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Forensic Method For Snakebites May Lead to Rapid MDx

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NEW YORK (GenomeWeb) ― An international team of tropical medicine researchers has developed a forensic procedure that can accurately identify the culprit in approximately one in four cases of snakebite. The method relies on PCR amplification and sequencing of a unique snake-specific mitochondrial DNA biomarker taken from swabs of bite sites.

The researchers are now seeking collaborators to build a rapid isothermal molecular diagnostic test to better identify venomous snakebites in the field.

The new protocol will also be used as a "gold standard" for epidemiological studies and for an upcoming clinical assessment of a venom immunoassay, according to developer Ulrich Kuch, head of the department of tropical medicine and public health at Goethe University Frankfurt in Germany.

"The key importance of DNA sequence-based identification using trace DNA from the bite site is, it enables us to have a larger number of patients with snake species identification, especially in clinical studies," said Kuch. An increase in sample sizes could be valuable in researching this understudied global health problem.

Snake bite envenomation is considered a neglected tropical disease, and an important public health issue, by the World Health Organization. There may be as many as 1.8 million envenomings per year worldwide. And, because most snakebite victims are young, the economic impact can be considerable, with mortality and morbidity having disproportionate and dire effects on rural economies.

The new bite identification method was presented at the American Society of Tropical Medicine and Hygiene annual meeting in New Orleans this week. It comprises data from research studies in Southeast Asia, most recently one in southeast Nepal, where a survey conducted in 2001 tallied 143 individuals with a history of snakebite and 20 fatalities over a 14-month period, which translates to an annual incidence of 1,162 snakebites and 162 snakebite deaths per 100,000 people.

Speedy care and proper triaging of snakebite victims can save lives as well as resources. In the aforementioned study, the authors found that an initial visit to a traditional healer and lack of available transportation were associated with increased risk of death, while fast transport to a clinic via motorcycle increased survival.

If patients make it to a clinic, however, some cases of envenomation respond to anti-venom, while others do not. And if the bite was from a non-venomous snake, knowing this immediately could help conserve precious resources.

In the study presented at ASTMH, species-identifying DNA was recoverable from 194 out of 749 wounds, 87 of which were from venomous snakes like the cobra, pit viper, and krait.

Previous research had to depend on patients bringing snakes to the clinic. "So far, [that] had been the gold standard for any epidemiological or clinical studies when it comes to identifying the snake species ― you always needed a dead snake, or sometimes a live snake, brought by the patient," Kuch said.

Now, "We're basically applying standard forensic techniques; collecting trace DNA with a swab stick, isolating DNA, and running PCR," he said. Importantly, in the recent study there was complete agreement between sequence-based identification and morphological ID when a snake was available.

In the study, bite swabs were collected from patients at three different clinics in Nepal. The work of the clinical teams made the overall study particularly innovative, Kuch noted, as running a Good Clinical Practices-conforming trial in a "rural, post-conflict nation" is an achievement.

Samples were collected along with clinical and epidemiological data, stored at the study site, and later shipped to Kuch's lab in Germany. The sample prep was straightforward phenol-chloroform extraction, but Kuch said he believes tweaking this step might lead to time savings, and be crucial for a rapid DNA-based test.

The method uses conventional PCR to amplify a portion of the cytochrome B gene, using primers that are generic for snakes covering "a very broad taxonomic range of different snake species," Kuch said. The primers were also designed to be maximally different from human gene sequences. "Human DNA … is always present in excess at the bite site, sometimes those bites are profusely bleeding and so on, so you get lots of human DNA and considerably less snake DNA," he said.

This step was followed by nested PCR, which "increased the number of positives quite substantially," Kuch said.

Once enough snake DNA was amplified, Kuch and his team sequenced it, then compared the results to a reference library of snake sequences they established for particular geographic regions. However, "if those sequences are already present in Gen-Bank, you can just BLAST them, of course, and get the result," he noted.

The new method now has many uses, according to Kuch. For epidemiological studies of which species of snakes bite people in a given geographic area, for example, this method is "a straightforward approach for getting this information, in addition to collecting whatever snakes people bring to the hospital."

At the moment, Kuch's group is also developing a rapid diagnostic test that uses lateral flow immunochromatographic methods to detect venom proteins in the blood of patients. They plan to validate this assay in a clinical study in Myanmar and Nepal. "To evaluate the sensitivity and specificity of such tests in clinical settings, we need an independent set of data about the snake species involved, and this is why the DNA-based test is so valuable for us," Kuch said.

However, Kuch and his colleagues have also been working in the lab with isothermal PCR methods for detecting snake DNA, such as loop-mediated isothermal amplification (LAMP), and are currently seeking partners to develop a faster, point-of-care molecular test for envenoming.

The group is now pursuing techniques for "speeding up isolation of DNA from the swab sticks and then running a rapid species-specific PCR, visualizing PCR products in some more rapid and robust way that can also be used in the settings of the rural tropics," Kuch said, adding that the group is actively seeking collaborators to develop the methods.

Preliminary results have been encouraging, and Kuch had confidence that such a diagnostic can be built, and can save lives. "If you have a LAMP test, and DNA isolation is accelerated, then you're in a time frame that's clinically useful," he said. "You get your test results early enough to make treatment decisions for the patient. That's also something that could be done without a lot of instrumentation ... that you can feasibly do in a rural clinic in Nepal."

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