Medical Mycology 2002, 40, 87±109 Accepted 9July 2001 Review article Utilizationof the internaltranscribed spacer regions as molecular targets to detect andidentify human fungal pathogens P.C.IWEN*, S.H.HINRICHS* & M.E.RUPP Downloaded from https://academic.oup.com/mmy/article/40/1/87/961355 by guest on 29 September 2021 y *Department ofPathology and Microbiology,University ofNebraska MedicalCenter, Omaha, Nebraska, USA; Internal Medicine, y University ofNebraska MedicalCenter, Omaha, Nebraska, USA Advancesin molecular technology show greatpotential for the rapiddetection and identication of fungifor medical,scienti c andcommercial purposes. Numerous targetswithin the fungalgenome have been evaluated, with much of the current work usingsequence areas within the ribosomalDNA (rDNA) gene complex. This sectionof the genomeincludes the 18S,5 8Sand28S genes which codefor ribosomal ¢ RNA(rRNA) andwhich havea relativelyconserved nucleotide sequence among fungi.It alsoincludes the variableDNA sequence areas of the interveninginternal transcribedspacer (ITS) regionscalled ITS1 and ITS2. Although not translatedinto proteins,the ITScoding regions have a criticalrole in the developmentof functional rRNA,with sequencevariations among species showing promiseas signature regionsfor molecularassays. This review of the current literaturewas conducted to evaluateclinical approaches for usingthe fungalITS regions as molecular targets. Multipleapplications using the fungalITS sequences are summarized here including those for cultureidenti cation, phylogenetic research, direct detection from clinical specimensor the environment,and molecular typing for epidemiologicalinvestiga- tions. Thebreadth of applicationsshows that ITSregions have great potential as targetsin molecular-basedassays for the characterizationand identi cation of fungi. Developmentof rapidand accurate ampli cation-based ITS assays to diagnose invasivefungal infections could potentiallyimpact care and improve outcome for affectedpatients. Keywords fungalPCR, fungalITS, internal transcribed spacer, rDNA gene complex Introduction been used in clinical microbiology applications for the classication and identication of bacteria and eukar- The ability to determine the nucleic acid sequence of yotic pathogens, such as fungi for many years [1]. The genomic DNAhas revolutionized mostareas of con- ribosomal DNA(rDNA) genes are found in all micro- temporary biomedical research. DNAand RNAhave organisms and known toaccumulate mutations at aslow constant rate over time. Nucleotide sequence hetero- geneity within this region can be used to phylogenetically classify microorganisms. Interspaced among the highly Correspondence:Peter C. Iwen,Department of Pathology and Microbiology,University of Nebraska Medical Center, Omaha, conserved sequences of the rDNAgenes are regions of NE68198-6495,USA. Tel.: 1402-559-7774;fax: 1402-559-4077; variable sequences called spacer regions. The function of ‡ ‡ e-mail:[email protected]. these regions is not completely known; however, they are ã 2002 ISHAM,ISHAM Medical Mycology , 40, 87±109 88 Iwen et al. historically referred to as spacers since they separate the gene) [19–22]. The comparison of nucleotide sequences functional DNAsequences of the various rDNAgenes. within these gene regions has been successful for the Since mutations within the spacer regions of the separation of fungal genera and species. However, rDNAgene complex occurwith greater frequency than limited sequence variability within these rDNAgenes, with the rDNAgenes, the sequence heterogeneity within together with aneed to compare large sequence regions, this area has been useful for the separation of both has led to ashift to evaluate the shorter spacer regions as genera and species. Early molecular studies showing the targets to separate fungal species. usefulness of the rDNAcomplex to classify bacteria The dramatic increase in the incidence of opportunis- were done by Gutel et al.[2]. In this group of ticfungal infections along with the development of new microorganisms, the operon organization consists of a antifungal agents with various spectra of activity and the Downloaded from https://academic.oup.com/mmy/article/40/1/87/961355 by guest on 29 September 2021 promoter region followed by asequence coding for the emergence of antifungal resistance has led to acritical 16S rDNAgene, aspacer (also referred to as the need for diagnostic methods that can rapidly and intergenic spacer), the 23S rDNAgene coding sequence, accurately identify fungal pathogens [23–25]. The re- another short spacer, and nally the sequence coding for ported molecular techniques, using areas within the 5S rDNAgene. With the subsequent development of the rDNAgene complex as atarget, have shown promise for polymerase chain reaction (PCR)technique in combina- the detection and identication of fungal pathogens. The tion with this previous knowledge about the rDNA purpose of this paper is to review amplication methods complex, techniques to identify bacteria using the 16S/ that use ITS regions as molecular targets to diagnose and 23S ribosomal spacer as atarget were developed [3,4]. classify fungal pathogens. The commercialand industrial This ability to amplify and to compare sequences within approaches using the ITS regions as targets have been the 16S/23S-spacer region has had an enormous impact evaluated in numerous other studies and will only be on the classication of bacteria [5,6]. Additionally, discussed in limited detail in this review [26–36]. Finally, utilization of primers that recognize universally con- for amore thorough discussion of PCRmethods for the served sequences within the rDNAgenes of prokaryotic diagnosis of invasive fungal infections, the reviews of cells which ank the variable signature sequence within Reiss et al.[37] and Walsh and Chanock [38] are the spacer region has allowed for the identication of recommended. pathogens which previously could not be cultivated [7– 9]. Similarly, it is also recognized that eukaryotic cells Biologyof theITS regions such as fungi have arDNAgene complex region with Introduction comparable characteristics. The organization of this complex in fungi includes asequence coding for the Afascinating feature of biological life is the commonuse 18S rDNAgene, an internal transcribed spacer region of the DNAgenetic code and its subsequent processing (ITS)1, the 5 8S rDNAgene coding region, another ITS into functional units of protein through the intermediate ¢ region (called ITS2) and the sequence coding for the 28S RNAmolecule. The transcription of DNAinto RNA rDNAgene. As in the case of the 16S and the 23S rDNA and translation of RNAinto protein are both highly genes, the coding regions of 18S, 5 8S and 28S nuclear regulated and compartmentalized in all living organisms. ¢ rDNAgenes evolved slowly, and are relatively con- The cellular factory responsible for the production of served among fungi, providing amolecular basis of protein is the ribosome. As the essential functions of establishing phylogenetic relationships [10]. Between ribosomes are critical for survival, their physical para- these rDNAgene-coding regions are the ITS1 and meters have been conserved in all formsof life, from ITS2 regions, which are similar to the spacer regions in bacteria to humans. Some components within the the bacterial rDNAthat evolved more rapidly, leading to ribosomal factories have, however, changed during the sequence variability among genera and species of fungi. evolutionary process. These similarities, as well as the Early work in molecular testing using the rDNA changes within genetic material can be used as tools for complex as atarget concentrated inthe region of the 18S the identication of microorganisms including fungi, rDNAgene (also referred to as the small-subunit rDNA which is the focus of this review. The sequence homology gene or the 16S-like rDNAgene) [11–16]. Other genes within the rDNAgenes of fungi (18S, 5 8S and 28S ¢ within the rDNAcomplex have also been used for the genes) and differences within the spacer regions (ITS1 molecular evaluation of fungi. These include the 5S and ITS2) are the genetic basis for the organization of rDNAgene [17], the 5 8S rDNAgene [18] and the 28S the fungi into taxonomic groups. After adetailed ¢ rDNAgene (also referred to as the large-subunit rDNA discussion of the principles that are known about the gene, the 25S to 27S rDNAgene, or the 28S-like rDNA biological aspects of the ITS regions, the application of ã 2002 ISHAM, Medical Mycology , 40, 87±109 ITS regionas a moleculartarget for fungi 89 The 18S-gene region is about 1800 bp in size with both conserved and variable domain sequences. Sequence variations within this region have been used to assess the taxonomic relationships of the major groups of Fig. 1 Representationof the rDNA genecomplex in fungi denotinggene order and position of theITS regions. living organisms and to separate genera and species based on sequence polymorphism [12]. However, the drawback to using this region for the identication of using these spacer regions as molecular targets will be species is the relative sequence homology among fungal considered. species and the need to sequence alarge number of bases in order to do comparative analysis. The 5 8S Downloaded from https://academic.oup.com/mmy/article/40/1/87/961355 by guest on 29 September 2021 ¢ rDNA complex region on the other hand is only about 160 bp long and The ITS regions are located in the rDNAgene complex highly conserved within major organism