Tropical Disease

A new test diagnoses patients with melioidosis within hours, rather than days, meaning they can receive the correct antibiotics faster. Globally, more than half of patients die after infection with the neglected tropical disease, melioidosis, often before they are diagnosed. A new rapid test could save lives by diagnosing patients in hours, rather than several days taken by current bacterial culture methods, meaning they receive the correct antibiotics faster. The test uses CRISPR to detect a genetic target that is specific to Burkholderia pseudomallei, the bacterium that causes melioidosis, with 93 per cent sensitivity. It was developed by researchers at the Mahidol-Oxford Tropical Medicine Research Unit (MORU), Chiang Mai University, Vidyasirimedhi Institute of Science and Technology (VISTEC) in Thailand, and the Wellcome Sanger Institute in the UK. The results, published today (14 March) in Lancet Microbe, mean more lives could be saved from melioidosis, with a rapid, easy-to-use diagnostic test that could be rolled out globally. Melioidosis is a neglected tropical disease that is estimated to affect 165,000 people worldwide each year, of whom 89,000 die from the disease. It is caused by the bacterium, Burkholderia pseudomallei, which lives in soil and water in tropical and subtropical regions, and enters human bodies via inoculation through skin abrasions, ingestion or inhalation. It is difficult to diagnose melioidosis as symptoms vary from localised abscess or pneumonia to acute septicaemia, or may present as a chronic infection. As a result of this, and the locations of isolated communities in rural areas that it mostly affects, the disease remains hugely underreported. Currently, melioidosis is diagnosed in patients after bacterial samples are cultured, which takes three to four days. In Thailand, approximately 40 per cent of patients with melioidosis die, many of whom die within the first one to two days following admission to hospital, while waiting for a diagnosis. There is no licensed vaccine for melioidosis, but patients can be effectively treated with intravenous antibiotics -- ceftazidime or carbapenem -- during the first intensive phase of treatment. However, current practices often involve initially treating patients with a range of unnecessary antibiotics to target the various symptoms the disease produces, which can waste time and resources. In a new study, the team set out to develop a new rapid test to reduce the time taken to correctly diagnose and treat patients with melioidosis. The researchers identified a genetic target specific to B. pseudomallei byanalysing over 3,000 B. pseudomallei genomes, most of which were sequenced at the Sanger Institute. They searched for conserved regions of the genome and screened the targets against other pathogens and human host genomes, to ensure their chosen target was specific to B. pseudomallei. Their test, called CRISPR-BP34, involves rupturing bacterial cells and using a recombinase polymerase amplification reaction to amplify the bacterial target DNA for increased sensitivity. Additionally, a CRISPR reaction is used to provide specificity, and a simple lateral flow 'dipstick' read-out is employed to confirm cases of melioidosis. To assess the efficacy of the test, the team collected clinical samples from 114 patients with melioidosis and 216 patients without the disease at Sunpasitthiprasong Hospital, a hospital in northeast Thailand where melioidosis is endemic. The CRISPR-BP34 test was then applied to these samples. The new test showed enhanced sensitivity at 93 per cent, compared to 66.7 per cent in bacterial culture methods. It also delivered results in less than four hours for urine, pus, and sputum samples, and within one day for blood samples. This is a significant improvement over the current bacterial culture diagnostic method, which typically takes three to four days. This new rapid diagnostic test will enable health professionals to prescribe the correct antibiotics faster, meaning fewer patients will die while waiting for a diagnosis. While saving precious time, the new test will also save resources and money, with fewer unnecessary antibiotics prescribed and less time for patients in hospital. In next steps for the team, they are currently designing randomised clinical trials to show the effectiveness of these tests in hospital settings. Plus, members of the team will begin investigating the role of human genetics in susceptibility and immune response to melioidosis infection. Dr ClaireChewapreecha, co-lead author at the Mahidol-Oxford Tropical Medicine Research Unit (MORU), Thailand, and Wellcome Sanger Institute International Fellow, said: "Working in rural Thailand has many limitations. But we have shown that limitations breed innovation, and what succeeds here can succeed anywhere. I am so proud of the team behind this new, robust rapid diagnostic test for melioidosis, and hope that it can potentially be used anywhere in the world to get the right treatments to patients faster, ultimately saving lives." Dr Somsakul Wongpalee, co-lead author at Chiang Mai University, Thailand, said: "We carefully designed the rapid diagnostic test based on CRISPR-BP34, with a robust algorithm, and tested its performance in vitro. We are thrilled that the CRISPR-BP34 test demonstrates outstanding diagnostic efficacy when tested on clinical samples, showcasing its potential to significantly impact patient outcomes and potentially save lives in the near future." Professor Nick Thomson, senior author and Head of Parasites and Microbes at the Wellcome Sanger Institute, said: "This research is a testament to international collaboration and how the application of genomics at scale leads to clinical intervention. Using a genetic target mined from a bank of thousands of bacterial genomes, the team was able to produce an incredibly sensitive test that is specific to the bacterium behind melioidosis. I look forward to seeing the clinical impacts of this research." Professor Nick Day, senior author and Director of the Mahidol-Oxford Tropical Medicine Research Unit (MORU), Thailand, and the Wellcome Trust Thailand Asia and Africa Programme, said: "Melioidosis has been neglected despite its high mortality rate and high incidence in many parts of Asia. Early diagnosis is essential so that the specific treatment required can be started as soon as possible. The new rapid diagnostic tool developed through this collaboration has the potential to be a game-changer."

Scientists discovered antibodies that protect against a host of lethal snake venoms. Scripps Research scientists have developed an antibody that can block the effects of lethal toxins in the venoms of a wide variety of snakes found throughout Africa, Asia and Australia. The antibody, which protected mice from the normally deadly venom of snakes including black mambas and king cobras, is described on February 21, 2024, in Science Translational Medicine. The new research used forms of the toxins produced in the laboratory to screen billions of different human antibodies and identify one that can block the toxins' activity. It represents a large step toward a universal antivenom that would be effective against the venom of all snakes. "This antibody works against one of the major toxins found across numerous snake species that contribute to tens of thousands of deaths every year," says senior author Joseph Jardine, PhD, assistant professor of immunology and microbiology at Scripps Research. "This could be incredibly valuable for people in low- and middle-income countries that have the largest burden of deaths and injuries from snakebites." More than 100,000 people a year, mostly in Asia and Africa, die from snakebite envenoming -- rendering it more deadly than most neglected tropical diseases. Current antivenoms are produced by immunizing animals with snake venom, and each generally only works against a single snake species. This means that many different antivenoms must be manufactured to treat snake bites in the different regions. Jardine and his colleagues have previously studied how broadly neutralizing antibodies against the human immunodeficiency virus (HIV) can work by targeting areas of the virus that cannot mutate. They realized that the challenge of finding a universal antivenom was similar to their quest for an HIV vaccine; just like quickly evolving HIV proteins show small differences between each other, different snake venoms have enough variations that an antibody binding to one generally doesn't bind to others. But like HIV, snake toxins also have conserved regions that cannot mutate, and an antibody targeting those could possibly work against all variants of that toxin. In the new work, the researchers isolated and compared venom proteins from a variety of elapids -- a major group of venomous snakes including mambas, cobras and kraits. They found that a type of protein called three-finger toxins (3FTx), present in all elapid snakes, contained small sections that looked similar across different species. In addition, 3FTx proteins are considered highly toxic and are responsible for whole-body paralysis, making them an ideal therapeutic target. With the goal of discovering an antibody to block 3FTx, the researchers created an innovative platform that put the genes for 16 different 3FTx into mammalian cells, which then produced the toxins in the lab. The team then turned to a library of more than fifty billion different human antibodies and tested which ones bound to the 3FTx protein from the many-banded krait (also known as the Chinese krait or Taiwanese krait), which had the most similarities with other 3FTx proteins. That narrowed their search down to about 3,800 antibodies. Then, they tested those antibodies to see which also recognized four other 3FTx variants. Among the 30 antibodies identified in that screen, one stood out as having the strongest interactions across all the toxin variants: an antibody called 95Mat5. "We were able to zoom in on the very small percentage of antibodies that were cross-reactive for all these different toxins," says Irene Khalek, a Scripps Research scientist and first author of the new paper. 'This was only possible because of the platform we developed to screen our antibody library against multiple toxins in parallel." Jardine, Khalek and their colleagues tested the effect of 95Mat5 on mice injected with toxins from the many-banded krait, Indian spitting cobra, black mamba and king cobra. In all cases, mice who simultaneously received an injection of 95Mat5 were not only protected from death, but also paralysis. When the researchers studied exactly how 95Mat5 was so effective at blocking the 3FTx variants, they discovered that the antibody mimicked the structure of the human protein that 3FTx usually binds to. Interestingly, the broad-acting HIV antibodies that Jardine has previously studied also work by mimicking a human protein. "It's incredible that for two completely different problems, the human immune system has converged on a very similar solution," says Jardine. "It also was exciting to see that we could make an effective antibody entirely synthetically -- we did not immunize any animals nor did we use any snakes." While 95Mat5 is effective against the venom of all elapids, it does not block the venom of vipers -- the second group of venomous snakes. Jardine's group is now pursuing broadly neutralizing antibodies against another elapid toxin, as well as two viper toxins. They suspect that combining 95Mat5 with these other antibodies could provide broad coverage against many -- or all -- snake venoms. "We think that a cocktail of these four antibodies could potentially work as a universal antivenom against any medically relevant snake in the world," says Khalek. This work was supported by funding from the National Institutes of Health (R35 CA231991, U01 AI142756, RM1 HG009490, R35 GM118062, R35 GM118069), the Damon Runyon Cancer Research Foundation (2406-20), the Jane Coffin Childs Fund, the Mark Foundation for Cancer Research and the Howard Hughes Medical Institute.

Researchers can accurately track where typhoid fever cases are highest by monitoring environmental samples for viruses called bacteriophages that specifically infect the bacterium that causes typhoid fever. Researchers can accurately track where typhoid fever cases are highest by monitoring environmental samples for viruses called bacteriophages that specifically infect the bacterium that causes typhoid fever. Senjuti Saha of the Child Health Research Foundation in Bangladesh and colleagues report these findings in a new study published February 15 in the open access journal PLOS Neglected Tropical Diseases. Typhoid fever is a common infection in many low- and middle-income countries and causes an estimated 135,000 deaths and 14 million infections globally each year. The World Health Organization has prequalified two typhoid vaccines, but for policymakers to plan effective vaccination strategies, they need accurate, high-resolution estimates of where the burden is highest. Traditionally, people have cultured the bacterium that causes typhoid fever from blood samples to determine where the infection is most common, but in the new paper, researchers tried a more cost-effective surveillance approach. They tested environmental water samples from sewage and other locations to detect bacteriophages specific to the water-borne pathogen that causes typhoid fever, Salmonella Typhi. The team tested 303 water samples from two locations in Bangladesh: the urban capital city, Dhaka, and a rural district, Mirzapur. They found that bacteriophages specific for Salmonella Typhi were present in 31% of environmental samples in Dhaka, compared to just 3% of samples from Mirzapur. This corresponds to results from more than 8,400 blood cultures, in which 5% of cultures from Dhaka and 0.05% from Mirzapur tested positive. The new results suggest that detecting bacteriophages specific for Salmonella Typhi may be a rapid environmental surveillance method that could help decision makers understand the presence of typhoid fever in the community. The researchers propose that environment monitoring of bacteriophage could be a simple, cost-effective and scalable tool to assist policy decisions on typhoid control. The authors add: ""Looking for bacteriophages in wastewater is a low-cost method for identifying typhoid hotspots without doing expensive blood cultures on thousands of people."