Fungi can directly harm as being opportunistic pathogen and/or indirectly by producing toxic secondary metabolites. During the past two decades, scientific advancements in the field of diagnostic microbiology have enhanced rapidly due to a technological revolution in molecular aspects of microbiology. In particular, speedy detection techniques for nucleic acid amplification and its mutual characterization with automated and user-friendly software have significantly widened the area of diagnostic detections for the clinical microbiologist. The available conventional methods were mostly labor-intensive and repeatedly requires days to weeks before results. Moreover, these examinations were usually performed at hospitals only, due to the complexity and length of such testing. This has led to the discovery of direct detection methods for the fungal pathogens. Molecular methods developed as diagnostic tools for detection of these fungal infections, are very sensitive and highly specific. Out of these, one that will be cost-effective and exhibit on-site applicability, sensitivity, and specificity will become a "gold standard" for fungal detections. In this chapter, we will discuss the recent advances in molecular methods and their applications for detection of human fungal pathogens.
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Pathogenic fungi cause or can cause disease to plants, animals and particularly humans, either directly or indirectly. There are a number of fungal species that specifically cause human diseases. Majority of the pathogenic fungi appears to be soil-inhabiting species where they exist as saprobes, but attacks aggressively under favorable conditions. Thus, fungal infections may be due to opportunistic fungi rather than fungi that specifically cause human diseases. These opportunistic pathogens are usually more harmful since these don't depend on living organism and have not evolved with any particular host, thus having a greater probability of killing their hosts. Besides, those secreting toxic secondary metabolites (extrolites) and those present in air acting as allergen [Vermani et al. 2010] can, indirectly, cause disease when they enter inside the body. It is believed that almost any fungus has the potential to cause an infection. Unlike bacterial diseases, fungal diseases are more complicated to treat because of its eukaryotic nature, by the virtue of which these make two types of cells. Also, under treatment stage, the drugs prescribed to the patients can't be capable of differentiating host cells that are also eukaryotic in nature, and thus damaging host cells in addition of removing fungal infections. There are chances that even after the treatment, diseases can recurre.
With mounting mortality of 90% in immunocompromised patients [Kontoyiannis and Bodey 2002; Kami et al. 2000] and cancer patients, at global scale, in the past two-three decades, life-threatening incidences of fungal infection had increased at an alarming rate and it had became a subject of medical importance. With such alarming situation, there is a need to improve the accuracy and speed of the available diagnostic methods for identifying the pathogenic fungi. In recent years, there has been a significant advancement in the innovative diagnostic methods for identification and detection of microorganisms at the molecular level. In this chapter we will review molecular methods currently used for typing species and strains of medical importance and discuss some new diagnostic methods that has been used for detecting other microorganisms. The content will emphasize the concept of each method, as well as its advantages and limitations.
II Molecular Methods: Need of Early Diagnosis
Early diagnosis of IFIs remains a major problem. Diagnosis of fungal pathogens in the early hours is imperative, as delayed or missed diagnosis increases mortality rate. The survival of the individual depends on early diagnosis and prompt initiation of effective antifungal treatment [Trama et al. 2005]. Diagnosis is often a difficult task as it is generally depends upon host factors, clinical and radiological findings, and mycological criteria. The detection tools accessible for the clinician usually include clinical signs and symptoms, culture, microscopy, radiography, serology, and histopathology [Stevens 2009]. Unfortunately, conventional laboratory tests, such as culture and galactomannan detection, lacks sensitivity, specificity, and are rarely conclusive, resulting in true-positive results only at advanced stages of infection [Denning 1998; Hope et al. 2005; Lagte 1999] and work on "one substance one assay" concept [Konietzny and Greiner 2003; Yong and Cousin 2001]. Chromatographic methods have limited sensitivity and require longer time for analysis while serological tests have a problem of cross reactivity [Kappe et al. 1996; Schonheyder 1987]. Furthermore, the emergence of clinical resistance to commonly used azole drugs impedes successful treatment [Denning 1997; Marr et al. 2002; Steinbach & Stevens 2003]. The limitations for invasive fungal infections (IFIs) have led to the development of molecular techniques to aid in the detection of IFIs [White et al. 2006]. Molecular techniques offer a faster and more precise estimation than classical methodologies. For this, multicopy genes are targeted, such as, 18S rDNA, internal transcribed spacer (ITS) regions, 28S rDNA, etc. Since these are present in greater numbers (>100 copy) in the genome, their amplification will be easy, rapid, thereby, providing a high sensitivity [Bialek et al. 2005]. Nucleic acid-based diagnostics are the fastest growing component of many clinical laboratories. These applications are gradually replacing or complementing culture-based, biochemical and immunological assays for the detection of a wide range of microorganisms.
III DNA-DNA Hybridization
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Charles Sibley and Jon Ahlquist pioneered this technique for examining the phylogenetic relationships in avains [Sibley and Ahlquist 1984]. The labeled DNA of one organism is mixed with the unlabeled DNA of next organism. The mixture is incubated for denaturation followed by annealing of the strands. The labeled single stranded DNA forms hybrids of double-stranded DNA with unlabeled single stranded DNA. The single stranded DNA sequences binds more firmly with only those strands that have a higher degree of similarity, therefore such hybrids would require more energy for separating the hybridized strands than dissimilar sequences. DNA microarray and oligonucletodie hybridization, are the two techniques that use this protocol, have became a powerful tools for molecular typings.
1. DNA Microarray
DNA microarray (DNA chip technology) has been a useful tool in detecting a variety of microorganisms, especially for those microroganisms where long protocols are required or difficult to distinguish by conventional methods [Adamczyk et al. 2003; Fukushima et al. 2003; Volokhov et al. 2002]. The principle involved is hybridization of the fluorescently labelled DNA sequences to complementary target sequences which generates a signal depending upon the amount of target sample binding to the probes (Fig. 1). An array of oligonucleotide probes, developed to detect airborne fungi, were designed by taking 18S rRNA gene sequences and were used to detect 31 species belonging to 15 genera [Wu et al. 2003]. Comparative genomics exposed the absence and divergence virulence based genes C. albicans and C. dubliniensis [Moran et al. 2004] while species-specific oligonucleotide probes were designed, using ITS regions, to diagnose 12 potential fungal pathogens [Leinberger et al. 2005].
The greatest advantage of this technology is that many genes can be checked in a single run. An additional benefit is that no prior information of target DNA sequences is not required to construct and use the DNA microarrays. They are relatively fast, adaptable, comprehensive, and user-friendly. There are obviously associated drawbacks of microarrays, such as for gene expression analysis. mRNA of good quality is necessary. Array fabrication is another problem for this technique besides its equipment and associated cost.
2. Oligonucleotide hybridization
The short piece (>20 bases) of nucleotide sequences are designed to binds to the complementary target DNA sequences. The oligonucleotides designed with known polymorphic sites can be end-labeled with dye or enzyme or radioactive tag. It labeled oligonucleotide acts as a probe are hybridized as Southern blot assay form either total genomic DNA or with specific amplicons amplified by PCR. The mismatched blots are washed off, therefore the matched blots produces a single for detection.
The signal generated provides direct results for the presence of positive sample. Thus, providing a highly specific result. They are also resistant from RNases. Being small in size. They can easily be used for in situ hybridization purposes. Moreover, because they are synthetically designed, it is probable to have the same GC content as G/C bases binds more strongly than A/U bases. Some disadvantages of these probes are due to the limited process of labeling oligonucleotides, limitations on label quantity per probes leads which reduces the sensitivity, and the hybrids formed are less stable because of the short length of the probes. An additional disadvantage comes with the necessary application DNA synthesizer.
IV Polymerase Chain Reaction Methods
PCR technology had markedly influenced the molecular diagnosis of fungal infectious diseases, as previous efforts were mostly hindered by having small amount of target DNA for diagnostic purposes. With the breakthrough of PCR, the problem was solved as this method amplifies the single copy of target DNA into millions in few minutes. It has become one of the easiest methods for the identification of any microorganism. The importance of this technology has been evident from the fact that PCR has been the widely used technique for the diagnosis or the study of any type of microorganisms. Several researchers have used the PCR/PCR-dependent methods to recognize the specific fungal infection in patients, mostly for Aspergillus, Candida, and Pneumocystis while for other fungal pathogens, there are a limited number of reports available.
To find a phylogenetic relationship between pathogenic species of A. fumigatus, A. flavus, A. niger, A. terreus, and Emericella nidulans, PCR amplification of the mitochondrial cytochrome b gene was performed followed by sequencing of the amplicons. All five species produced a species-specific band of 426 bp except of E. nidulans [Li et al. 1998]. Addition of an extra primer showed increase in identification sensitivity of detecting human pathogenic fungus [Kappe et al. 1998]. Targeting 18S rDNA, A. fumigatus was amplified from pulmonary tuberculosis in patients by a two-step PCR [Bansod et al. 2008]. The species-specific primers and probes were developed by comparing the 18S rRNA gene sequences of Aspergillus, Candida, Mucor, Penicillum, Trichosporon cutaneum, and T. glabrata pathogens. However, the specificity of these probes was not 100%, the sensitivity was much more than the conventional PCR system. Heating the DNA isolating buffers to raise the DNA yield and amplifying a multicopy gene achieved the higher sensitivity [Einsele et al. 1997]. The air fungal concentration was measured by mold-specific quantitative PCR (MSQPCR) where Aspergillus penicillioides followed by A. versicolor was found to be present more than any other Aspergillus species in Cincinnati, USA [Meklin et al. 2007]. To identify the pathogenic fungi directly from cultures and speedy detection of A. fumigatus, Candida albicans, C. glabrata, C. parapsilosis, and C. tropicalis, multiplex PCR has been used where ITS regions were selected to rose the sensitivity upto 100-1000 DNA molecules [Luo and Mitchell 2002].
V PCR Based Methods
1. Microsatellite Typing
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Microsatellites are short sequence repeats (SSRs) that showed a considerable point of inter- and intra-specific polymorphism (~10-2 to 10-5) [Richard et al. 1999]. This developing technology makes use of hypervariable DNA regions composed of ten or more tandem repeats consisting of two, three or four nucleotides. Hypervariability results due to the mutations which have occurred during DNA replication of single strand or during recombination in meiosis. Microsatellites are generally amplified by PCR and is technically called as microsatellite length polymorphism (MLP) [Abdin et al. 2010] while multilocus microsatellite typing (MLMT) is combination of MLP with DNA sequencing.
A. fumigatus [Bart-Delabesse et al. 1998] strains were characterized with microsatellite-based multiplex PCR [Araujo et al. 2009] while A. flavus [Hadricha et al. 2010] and A. sydowii [Rypien et al. 2008] were identified by microsatellite typing, both techniques had shown high discriminatory power with excellent reproducibility [Hadrich et al. 2010; de Valk et al. 2008]. MLP was carried out to show the different clades in C. albicans [Chávez-Galarza et al. 2010] while MLMT of a highly polymorphic microsatellite locus (CAI) of C. albicans was defined outside the known coding region [Sampaio et al. 2003] and the same locus was further amplified by multiplex PCR for greater sensitivity to observe the microevolutionary changes due to strand slippage by Taq DNA polymerase and loss of heterozygosity [Sampaio et al. 2005].
The main gain of this technology is its distinguishing ability due to the presence of variability in microsatellite regions. This provides another benefit of specific amplification in mixed cultures as strain specific primers are available. Another important advantage of the method is to evaluate the results from other groups on computer based softwares [Hennequin et al. 2001]. Although the shortcoming of occurrence of null alleles or homozygous condition in place of heterozygous genotyping, this technique can provide best strain typing method for pathogenic fungal agents that can cause outbreak.
2. Restriction Fragment Length Polymorphism (RFLP)
With the discovery of restriction endonucleases, they have continuously been used to discriminate the species and strains of fungi along with other organisms. The basic principle is restriction of genomic DNA with endonucleases and its examination on agarose or polyacrylamide gels upon electrophoresis. Depending upon the band size and number of restriction sites present, it is possible to evaluate the different banding patterns for comparing the polymorphisms [Tait 1999].
RFLP has been regarded as "gold standard" for fingerprinting of A. fumigatus [Bart-Delabesse et al. 2001]. This method for analyzing of A. fumigatus isolates of geographically and epidemiologically diverse origin, after digesting the total cellular DNA with SalI and XhoI endonucleases [Denning et al. 1990] while a pattern of DNA marker for opportunistic fungal pathogens (A. fumigatus, A. flavus, A. niger, A. nidulans, and A. terreus) was produced by restriction of the amplicons by HhaI [Mirhendi et al. (2007]. A combination of RFLP and RAPD markers show greater discriminatory power [Semighini et al. 2001] for differentiating isolates of A. fumigatus. Recent studies have suggested that several A. fumigatus strains had been misidentified on morphological basis, which when tested at molecular level proved to be A. lentulus and Aspergillus udagawae besides A. fumigatus [Balajee et al. 2006]. On the basis of 18S rDNA, Candida spp. were differentiated from A. fumigatus and A. niger [Isik et al. 2003] while intraspecific differentiation between Candida spp. were done by digesting amplified ITS regions with MspI [Mirhendi et al. 2006, Mousavi et al. 2007]. Only two species, C. albicans and C. dubliniensis showed similar pattern after digesting with MspI [Mirhendi et al. 2006]. In a recent report, high infection risks Candida species, in particular C. albicans, to HIV-infected Ethopian patients was seen [Isogai et al. 2010]. Candida orthopsilosis and Candida metapsilosis were recently described as separate species after dividing them [Mirhendi et al. 2010].
From the above points, it has became clear that this technology was the basis of early methods for developing genetic fingerprintings at species level in addition of identification of newer species from phenotypically similar organisms. RFLPs can be used in many different settings to accomplish different objectives like with pulse field gel electrophoresis [Arshad et al. 1993], studying molecular epidemiology of microorganisms such as viral, bacterial, and fungal pathogens [Weber et al., 1997; Lipuma, 1998; Soll, 2000; Erdman et al., 2002]. Analysis of RFLP variation in genomes was a vital tool in genome mapping and genetic disease analysis.
3. Random Amplified Polymorphic DNA (RAPD)
As the name pronounce, the genomic or template DNA is randomly PCR amplified to show some polymorphisms. Template DNA with several short primers (8-12 bases) is allowed to anneal at low temperature (30-38 °C) for generating multiple PCR amplicons. These fragments are then allowed to move as their electrophoretic mobilities. RAPD only checks variations in the length across the two primer binding sites [Williams et al. 1990]. The change in nucleotides in primer binding region, particularly at the 3' ends, can avert primer binding to the template DNA with no PCR amplification, and thus absence of band, resulting in a different pattern of amplified DNA segments on the gel. Similarities in total band number and band mobility among the strains can be inferred for epidemiological studies [Beokhout et al. 1997]. While using array of primers, RAPD sensitivity increases to detect variations between strains which can be obtained only by a combination of two methods [Mondon et al. 1997, Brandt et al. 1995].
A specific-specific fingerprint was developed for A. fumigatus, A. niger [Mirhendi et al. 2009]and A. flavus [Raclavsky et al. 2006] isolates by six and five primers respectively, while a RAPD was performed to differnetiate the anamorphs and telomorphs of A. chevalieri, A. nidulans, A. tetrazonus (quadrilineatus) and their corresponding teleomorphs [Abu Seadah and El Shikh 2008]. For observing candida organisms in buccal cavity, 8 of 10 patients showed same genotypes of C. dubliniensis suggesting that these have originated from endogenous commensal strains [Jewtuchowicz et al. 2009] whereas at two Hungarian hospitals, C. parapsilosis sensu lato [Kocsube et al. 2007] alongwith C. metapsilosis, was found to be the cause of hospital infection. A characteristic molecular fingerprint was developed for each Candida species. In addition to that different C. albicans strains was shown to get differentiated by specific PCR based amplification of secreted aspartic proteinase (SAP) genes and dipeptidyl aminopeptidase (DAP2) gene [Bautista-Munoz et al. 2003]. RAPD profiles of C. glabrata showed a genetic shuffling in HIV infected patients [Samaranayake et al. 2003].
Although the RAPD method is fast and simple, there are few drawbacks of it. One of the major disadvantages is its reproducibility due to the low annealing temperatures that may result in non-specific amplifications. Even a single base change can hinder the amplification and thus absence of characteristic band. The method is very susceptible to reaction conditions, DNA quality and temperature profiles. It is vital for RAPD to maintain constant reaction conditions for achieving achieve reproducible results. Moreover, PCR buffer, master mix and thermal cycler should be of one type in order to get the amplifications of strains at the same time while comparing the multiple strains of a species. Another disadvantage is the two bands may co-migrate but differ in their nucleotide sequence, causing a major setback in interspecific studies. By RAPD profiles, heterozygous genotypes cannot be detected as this marker is for haploid organisms showing dominant or null alleles.
4. Amplified Fragment Length Polymorphism (AFLP)
Amplified fragment length polymorphism is a highly sensitive technique for detecting polymorphisms in DNA of any organisms. This method is based on PCR for fast identification of genetic diversity (Mueller and Wolfenbarger 1999). It is basically a two step procedure, the genomic DNA is restricted first by one or more restriction enzymes followed by the ligation of adapters to the sticky ends of the restricted fragments. Two PCR primers having corresponding adaptors and restriction site-specific sequences perform amplifications of selective fragments. Finally, the amplified products are seen by electrophoretic separation of amplicons on agarose gels (Vos et al. 1995). It has become a commomly used techique in laboratory for the identification of genetic variation in strains or closely related species of plants, fungi, animals, and bacteria.
AFLP was compared with short tandem repeats (STR) to find the genetic relatedness of A. fumigatus. Both methods had shown that interpatient isolates belonged to different genotypes whereas the isolates from intrapatient deep sites were all of the same genotype. On the contrary, isolates among respiratory samples shown difference in genotypes within the individual patients [de Valk et al. 2007].
A modified the AFLP method with addition of fluorescent dye (bromophenol blue) to clearly differentiate between genus and species. AFLP patterns, generated by a modification, are highly specific for each for species of Candida [Kantardjiev et al. 2006]. A combination of methylation specific PCR (MSP-PCR) and AFLP has been shown as a good source of identification and discrimination between the fungal species [Lopes et al. 2007]. In a recent report, a genotypic study was carried out on C. parapsilosis strains isolated from different geographical regions of the world and from different body sites. A considerable variation and geographical clustering among the isolates was found when the relative intensity for each fragment of AFLP genotypic profiles was considered. Using same technique, Hensgens et al. (2009) had shown a recombination of genes had occurred in between the C. parapsilosis and C. metaplisosis [Duarte-Escalante et al. 2009; Tavanti et al. 2010].
Despite having good discriminatory power, there are still some drawbacks with this method. Like the RAPD, AFLP also lacks the agreement of homology between two bands of similar molecular weight (MW), thus producing complications in phylogenetic analyses studies while the reverse may be true for non-homologous bands. Similarly, AFLP markers exhibit dominance. Furthermore, AFLPs need automated analysis due to the huge amount of information is created them. In genetic mapping, AFLPs often collect at the centromeres and telomeres and finally they need a good laboratory, especially for data analysis.
Comparitively with other marker techniques including randomly amplified polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP), and microsatellites, there are many advantages of AFLP. Besides amplifying 50-100 fragments at a time, AFLP has higher reproducibility, resolution, and sensitivity [Mueller and Wolfenbarger 1999; Klaassen and Osherov 2007]. Also, no prior knowlegde of sequence is needed for the amplification [Meudt and Clarke 2007]. Thus, resulting the AFLP as extremely beneficial technique in identification of taxa where very little is known about the genomic makeup of various organisms.
In recent decade, several additional innovative techniques have emerged to improve the protocols for diagnosis of pathogens by overcoming the previous drawbacks. But before using the molecular detections, it is crucial to extract the nucleic acid from the samples from medical sample material. For these, no universal method has yet been obtained for the source of sample collection and nucleic acid extraction protocol. DNA isolation is thought as the target of choice due to its relative stability and ease of extraction; still the extraction methods provide variations in DNA concentration. Abundant genetic information has been made public which can be used to design primers. The new assays should be first checked through in silico method for the cross-reactivity and the feasibility of the reaction to obtain good results.
In a recent research, use of ITS regions as a convenient universal marker for fungal species identification was recommended [Balajee et al. 2007 and Samson et al. 2007b]. Microsatellites produced a detectable signal in any circumstances. This assay had proved to be an extremely robust method [de Valk et al. 2007; Klaassen and Osherov 2007] to verifying the presence of fungal species and therefore this can be treated as 'gold standard' for molecular detections and fungal identifications. There are certain new techniques that have been developed can also be used for identification purposes. Pyrosequencing (Fig. 2) with inversion probes has shown much greater sensitivity in case of Mycobacterium identification [Novais et al. 2008]. Sequence-enabled reassembly with green fluorescent protein (GFP), b-lactamase (LAC), or mCpG can also be utilized directly to quantify the dsDNA [Ghosh et al. 2006]. Some isothermal amplification methods such as loop mediated isothermal amplification (LAMP) and nucleic acid sequence based amplification (NASBA) can also be used for this purpose.
These recent genomic advances provided the main impetus which will further enhance the sensitivity of identification. Though these methods are quite superior and efficient than others, yet they are to be modified and improved as per fungal genomes for diagnosis purposes. Further, a thorough revision of all pathogenic Aspergillus species is much needed for developing better understanding about the virulence and variations present in the natural isolates.