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Nucleic acid-based diagnostic for leptospirosis

Early diagnosis of leptospirosis is vital for better treatment, control and prevent wide spread of the disease. However, current diagnostic methods are not useful in early diagnosis or they are often costly and not readily available in many resource-limited settings. Thus, the absence of an adequate laboratory test remains the key barrier for diagnosis of this disease and there is a need for a simple, accurate and rapid diagnostic for this disease. Loop-mediated isothermal amplification (LAMP) technology is a potential candidate to realizing this. Our invention focuses on the utilization of LAMP for the detection of Leptospira by targeting two important genes of Leptospira, one for the detection of all Leptospira and another for the detection of only the pathogenic strains of Leptospira. The invention here has been demonstrated on various types of samples ranging from human to animal to environmental samples, further expanding its applicability compared to other technologies. Canonically, LAMP reaction is usually gauged and assayed by several simple methods, such as colorimetric indicators calcein and hydroxynapthol blue. We have preliminarily tested three different detection methods namely tapered optical fiber biosensor, nanoparticle, and lateral flow biosensor for a more accurate detection. The ultimate outcome of this study will be the development of a point-of-care test that allows rapid and sensitive testing in a primary care setting without the need of trained personnel. This will ultimately translate to better diagnosis of the disease with improved management of infection. The device can also be used in disease surveillance and monitoring.

 

Leptospirosis, currently a worldwide zoonotic disease, has been recognized as an important emerging infectious disease in the past few decades. The clinical manifestations of leptospirosis are nonspecific and may resemble those of other diseases, such as dengue fever, often causing misdiagnosis. There was report from Malaysia showed about 38% cases were misdiagnosed as dengue. Early diagnosis of the disease is crucial for early interventions, but current diagnostic methods are not useful in early diagnosis, costly or often not readily available in many resource-poor countries. Thus, no diagnostic technique is completely satisfactory, and the absence of an adequate laboratory test remains the key barrier for diagnosis of this disease. Therefore, availability of a rapid, sensitive and cost-effective point-of-care diagnostic test is essential to diagnose the disease for early treatment regimen before the disease aggravate.

 

This invention focuses on the use of loop-mediated isothermal amplification (LAMP) technology for the detection of Leptospira. The originality of the invention here is the use of two sets of primers targeting two important Leptospira genes (secY and lipL32) and their combination in a LAMP assay. Targeting secY gene allows detection of both pathogenic and saprophytic Leptospira as it is a housekeeping gene that are common to all species of Leptospira. Meanwhile, targeting lipL32 gene allows the detection of pathogenic strains of Leptospira as this gene is only present in pathogenic strains but not in saprophytic strains. Collectively, this invention allows the detection and differentiation of both pathogenic and saprophytic Leptospira simultaneously, further improving both its specificity and sensitivity. Most current technologies target only the pathogenic Leptospira, which may miss out the saprophytic ones that causes infection in human

Currently there are no similar product available in the market. The closest direct competitors were diagnostics based on real-time PCR technology such as the PUMA Lepto kit (France), Techne™ PrimePRO QPCR DNA detection Kit, Leptospirosis (Fisher Scientific) and ViPrimePLUS leptospirosis qPCR kit (Vivantis, Malaysia). However, these diagnostics were based on the detection on a single Leptospira gene (usually the pathogenic gene) and thus, is not able to detect and differentiate between the pathogenic and saprophytic serovars of Leptospira. The approach in our invention not only allows the detection but also the differentiation of the saprophytic and pathogenic serovars of Leptospira. On the other hand, though diagnostics based on real-time PCR technology had its supremacy over other methods, they are often hindered by the presence of PCR inhibitory substances in the biological samples and the use of equipment that are not often available in remote and resource-poor countries. The LAMP technology utilized here is an outstanding alternative gene amplification procedure, wherein the amplification can be completed in less than one hour under constant temperature by one type of enzyme in conjunction to its simplicity and resistant to inhibitors. Therefore, LAMP does not require the use of a thermal cycler and can be performed simply with a heating block or water bath. On a different note, existing technologies are usually focused on human and/or animal samples. The invention here was demonstrated on human samples, animal samples, rat kidney samples and environmental samples such as soil and water which will eventually expand its area of applicability compared to current technologies.

 

Leptospirosis has often been misdiagnosed as dengue due to the former poses early clinical manifestations akin to dengue, two of them being ranked as the most important infectious diseases in Malaysia. These eventually cause improper medical management and treatment of patients, which is why there is a need for a new simple, low cost and accurate nucleic acid diagnostic for this disease. The outcome of this project will be a potential game-changer that may eventually aid in the accurate diagnosis of leptospirosis through improvements to the LAMP system, translating to earlier interventions. In Brazil, USD 11.8 million were lost due to the disease and in New Caledonia, close to Euro 1 million were spent on the diagnostic, prevention and control of the disease. The invention here will not just be beneficial to Malaysia but also other resource-limited countries and developing countries, especially those where leptospirosis is endemic. With the developed prototype, it will aid in better patient management, prevent unnecessary drug usage, and reduce health care utilisation costs. This will ultimately, minimize the economic loss and overall cost caused by the disease. In terms of environment, because leptospirosis is also an environmental-associated disease, the invention here can also aid in the environmental surveillance of the bacteria since it has been demonstrated to work on environmental samples as well. This can help to monitor, predict and control the outbreak of the disease from the environment.

 

 

 

Assoc. Profesor Dr. Chee Hui Yee

Department of Medical Microbiology and Parasitology

Faculty of Medicien and Health Sciences

Tel: 03-89472576

Email: cheehy@upm.edu.my

Date of Input: 05/09/2019 | Updated: 05/09/2019 | asrizam

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