of a dipstick dot-ELISA with commercial assays for anti-dengue virus IgM antibodies.

Dengue virus infections have undergone dramatic expansion in range, affecting several tropical and subtropical regions of the world. Dengue virus causes life-threatening complications characterized by dengue hemorrhagic fever and dengue shock syndrome. No standard validated test systems are available for serological diagnosis of dengue virus infection. This creates problems in the diagnosis and proper management of patients. Evaluation of a Defense Research and Development Establishment (DRDE) dengue virus dipstick dot-ELISA test, developed in house, vis-à-vis commercially available immunodiagnostic kits was carried out for detection of IgM antibodies. The DRDE dengue dipstick dot-ELISA was performed on the basis of the dot-ELISA principle. Commercial tests, namely, the Panbio Dengue IgM Capture ELISA system (Panbio, Sinnamon Park, Australia) and Pathozyme Dengue M (Omega Diagnostics, Alva, UK), were performed according to the manufacturers' protocols. Ninety-one serum samples collected from the states of Kerala and Delhi, India during August and November of 2004 were used in the present study. The overall agreement among all three tests was found to be only 72.16% for IgM antibodies. Correlations between the DRDE dipstick dot-ELISA and the Panbio kit, between the DRDE dipstick dot-ELISA and the Pathozyme Dengue M kit, and between the Panbio kit and the Pathozyme Dengue M kit were found to be 96, 93, and 94%, respectively. Although the Panbio kit is widely used in various laboratories in India, the DRDE dipstick dot-ELISA promises to be a useful kit because of its field applicability and comparable sensitivity.

Inhibitory potential of neem (Azadirachta indica Juss) leaves on dengue virus type-2 replication.

In the present study we report in vitro and in vivo inhibitory potential of crude aqueous extract of neem leaves and pure neem compound (Azadirachtin) on the replication of Dengue virus type-2. In vitro antiviral activity of aqueous neem leaves extract assessed in C(6/36) (cloned cells of larvae of Aedes albopictus) cells employing virus inhibition assay showed inhibition in dose dependent manner. The aqueous extract of neem leaves at its maximum non-toxic concentration of 1.897 mg/ml completely inhibited 100-10,000 TCID(50) of virus as indicated by the absence of cytopathic effects. The in vivo protection studies with neem leaves extract at its maximum non-toxic concentrations 120-30 mg/ml resulted in inhibition of the virus replication as confirmed by the absence of Dengue related clinical symptoms in suckling mice and absence of virus specific 511 bp amplicon in RT-PCR. The pure neem i.e. Azadirachtin did not reveal any inhibition on Dengue virus type-2 replication in both in vitro and in vivo systems.

Serological & virological investigation of an outbreak of dengue fever in Gwalior, India.

BACKGROUND & OBJECTIVES: An outbreak of febrile illness occurred between September to November 2001 in Gwalior, Madhya Pradesh affecting individuals mostly in the age group < 30 yr. A total of 312 febrile indoor patients suspected to have dengue infection were investigated. METHODS: The investigation included examination of blood samples from patients for dengue specific IgM and IgG antibodies, isolation of virus in suckling mouse pups and in C(6/36) cell line followed by confirmation and typing through reverse transcriptase-PCR and nested PCR. RESULTS: The serological analysis of the 312 samples indicated 65 per cent positivity of which 21 per cent are of recent infection as indicated by the presence of IgM antibody and 78 per cent are found to be secondary in nature by showing the presence of IgG and/or IgM antibodies. The RT-PCR analysis of patients' sera employing dengue virus group specific conserved amplimer confirmed the etiological agent as dengue complex by showing the characteristic 511 bp amplicons. None of the antibody positive samples were found to be positive by RT-PCR. A total of 13 (6%) samples positive by RT-PCR, were processed for virus isolation in mouse pups and in C(6/36) cells. Of these 9 samples (80%) were confirmed positive for virus isolation as identified by RT-PCR. INTERPRETATION & CONCLUSION: The typing of isolates by nested PCR employing serotype specific amplimer revealed 119 bp amplicon characteristic of dengue virus type-2 and thus confirming the outbreak attributed to dengue virus type-2.

Evaluation of a dipstick ELISA and a rapid immunochromatographic test for diagnosis of Dengue virus infection.

Here we report standardization of a dipstick enzyme-linked immunosorbent assay (ELISA, Dipstick ELISA) and its comparative evaluation with a commercial Rapid PanBio Immunochromatographic test (IC test) for detection of Dengue (DEN) virus-specific IgM and IgG antibodies in patient sera. Among crude and purified viral antigens prepared from mouse brains or cell cultures, a DEN virus type 2 antigen purified from cell cultures by sucrose density gradient centrifugation was found superior in terms of the signal/ noise (S/N) ratio in the assay system. The sensitivity of detection of the virus by specific IgM antibody was improved by removal of IgG from patient sera prior to testing. The evaluation of the Dipstick ELISA by use of 156 serum samples revealed an overall accordance of 96% and 93% with the IC test in detection of IgM antibodies to DEN viruses (IgM antibodies) and IgG antibodies to DEN viruses (IgG antibodies), respectively. The sensitivity of the Dipstick ELISA and the IC test with reference to the mu-capture ELISA was 83% and 87%, respectively, with a specificity of 98% in both cases. The sensitivity of the Dipstick ELISA with reference to the IC test in detecting IgM and IgG antibodies was 84% and 94%, respectively, and the specificity of the Dipstick ELISA was 98% and 92%, respectively.

Anti-microbial activity of a new vaginal contraceptive NIM-76 from neem oil (Azadirachta indica).

Efficacy of NIM-76, a spermicidal fraction from neem oil, was investigated for its antimicrobial action against certain bacteria, fungi and Polio virus as compared to whole neem oil. The NIM-76 preparation showed stronger anti-microbial activity than the whole neem oil. It inhibited growth of various pathogens tested including Escherichia coli and Kleibsiella pneumoniae which were not affected by the whole neem oil. NIM-76 also exhibited antifungal activity against Candida albicans and antiviral activity against Polio virus replication in vero cell lines. It also protected mice from systemic candidiasis as revealed by enhanced % survival and reduced colony forming units of C. albicans in various tissues. This shows that NIM-76 has a potent broad spectrum anti-microbial activity.

Comparison of two colorimetric assays to determine viral infectivity in micro culture virus titration.

Efficacy of two colorimetric assays, viz. MTT (3-4,5-dimethylthiazol-2-(yl-2,5-diphenyl tetrazolium bromide) and neutral red (NR) assays, performed by integrating them to micro culture virus titration (MCVT), was compared with the conventional MCVT method in terms of percentages of infectivity and 50% infectivity end points by employing Polio virus type-3 and Dengue virus type 4 as the candidate viruses. The results suggested that MTT assay has an edge over NR assay as well as conventional MCVT method. For the first time, NR assay has been successfully employed for the determination of virus infectivity titre.

Freshwater cyanobacterium Microcystis aeruginosa (UTEX 2385) induced DNA damage in vivo and in vitro.

Microcystins are a family of potent hepatotoxins and liver tumor promoters produced by several genera of cyanobacteria including Microcystis, Nodularia, Anabena, Nostoc, etc. They are chemically very stable and represent a public health threat when they occur in water used for human consumption. We investigated the DNA damage effects of M. aeruginosa UTEX 2385 in mouse liver in vivo and also in mammalian cells in vitro. The DNA damage effect is compared with purified toxin microcystin-LR (MCLR) in non-hepatic cells viz. baby hamster kidney cells (BHK-21) and mouse embryo fibroblasts primary cells (MEF). Cell-free extracts of UTEX 2385 induced significant DNA fragmentation at 0.5, 1 and 2 LD(50) (32.7, 65.4 and 130.8 mg/kg, respectively) and it was also time dependent. M. aeruginosa UTEX 2385 and MCLR induced significant DNA fragmentation in BHK-21 and MEF cells at 100 and 1.0 µg/ml concentration. Electrophoretic analysis revealed necrotic DNA damage by UTEX 2385 in vivo. Both the toxins caused smear in agarose gel electrophoresis indicating the necrotic DNA damage in MEF cells, whereas, multiple DNA fragments in BHK-21 cells. The DNA damage effect of the toxin is supported by data on hepatotoxicity in vivo and cytotoxicity in vitro.

Enterovirus specific IgM responses in children with acute and chronic paralytic syndrome.

Enterovirus specific IgM responses in 51 children aged 0-7 years with acute, clinically diagnosed paralytic syndrome and 8 children with chronic paralysis were detected by IgM antibody capture enzyme immunoassay. Twenty-nine out of 51 (56.86%) acute phase sera were positive for enterovirus (Polio, CVB3 and CVA7) IgM antibodies, in 21 of whom poliovirus antibodies were found in close association with CVB3 and CVA7. On the other hand, preponderance of CVA7 specific IgM was detected in 6 out of 8 sera samples of chronically paralytic children.

Relative efficacy of egg albumin as blocking agent in dot immunobinding assay for detection of group B coxsackieviruses.

Non specific binding (NSB) is an important factor affecting sensitivity and specificity in dot immunobinding assay (DIA). Several blocking agents e.g. egg albumin, casein, gelatin, milk powder and goat serum were evaluated for their relative efficacy vis-a-vis bovine serum albumin (BSA) for DIA system purported for detection of group B coxsackieviruses (CVB). The results suggest that egg albumin (5%) which is economical and readily available may act as an effective blocking agent in DIA system.

Evaluation of indirect enzyme linked immunosorbent assay for the detection of coxsackieviruses in clinical samples and its comparison with dot-immunobinding assay.

Indirect enzyme linked immunosorbent assay (ELISA) was applied for the direct detection of coxsackieviruses in clinical samples viz. rectal swabs (RS) and throat swabs (TS) collected from patients admitted to various nursing homes and local hospitals. Results indicate the presence of different CVA types in 65 (62.5%) out of 104 RS, and 18 (52.9%) out of 34 TS samples. Dot-immunobinding assay was also standardized for the identification of CVA types employing 52 RS samples and the results compared with indirect ELISA. Dot-immunobinding detected more CVA types in a relatively larger number of specimens than indirect ELISA.

Comparison of indirect and sandwich ELISA for the identification of ECHO viruses.

An indirect enzyme linked immunosorbent assay (ELISA) was standardized for the identification of ECHO viruses isolated in buffalo green monkey (BGM) kidney cell culture on inoculation of 113 sewage samples. Comparable results were obtained with both indirect and sandwich ELISA for the identification of ECHO viruses in respect of 15 out of 34 sewage samples which showed 75-100% CPE in BGM monolayers.