MOLECULAR DETECTION OF FUNGAL DISEASES
With the rise of many new diseases caused due to viruses, bacteria and fungi; it is essential for the rapid detection of such diseases. The severity of such diseases can be reduced by its rapid detection carried out by different methods. The conventional methods may include the idea of just identifying the disease symptoms, identification of these pathogens in the laboratory by different morphological and biochemical tests, etc . The conventional methods may include certain disadvantages like incorrect identification which would eventually lead to an incorrect diagnosis and a wrong treatment. Certain other factors also need to be kept in mind like:-[1,3] Requirement of highly skilled laboratory personnel,
High risk of contamination,
Antigenic cross reactivity between species and genera,
Possibly hazardous cultures, and,
Non – quantitative.
Keeping the above points with respect to the conventional methods, rapid molecular diagnostic tests have been developed. Molecular diagnostic tests include PCR, immunoassays, and DNA/RNA probe technology .
Unlike viral pathogens which are breaking headlines in the news, it has often been said that fungal diseases are a silent epidemic . Fungal diseases normally affect immune – compromised hosts, patients requiring complex surgical procedures like in trauma cases, or in patients hospitalized with severe diseases like acute myelogenous leukemia. Aspergillus fumigatus and Candida albicans are the major cause invasive filamentous infections and other yeast related infections . The centre for disease control and prevention has categorized fungal infections into three main categories – 1) Opportunistic infections – These infections affect people suffering from severe diseases which have weakened the immune system. Examples include cancer, HIV/AIDS, transplant recipients etc. Such people usually suffer from cryptococcosis and aspergillosis. 2) Hospital associated infections - Certain changes in modern healthcare practices could lead to the development of drug resistant fungi. An example of such an infection includes candidemia which is the major cause of bloodstream infections. 3) Community acquired infections – Such types of infections are endemic only to a particular area. Fungal infections of such types are caused due to certain climatic conditions, or changes in moisture and temperature. Examples of such types of infections include coccidioidomycosis also known as valley fever, blastomycosis, and histoplasmosis.
Keeping in mind the points discussed above, it is therefore essential to come up with detection methods that can rapidly identify molecular pathogens. Hence, molecular detection techniques have been developed.
MOLECULAR DETECTION AND PHYLOGENETIC ANALYSIS USING WHATMAN FTA
This tool has greatly helped in identifying pathogenic fungi especially Madurella grisea which causes Mycetoma (Madura foot). It is an infection of the skin and subcutaneous tissue which is the result of implantation of fungal spores. IT is common in India, Africa and other tropical areas. Unfortunately conventional detection techniques could not identify the causative agent behind this infection. Hence molecular detection techniques were employed .
Isolated from people infected with Mycetoma were cultured and the fungal genomic DNA was extracted using Whatman FTA papers. Whatman FTA papers are pre treated with certain chelators and denaturants. The chelators and denaturants help in lysing the microorganisms that come in contact with the filter paper. The nucleic acids that get released after lysis get entangled in the fibers of the filter paper while the cellular debris pass through. Scientists tested this similar method with aqueous fungal suspensions and found similar results. The fungal genomic DNA that was extracted was of PCR grade. PCR was used to amplify the ITS (Internal...
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