Development & evaluation of biotinylated DNA probe for clinical diagnosis of chikungunya infection in patients’ acute phase serum & CSF samples.

Background & objectives: The resurgence of chikungunya virus (CHIKV) in the Indian Ocean Islands and India has drawn worldwide attention due to its explosive nature, high morbidity and complex clinico-pathological manifestations. The early confirmatory diagnosis of CHIKV is essential for management as well as control of unprecedented epidemics. The present study describes the development and evaluation of a highly sensitive and specific E1 structural gene specific biotinylated DNA probe for detection of chikungunya virus in clinical samples using a dot blot format. Methods: The complementary DNA (cDNA) of CHIKV was spotted on to nylon membrane. The membrane was subjected to prehybridization and hybridization and developed using a colour development solution containing DAB chromogen. Results: The CHIKV E1 specific DNA probe was highly sensitive detecting picogram levels of target nucleic acid. The comparative evaluation with SYBR Green I based real-time RT-PCR revealed 99 per cent accordance with a sensitivity and specificity of 99 and 98 per cent, respectively. The specificity of this assay was further confirmed through cross-reaction studies with confirmed dengue and Japanese encephalitis (JE) patient serum samples along with infected culture supernatant of Ross River and Saint Louis encephalitis and plasmid DNA of O’Nyong Nyong, Semlinki forest and Sindbis viruses. Interpretation & conclusion: The DNA probe reported in this study may be useful for specific, sensitive and confirmatory clinical diagnosis of chikungunya infection in acute phase human patient serum and CSF samples. This assay can also be used in the laboratory for quantification of viral antigen in cell culture supernatant for research purpose.

Evaluation of a commercial Dengue NS1 enzyme-linked immunosorbent assay for early diagnosis of dengue infection.

Purpose: Dengue is one of the most serious mosquito-borne viral infections affecting tropical and subtropical countries in the world. Since there is no immunoprophylactic or specific antiviral therapy available, timely and rapid diagnosis plays a vital role in patient management and implementation of control measures. This paper evaluates a commercially available NS1 antigen capture ELISA vis-a-vis SD bioline Dengue NS1 antigen test for early detection of dengue virus. Materials and Methods: To evaluate a commercial NS1 antigen detection kit vis-a-vis SD bioline Dengue NS1 antigen test, a total of 91 clinical samples were tested. Virological investigations with regard to dengue virus, viz. NS1 antigen capture ELISA (Panbio, Australia), SD bioline Dengue NS1 antigen test, RT-PCR and virus isolation were performed. Results: Out of 91 samples, 24 (26%) were positive by NS1 antigen capture ELISA, 15 (16%) by SD bioline Dengue NS1 antigen test and 11(12%) positive by RT-PCR analysis. The RT-PCR-positive samples were further subjected to virus isolation and resulted in three isolates. The results of the Panbio NS1 antigen capture ELISA, SD bioline Dengue NS1 antigen test, RT-PCR and virus isolation were correlated among themselves. Conclusions: The present study comprehensively established the utility of NS1 antigen ELISA in early diagnosis of dengue infection.

Evaluation of different granulated active carbons for removal of Microcystin-LR from contaminated water.

Most frequently encountered freshwater cyanobacterial toxin is Microcystin-LR (MC-LR). Microcystins released from cells into water have been responsible for the death of humans, domestic and wild animals. Removal of microcystin by active carbon has been one of the best methods available so far. This study evaluates three grades of active carbon namely 40, 60 and 80 CTC for their removal efficiency of MC-LR from contaminated water. Kinetics of toxin removal was studied in time course experiments. Protection in mouse model was studied for the samples after the adsorption. Toxin quantitation was done by HPLC method. The MC-LR concentration after 24 hr treatment with 40, 60 and 80 CTC carbons were 4.8, 3.3 and 1.3 microg/ml respectively from an initial concentration of 5.2 microg/ml. Protection in mouse bioassay was seen after 48, 24 and 2 hr of adsorption time respectively for 40, 60 and 80 CTC carbons. 80 CTC carbon was found to be most efficient in removing MC-LR from contaminated water.

Toxins and bioactive compounds from cyanobacteria and their implications on human health.

Many species of cyanobacteria (blue-green algae) produce secondary metabolites with potent biotoxic or cytotoxic properties. These metabolites differ from the intermediates and cofactor compounds that are essential for cell structural synthesis and energy transduction. The mass growth of cyanobacteria which develop in fresh, brackish and, marine waters commonly contain potent toxins. Cyanobacterial toxins or cyanotoxins are responsible for or implicated in animal poisoning, human gastroenteritis, dermal contact irritations and primary liver cancer in humans. These toxins (microcystins, nodularins, saxitoxins, anatoxin-a, anatoxin-a(s), cylindrospermopsin) are structurally diverse and their effects range from liver damage, including liver cancer to neurotoxicity. Several incidents of human illness and more recently, the death of 60 haemodialysis patients in Caruaru, Brazil, have been linked to the presence of microcystins in water. In response to the growing concern about the non-lethal acute and chronic effects of microcystins, World Health Organization has recently set a new provisional guideline value for microcystin-LR of 1.0 microg/L in drinking water. Cyanobacteria including microcystin-producing strains produce a large number of peptide compounds, e.g. micropeptins, cyanopeptolins, microviridin, circinamide, aeruginosin, with varying bioactivities and potential pharmacological application. This article discusses briefly cyanobacterial toxins and their implications on human health.