Molecular detection of fungal pathogens

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 [1]. 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, Time consuming, 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 [1]. FUNGAL DISEASES Unlike viral pathogens which are breaking headlines in the news, it has often been said that fungal diseases are a silent epidemic [2].

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 [2]. 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 [4]. 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 Transcribed Spacer) region and the D1/D2 region of the large ribosomal units of the isolates cultured earlier. The ITS region is the most widely sequenced DNA in fungi and hence are used as molecular targets to detect fungal pathogens.

The D1/D2 regions also are associated with the ITS which help in the detection of fungal pathogens. Such a detection technique helped in identifying the causative agent of this infection [4]. MOLECULAR DETECTION USING PCR The development of PCR by Kary Mullis has revolutionized molecular biology. Its use has been widespread. PCR essentially is based on the principle of amplification of a target region of DNA using short primers specific to that region. The utilization of PCR to identify pathogenic fungal species is based on three steps [1. 5] – A method that would help in identifying the presence of DNA or RNA in the sample.

The extraction of the DNA or RNA from the sample. And finally, in order to identify the fungus, a particular or a specific target region of DNA or RNA needs to be selected. The first step involves the using of DNA probe technology. It uses specific probes that have homology to the target DNA. Recently, microarray technology has been used to a great extent to identify fungal pathogens. Oligonucleotide arrays have been developed to identify the presence of certain fungal species. The DNA from a suspected fungal pathogen can be screened across many oligonucleotide probes bound to a membrane.

The DNA from the fungal pathogen is fluorescently labeled and is compared with the pattern shown by a known set of organisms [1,5]. Figure 1 shows the use of a DNA based macroarray to identify fungi causing cranberry rot [7]. Another method is the use of Fluorescent in – situ hybridization (FISH). [1]. Figure 1 – A DNA macroarray showing the results of cranberry rot causing fungi. The top left and botton right spots are positive controls. A positive result is seen for Phyllosticta, Coleophoma, Epicoccum, Godronia, Alternaria, Pestalotia, and Pilidium [7].

In order for the second step to be successful, care needs to be taken to extract high purity DNA and rapid extraction techniques need to be designed. One example of a rapid extraction technique includes the use of an extraction buffer to extract DNA from isolated fungal colonies on an agar plate. Although this method is rapid, it does not result in high purity DNA. Hence, DNA is extracted by various physical methods like using liquid nitrogen, bead beating, and the use of certain cell wall degrading enzymes. The DNA is then purified using phenol to remove DNA degrading enzymes. The DNA is precipitated using isopropanol and ethanol [1].

For the final step, different bioinformatics tools can be used to identify specific regions of DNA of a fungal species. NCBI has developed a Primer design tool. As mentioned in the first detection technique, the fungal large ribosomal unit and the ITS regions are ideal for primer design. This is due to their regions of high conservation and regions of high variability [1,5]. Apart from this other regions are also being explored for primer design to aid fungal diagnostics. Scientist have identified the ? – tubulin and other mating type genes as regions that can be used in developing taxon specific primers.

Once the primers have been designed, it is essential to check if they are specific for a given taxon [1]. MOLECULAR DETECTION BY PCR ENZYME IMMUNOASSAY Invasive aspergillosis caused by Aspergillus fumigatus affects patients undergoing hematopoetic transplants and various other kinds of transplants and hematopoetic malignancies. In order to rapidly detect this infection, a PCR enzyme immunoassay was developed. Scientists developed a method to identify Aspergillus species from other opportunistic molds by designing fungal specific primers and DNA probes.

The ITS region was again used as a target region to design primers. Identification of fungal species of Aspergillus was achieved within a day and as little as 0. 5pg could be detected using this method [6]. PADLOCK PROBE TECHNOLOGY This method is used to detect single nucleotide polymorphisms and also for the detection of fungal pathogens. Figure 1 shows the overview of this technology. Padlock probes are considered to be long oligonucleotides. They carry a non-target complimentary segment flanked by a target complimentary region at their 5’ and 3’ regions.

Hence, on hybridization the ends of the probes can be joined by ligases. This happens only when the end segments recognize the target sequence. The non-hybridized segment is removed by exonucleolysis. A real time PCR is performed to identify the amplified DNA regions [7]. Figure 2 – It shows the steps of the padlock probe technology [7]. OTHER APPROACHES Most of the other approaches involves the use of PCR as the base. Multiplex PCR has been developed to identify many fungal pathogens in a single reaction [8]. A real time light cycler assay has also been developed to identify medically important fungi [9].

Real time PCR has also been developed to identify Zygomycete infections in immune compromised hosts [10]. CONCLUSION With the development of these technologies, science has taken a step in the right direction. However, certain areas still need to be considered to develop it into an efficient area of detection of fungal pathogens. These areas include sensitivity, accuracy ,frequency of testing, and cost. With the need to precisely identify fungal pathogens, the development of next generation sequencers and pyrosequencers are a bright future prospect.

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