Use of molecular diagnostic tools for the identification of species responsible for snakebite in Nepal: a pilot study

Abstract: Snakebite is an important medical emergency in rural Nepal. Correct identification of the biting species is crucial for clinicians to choose appropriate treatment and anticipate complications. This is particularly important for neurotoxic envenoming which, depending on the snake species involved, may not respond to available antivenoms. Adequate species identification tools are lacking. This study used a combination of morphological and molecular approaches (PCR-aided DNA sequencing from swabs of bite sites) to determine the contribution of venomous and non-venomous species to the snakebite burden in southern Nepal. Out of 749 patients admitted with a history of snakebite to one of three study centres, the biting species could be identified in 194 (25.9%). Out of these, 87 had been bitten by a venomous snake, most commonly the Indian spectacled cobra (Naja naja; n = 42) and the common krait (Bungarus caeruleus; n = 22). When both morphological identification and PCR/sequencing results were available, a 100% agreement was noted. The probability of a positive PCR result was significantly lower among patients who had used inadequate “first aid” measures (e.g. tourniquets or local application of remedies). This study is the first to report the use of forensic genetics methods for snake species identification in a prospective clinical study. If high diagnostic accuracy is confirmed in larger cohorts, this method will be a very useful reference diagnostic tool for epidemiological investigations and clinical studies. Author Summary: Snakebite is an important medical problem in sub-tropical and tropical regions, including Nepal where tens of thousands of people are bitten every year. Snakebite can result in life-threatening envenoming, and correct identification of the biting species is crucial for care providers to choose appropriate treatment and anticipate complications. This paper explores a number of methods, including molecular techniques, to assist care providers in identifying the species responsible for bites in rural Nepal. Out of 749 patients with a history of snakebite, the biting species could be identified in 194 (25.9%). Out of these, 87 had been bitten by a venomous snake, most commonly cobras (n = 42) and kraits (n = 22). This study is the first to report the use of molecular techniques for snake species identification. The diagnostic accuracy of this method appears high but needs to be confirmed in larger studies

Circumstances of the bite and baseline characteristics of victims of snakebite in southern Nepal between April 2010 and October 2012 (n = 194).

<p>Circumstances of the bite and baseline characteristics of victims of snakebite in southern Nepal between April 2010 and October 2012 (n = 194).</p

Flow diagram showing numbers of individuals screened and included in each study centre.

<p>Between 01/04/2010 and the 31/10/2012, 749 victims of snakebite were included in the study and the snake species responsible for the bite could be ascertained in 194 cases.</p

Clinical features on admission of victims of snakebite in southern Nepal between April 2010 and October 2012 (n=194).

<p>Initial assessment of snakebite victims included vital signs, patients’ complaints and standardized evaluation of envenoming signs.</p

Factors associated with a positive PCR among 565 snake bite victims.

<p>Unadjusted Risk Ratio (RR) and their 95% Confidence Interval (95% CI) were calculated with respect to the baseline category, i.e., absence of the risk factor (RR = 1).</p

List of species responsible for snakebite in southern Nepal between April 2010 and October 2012 (n = 194).

<p>Species were identified either by morphological examination of preserved specimen or through PCR and DNA sequencing performed using bite-site swabs.</p

Epigenetic silencing of tumor suppressor candidate 3 confers adverse prognosis in early colorectal cancer.

Colorectal cancer (CRC) is a biologically and clinically heterogeneous disease. Even though many recurrent genomic alterations have been identified that may characterize distinct subgroups, their biological impact and clinical significance as prognostic indicators remain to be defined. The tumor suppressor candidate-3 (TUSC3/N33) locates to a genomic region frequently deleted or silenced in cancers. TUSC3 is a subunit of the oligosaccharyltransferase (OST) complex at the endoplasmic reticulum (ER) which catalyzes bulk N-glycosylation of membrane and secretory proteins. However, the consequences of TUSC3 loss are largely unknown. Thus, the aim of the study was to characterize the functional and clinical relevance of TUSC3 expression in CRC patients' tissues (n=306 cases) and cell lines. TUSC3 mRNA expression was silenced by promoter methylation in 85 % of benign adenomas (n=46 cases) and 35 % of CRCs (n =74 cases). Epidermal growth factor receptor (EGFR) was selected as one exemplary ER-derived target protein of TUSC3-mediated posttranslational modification. We found that TUSC3 inhibited EGFR-signaling and promoted apoptosis in human CRC cells, whereas TUSC3 siRNA knock-down increased EGFR-signaling. Accordingly, in stage I/II node negative CRC patients (n=156 cases) loss of TUSC3 protein expression was associated with poor overall survival. In sum, our data suggested that epigenetic silencing of TUSC3 may be useful as a molecular marker for progression of early CRC.This work was supported by grants to ME from the State of Baden-Württemberg for “Center of Geriatric Biology and Oncology (ZOBEL) - Perspektivförderung” and “Biology of Frailty - Sonderlinie Medizin”. EB received funding from the Deutsche Krebshilfe (#108287, #111086), the Deutsche Forschungsgemeinschaft (DFG, BU2285) and the German Cancer Research Center (DKFZ-MOST, Ca158)