Portable Molecular Diagnosis
About the Practice
Leptospirosis is a Zoonotic infection common in both wild and domestic animals which can spread to humans. There are many different types of leptospires, each tending to be associated with particular animal species. The most common Leptospires resulting in human infection are Leptospira icterohaemorrhagiae. Leptospirosis is a notifiable infectious disease in humans. However, under-reporting is likely particularly for those suffering from the milder form of the infection. The main reservoirs of infection are stagnant water and wild rodents. Current diagnostic methodologies for Leptospirosis include the use of MAT and ELISA, which is time-consuming and labour intensive. The use of PCR is advantageous over ELISA and MAT, but the sophistication in the use of instrument and reagents make them inadequate under small-scale settings. An alternative to this for the detection of Leptospira is Loop-Mediated Isothermal Amplification which amplifies Dunder isothermal conditions, requiring only a regular water bath or heating block for maintaining the temperature at 60 – 65°C. The LAMP reaction may be followed by the conventional gel electrophoresis or staining by fluorescent dyes like Propidium iodide and SYBR GREEN for easy visual detection. It could specifically amplify even with 10,000 fold diluted serum Dconfirming the superiority of LAMP as a more sensitive diagnostic tool for detecting Leptospira in serum samples especially where these diseases are endemic. The study further proposes the development of a LAMP based 'dip-stick' which can be used for multiple diseases in a single evaluation using pathogen-specific primers. Using the pathogen-specific probes for different pathogens when exposed under UV or by application of selective dyes like Propidium iodide or SYBR green could give a coloured indication. Under-reporting is another major hurdle and it significantly contributes to the higher rate of incidence. Water logging and rodent contact are more frequent in villages and remote areas where proper measures are not taken to prevent these classes of infectious dieases. The lack of awareness and also the inaccessibility of health officials to these areas add to the incidence numbers. Using a portable system such as LAMP dip-stick can easily tackle these problems. It makes these preventive measures much more effective as the diagnosis is done at the housing spots and measures can be prescribed depending upon the severity of the infection. The LAMP based system fits into a pocket of a health official and a conventional dry bath powered by solar or even by battery back up could help in the diagnosis. A one-time diagnosis could help us identify differnet pathogens involved irrespective of their origin. It can be used without any need of invasive techniques, the source of the sample can be either urine of the patient on a drop of blood. The canonical measures for diagnosis for some of the most common infections like typhoid, Leptospirosis, Malaria could account for around 2000-3000 INR depending upon the hospital setup while the total cost of the proposed LAMP based dipstick would come around 100-200 INR benefitting the society even in the weaker parts. This brings down the cost of healthcare drastically, heling even the government to take care of the financially backward classes of the society without being a burden for the society.
Detail:
Leptospirosis, also known as Weil disease, Icterohemmorrhagic fever or Canicola fever, is a rare and severe zoonotic infection caused by the genus Leptospira, common for both wild and domestic animals which can spread directly to humans. There are many different types of leptospires, each tending to be associated with a particular animal species. The most common leptospires resulting in human infection are Leptospira icterohaemorrhagiae. Leptospires are bacteria which can be either pathogenic (i.e. having the potential to cause disease in animals and humans) or saprophytic (i.e. free living and generally considered not to cause disease). Pathogenic leptospires are maintained in nature in the renal tubules and genital tracts of certain animals. Leptospirosis is a notifiable infectious disease in humans but human to human transmission occurs only very rarely. The main reservoirs of infection are standing water and wild rodents. The clinical manifestations include mild, influenza-like illness; Weil’s syndrome characterized by jaundice, renal failure, haemorrhage and myocarditis with arrhythmias; meningitis/ meningoencephalitis; pulmonary haemorrhage with respiratory failure. Exemplified by the large outbreaks in Nicaragua, Brazil, India, Southeast Asia, the United States and Malaysia, a rapid, accurate method for the diagnosis of leptospirosis is important to both the clinician and the patient. The current methods available for the detection of Leptospira include MAT (Microscopic Agglutination Test), ELISA technique and PCR based detection. Furthermore, there are other methods of detection as well like complement fixation test, Fluorescent Antibody test and Dhybridization assay. However, MAT is inadequate for rapid case identification since it can only be performed in a few reference laboratories and requires analysis of paired sera to achieve sufficient sensitivity and is tedious and time-consuming. On the otherhand, ELISA has comparatively less sensitivity as the detection titre is higher. While PCR, a molecular technique, is advantageous and produce fast and reliable results, but the sophistication in laboratory setup and the requirement of skilled labour makes it a less applicable for clinical diagnosis in small laboratory setups. It may also give false-negative results because of the presence of endogenous inhibitors in the clinical materials being examined. An alternative to this is Loop-Mediated Isothermal Amplification (LAMP), a single tube nucleic acid amplification method performed at the isothermal condition, which offers a rapid, accurate and cost-effective diagnosis of infectious diseases. A few LAMP methods for leptospiral Ddetection have been published. Sonthayanon P, et al. showed that LAMP was sensitive enough to detect only half of the analyzed Leptospira positive serum samples. Also, the primers they analyzed showed cross-reactivity with some other organisms indicating imperfect specificity. On the contrary, studies by Koizumi et al showed a high sensitivity and specificity for LAMP assay when compared with the current diagnostic methods for leptospirosis. We have developed a loop-mediated isothermal amplification (LAMP) assay for the rapid and sensitive detection of Leptospirosis. In the present study, a LAMP test assay was designed using a LAMP primer set designed against the conserved regions of a surface exposed lipoprotein (LipL41) from Leptospira interrogans australis for the detection of Leptospira species. The results showed that the LipL41 primers were highly sensitive and specific for Leptospira. It could specifically amplify even with 1x104 fold diluted serum Dconfirming the superiority of LAMP as a more sensitive diagnostic tool for detecting Leptospira in serum samples especially where these diseases are endemic. This study validates LAMP as an alternative molecular diagnostic tool and confirms the utility of the LipL41 primer set for the rapid and effective detection of Leptospira infections.
About the Innovator
Address: Cardiovascular Disease Biology Program, Rajiv Gandhi Centre for Biotechnology Thycaud PO, Jagathy, Thiruvananthapuram
City: Thiruvananthapuram
State: Kerala
PIN Code 695014
Email: sudheeshap@rgcb.res.in
Contact No: 919745993958.0
Practice Details
Link: The following are the unique feature of the innovation:
1. Specificity, reliability and speed in detection
2. Portability for endemic regions
3. Cost-effective method
4. Requires less technical expertise
5. Use of non-invasive samples- Urine/a drop of blood
Problem Scale: Worldwide
News Link 5: