Molecular Detection and Identification of *Candida*

*Muataz Mohammed Al-Taee*

#### **Abstract**

Human opportunistic yeast infections have become more common in recent years. Many infections are difficult to treat and diagnose due to the large number and diversity of organisms that can cause sickness. In addition, infectious strains eventually develop resistance to one or more antifungal medicines, severely limiting treatment choices and emphasizing the need of early detection of the infective agent and its drug sensitivity profile. Current techniques for detecting species and resistances are insensitive and specific, and they frequently need pre-cultivation of the causal agent, which delays diagnosis. New high-throughput technologies, such as next-generation sequencing or proteomics, make it possible to identify yeast infections more sensitively, accurately, and quickly. Opportunistic yeast pathogens, cause a wide spectrum of superficial and systemic infections, many of which are lethal. In this work, we give an overview of current and newly created approaches. It may be used to determine the presence of yeast infections as well as their medication resistance. Throughout the book, we highlight the following points: Explaining the benefits and drawbacks of each strategy, as well as the most promising advancements on their route to success.

**Keywords:** yeast pathogens, diagnosis, *Candida*, candidemia, sequencing, proteomics

#### **1. Introduction**

Various infections, ranging from superficial to systemic, are caused by opportunistic yeast pathogens, which are often deadly [1]. These viruses have become more common in recent years, making them a leading source of life-threatening illnesses. This is due in part to medical advancements, which have increased the survival rate of patients who are particularly vulnerable, such as premature babies, the elderly, and those with compromised immune systems. Furthermore, the widespread use of catheters, antibiotics, and abdominal surgery promotes opportunistic yeast expansion outside of their natural symbiont habitats [2]. Despite recent advances, death rates from invasive candidiasis remain high, at over 40%, and treatment is complicated by antifungal resistance and the advent of novel infections [3, 4]. Non-candida species such as *Candida dubliniensis, Candida glabrata, Pichia kudriavzevii, Candida parapsilosis,* and *Candida tropicalis* are becoming increasingly widespread. *Candida auris* has been around for a long time [5, 6].

*Candida spp.* have been identified as the cause of candidiasis [7–9]. Crossing pathogenic and non-pathogenic strains can result in the emergence of new virulent

variants [10]. *Candida spp.* does not belong to a single genus in the phylogenetic sense, as different *Candida* species may be found across the Saccharomycotina tree [11, 12].

Many therapeutically significant *Candida* species may be renamed as a result of current work on yeast genes and taxonomy, and physicians should be aware of this potential. Because virulence and antifungal resistance differ between species [13] and even between strains of the same species [14, 15], making treatment decisions at the species level (or even higher) is critical. As a result, it's vital to identify the infection's causal agent precisely, accurately, and rapidly so that proper antifungal medication may be started right once, especially in those with life-threatening candidiasis.

*Candidasis* is diagnosed using microscopy, selective culture, and/or biochemical methods [16, 17]. All of these approaches require isolating and cultureing the infectious agent from clinical samples, which takes around 48 hours for most pathogenic yeasts but may take longer for other samples or species. Furthermore, identification procedures need specialized expertise, can provide perplexing findings, and are timeconsuming, all of which add to the time it takes to achieve an accurate diagnosis. As a result, alternative techniques based on direct detection of diagnostic compounds are gaining popularity [18].

#### **Figure 1.**

*An overview of fungal infection detection methods. This graph depicts the many ways for identifying fungal species. It's possible to utilize mass spectrometry (blue backdrop), nucleic acid (red background), or antibodybased approaches (orange background). Techniques that combine more than one of these characteristics are represented in the section borders.*

#### *Molecular Detection and Identification of* Candida *DOI: http://dx.doi.org/10.5772/intechopen.107899*

Proteomics-based methods and targeted DNA sequencing are two examples of molecular diagnostic approaches that might be used directly on clinical samples. The need for infectious agent culture, the possibility to utilize a direct clinical sample, sensitivity and accuracy, cost, time, and knowledge requirements, as well as the spectrum of species that may be identified, all differ between the current and future techniques. Some sophisticated approaches promise quick identification of both types of infectious agents as well as the emergence of treatment resistance. The existence of infected cells does not necessarily correspond to DNA detection, which is a common flaw in DNA-based approaches [19].

As a result, several modern approaches concentrate on identifying RNA from actively transcribed genes, which is a better proxy for active cells and can also provide indications that differentiate invasive from commensal activity [20].

The field of yeast infection diagnosis has substantially advanced in the last decade, and is presently experiencing a revolution, thanks to the advent of sophisticated sequencing and proteomics methods. However, there is still a long way to go between the novel diagnostic method's effective proof of concept and its acceptability for broad clinical application. Diagnostic tools should be low-cost, quick, sensitive, accurate, and simple to use [20].

Currently, there are several molecular diagnostic approaches for yeasts on the market. They do, however, concentrate on the most prevalent pathogenic yeast species, leaving the rare and emerging pathogenic yeast species to be found later. Massive outbreaks of drug-resistant Corynebacterium auris isolates in hospital settings have underlined this fact, which were first misread by existing commercial approaches [3, 4]. In this article, we give a comprehensive review of the existing approaches for characterizing yeast infection and treatment resistance profiles. During the review, we underline the advantages and disadvantages of each approach, as well as the prospective new advances brought about by modern technology. The primary accessible techniques and strategies are shown in **Figure 1**.

#### **2. Molecular identification of targeted DNA regions**

It was found that polymerase chain reaction (PCR) allows for the selective amplification of a specific segment of DNA, yielding millions of copies of the sequence (amplicon) in a matter of hours. This method has a lot of diagnostic potential since it allows for the detection of small quantities of target DNA using specific oligonucleotides. To make the diagnosis, the existence of the amplicon (if unique to the target species), its size, or its exact sequence, which may be determined by sequencing or hybridization to a particular probe, can be employed. The combination of particular PCR designs with post-analysis has resulted in several alternative PCR-based approaches that are increasingly being employed in the diagnosis of yeast infections (**Figure 2**).

Furthermore, specific patterns in the DNA of infectious microorganisms can be detected without the use of selective PCR amplification, for example, by direct hybridization with specific probes or by recognizing patterns in the length of fragments resulting from enzymatic digestion of DNA by exonucleases. These strategies will also be discussed in this section [21].

#### **2.1 End-point PCR-based amplification**

A typical approach for detecting and identifying infectious agents in cultures or clinical samples is the endpoint [22]. Primers that preferentially amplify the target

#### **Figure 2.**

*PCR-based techniques for fungal diagnoses are depicted in this diagram. In diagnostics, there are two types of PCR-based procedures: (A) traditional PCR-based methods and (B) real-time PCR-based methods.*

locus have historically been used to detect and identify infections. The locus might be species-specific, producing an amplicon only if the target species is present, or it could have a broader range, producing an amplicon from several species. In the latter situation, differences in length, melting temperature, or sequence between the amplicons may allow for a more precise identification. A target location for a conserved rDNA gene found in multiple copies has been frequently utilized [23].

The existence of numerous copies, which allows amplification of even a small number of cells, and the intrinsically high degree of variation found in some locations, which allows the construction of species-specific tests, are two aspects that make this site excellent for diagnosis. The internal transcription spacer (ITS) of the rDNA locus has been acknowledged as the worldwide gold standard for fungal species identification [24, 25], and various global primers amplify this region. Other parts of the rDNA locus, such as Trichosporum's Intergenic Spacer region 1 (IGS1) may be more useful for identifying certain clusters or species [26].

Additional markers, such as beta-tubulin or translation elongation factor genes, can also be utilized in other fungal species [27]. It is now simpler, quicker, and more specific to find particular or diagnostic areas because to advances in bioinformatics and the availability of whole-genome sequencing data [28, 29].

In normal laboratories, PCR primers for common fungal species belonging to the major human pathogenic genera, such as *Candida, Aspergillus, Cryptococcus* and *Molecular Detection and Identification of* Candida *DOI: http://dx.doi.org/10.5772/intechopen.107899*

*Pneumocystis,* are used more frequently than broad-spectrum primers. The lack of species-specific commercial testing for less common species within those genera, as well as for other new fungal genera that often cause severe and rapidly progressing infections [30], is a drawback. Fungal or broad-spectrum PCR primers have the advantage of being able to recognize both common and unusual fungi. However, due to the sensitivity of the test, even a non-pathogenic fungus, symbiont fungi, or mycorrhizae may provide a positive result, the results should be evaluated by experts [31].

The YEAST panel is a newly constructed multiplexer panel that can identify 21 clinically significant yeast species from the genera *Candida, Trichosporon, Rhodotorula, Cryptococcus,* and *Geotrichum*, which account for 95% of yeast infections [32]. In many circumstances, amplicon sequencing is necessary to make a particular diagnosis. PCR's potential goes beyond species identification to the detection of more subtle genetic variations, such as those that contribute to a particular resistance profile.

Due to sensitivity limits and a lack of specialized techniques and commercial assays for many rare and developing fungal diseases, endpoint PCR is frequently not included in normal investigations to detect fungal pathogens on clinical samples [33]. However, in order to employ this excellent methodology for direct diagnosis utilizing patient samples, additional strategies for increasing sensitivity are being developed. Given the small number of infectious cells present in the test samples, the high sensitivity and specificity that PCR may theoretically give is an attractive prospect. There are various additional restrictions that may render PCR inefficient when DNA templates are acquired from clinical samples [34].

Amplification of DNA-extracted blood samples is hampered by the presence of hemoglobin and anticoagulants [35, 36]. Some DNA extraction businesses address this issue by incorporating treatment stages to eliminate potential inhibitors, which can be a problem with other methods. Modified PCR methods are being developed to overcome concerns such as low specificity. Using two overlapping primer pairs in nested PCR, for example, can enhance both specificity and sensitivity [37].

#### **2.2 Analysis of fragment length polymorphisms**

The fact that sequence differences can be identified after digestion with a sequence-specific restriction endonuclease is exploited by restriction fragment length polymorphism (RFLP). After amplification of the appropriate DNA fragments, this method is frequently used in combination with polymerase chain reaction. *Candida palmiolate, fermented Candida, Candida albicans, Candida duplexensis, Candida refractory,* and *C. albicans* were effectively identified using PCR-RFLP [38–40]. RFLP analysis requires large data sets, which limits its application in the clinic. Amplification fragment length polymorphism (AFLP), a related technique, reverses the order of polymerase chain reaction (PCR) and restriction cleavage [41].

This technique was utilized to analyze interspecific variability and identify various fungi in clinical isolates, such as Cryptococcus neoformans/gattii complex species and *Candida* species [42]. Despite the fact that AFLP takes longer and costs more than RFLP, it has been proven to be reliable, fast, and highly repeatable under controlled settings [43].

#### **2.3 Real-time PCR**

Quantitative PCR (qPCR), originally known as real-time PCR, quantifies the quantity of PCR product using fluorescent probes or interfacial dyes [44]. Dyes (for example, SYBR Green) are less costly than probes, but they have the drawback of attaching to dsDNA in non-specific ways, such as primer dimers and non-targeting DNA [45].

Primer-probe hairpins (e.g., Scorpion probes), hybridization probes (e.g., Molecular Beacons), hydrolysis probes (e.g., TaqMan), unnatural bases (PlexorTM primer), and synthetic-based probes are all now available. Peptide nucleic acids (PNAs) and locked nucleic acids (LNAs) (Faltin, Zengerle, and von Stet hydrolysis and hybridization probes) are being employed frequently in clinical diagnostics [46].

There are numerous categories for identifying main *candida* species. With the support of criteria such as the minimal information required to publish qPCR experiments, these approaches have been standardized [47]. The key benefit of qPCR over traditional PCR is that it can identify the payload of infectious diseases, although at a higher cost. Although in the clinic, simple positive or negative testing for the presence of the pathogen is frequently required, knowledge of pregnancy can be useful in monitoring the effect of treatment or identifying infection in a non-sterile human environment where overgrowth rather than simple presence is required [48].

Another clinical use of qPCR is to track the level of azole resistance in *Candida* species. Because significant levels of transcription are required when the predominant route of resistance is up-regulation of the gene encoding drug target or drug efflux pumps [48–50], these genes are linked to azole resistance.

#### **2.4 MCA**

MCA uses the temperature-dependent dissociation kinetics of dsDNA to discriminate PCR amplicons. The temperature at which half of a dsDNA molecule splits into single DNA is known as the melting point (Tm). Because the G-C base pairs produce three hydrogen bonds vs. two in the A-T base pairs, the Tm is sequence dependent, needing more energy to solve the first. As a result, a higher Tm level corresponds with a higher G/C concentration. Using split fluorescent dyes that glow only when bound to dsDNA, the dissociation process may be monitored as a reduction in fluorescence during progressive heating [51].

HRMA (High Resolution Melt Analysis) is a modernized version of classic MCA [52]. HRMA employs more advanced algorithms and fluorescence sensors, as well as brighter pigments in higher concentrations.

HRMA can detect and monitor minor fluorescence variations induced by changes in Tm below 0.5°C, allowing one base pair precision detection of sequence discrepancies. A Tm change of 41/length of sequence C occurs when a single G-C is substituted with an A-T [53].

As a result, amplicon length is an important consideration when organizing HRMA studies. Short fragments (50–300 bp) give a single, well-defined fusion region and simple profiles, but bigger fragments may represent several peaks and reduce discriminatory power [54].

Furthermore, selecting a suitable fluorescent dye is critical. Unsaturated colors (such as SYBR Green) hinder polymerization at maximum brightness dosages. Saturated dyes from the most recent generation (such as SYTO9 and ResoLight) do not have this inhibitory effect and can thus be utilized when saturated. Unsaturated dyes, on the other hand, can re-link to free sites during dissolution, resulting in more fuzzy forms [55].

Decath et al. (2013) effectively differentiated cultivated strains of 16 *Candida* species, including pathogenic primary *Candida* species, in 6 hours using MCA in the ITS2 region [56]. MCA is also utilized in the commercial multiplexed qPCR kit kiAsperGenius R. [56].

*Molecular Detection and Identification of* Candida *DOI: http://dx.doi.org/10.5772/intechopen.107899*

This group not only finds and distinguishes Aspergillus fumigatus, Aspergillus terreus, and Aspergillus spp., but also gives information on Ammophilus fumigatus resistance by detecting resistance-related mutations in the cyp51a gene [57].

Different approaches such as differential media culture (*Candida* ID, CHROMagar), MALDI-TOF mass spectrometry, and DNA sequencing have been compared to HRMA [58]. Because MCA and HRMA employ G/C content to differentiate two unique DNA fragments, they are limited in their ability to detect all amplicon sequence changes. The species pairings of *Candida orthopsilosis* and *Candida metapsilosis* [59], and *Candida fabianii* and *Meyerozyma guilliermondii* are indistinguishable due to similar G/C structure and Tm overlap [60].

The HRMA approach is inexpensive, employs generic tools, takes a short amount of time to perform, is straightforward, and uses a closed tube format, which eliminates the danger of PCR contamination [61]. As a consequence, HRMA offers a quick and low-cost method for measuring and identifying the most common clinical forms of *Candida*, as well as detecting co-infections with these species, straight from clinical samples [62].

#### **2.5 Detection of SNPs**

Detecting alterations at the single nucleotide level can be extremely important in the clinic, especially if the mutation is linked to medication resistance. Polymorphisms can be detected with a high degree of specificity using PCR-based methods. In these strategies, the following tactics are typically used: MCA is collected using real-time PCR with hydrolysis probes, hybridization probes, or fluorescent dye coupled to dsDNA; (ii) PCR (ASP) selectively amplifies target alleles using Allelespecific Taq DNA polymerase and 3-end allele-specific primers [63].

ASP can identify single core alterations, as well as modest insertions and deletions. The amplification thermal mutagenesis system (ARMS) and PCR amplification are two techniques that are comparable [64]. The combination of ASP with quantitative PCR (AS-qRT-PCR) and droplet PCR (AS-droplet-PCR) may improve genotyping and quantification of chimerism in recipients as compared to a standard short tandem polymerase chain reaction [65]. Hybridization using SNP-specific probes is another possibility. DNA array devices that combine parallel hybridization with many probes may provide a quick and simple testing platform. All of these methods, however, have the limitation of requiring extensive knowledge of the most critical SNPs [66].

All of these methods, including group systems, were utilized to find resistance mutations in a variety of fungal infections. To differentiate C. albicans isolates with and without hotspot mutations in ERG11, which provide azole resistance, MCA was utilized. PCR-based technologies are used in a variety of ways [67].

It was tweaked to detect SNPs in clinical samples. For Resistance mutations in the FKS1 and FKS2 genes in *C. glabrata*, and in FKS1 in *C. albicans* [66] have been developed PCR tests to detect echinocandin. Mutations in FKS1 and FKS2 in *C. glabrata* were also studied utilizing MCA and Luminex technology [65]. Finally, there are a variety of SNP detection technologies that may be utilized to uncover variants that cause resistance [67].

#### **3. Conclusions**

To summarize, molecular approaches for quantifying resistance in clinical samples take a significant amount of effort. Only a few commercially accessible diagnostic procedures include clinical testing. However, with resistance rates on the rise, clinical

specimen resistance screening is becoming more important. Furthermore, molecular approaches may only confirm the existence of known resistance mutations; they cannot rule out resistance based on unreported mutations or other biological processes like biofilm formation. As a result, traditional susceptibility testing will continue to be an important method for detecting resistance variations.

A trustworthy, speedy, and user-friendly application approach for correctly identifying *Candida* species, particularly in clinical specimens, is real-time polymerase chain reaction. It has a high sensitivity and specificity and can identify fungal DNA in blood, different bodily fluids, and biopsy samples within six hours. Because antifungal susceptibility patterns vary between different species, accurate identification of *Candida* morphologies is crucial. Correct identification facilitates the choice of antifungal medications for both prevention and therapy. More clinical studies are required to determine the full potential of these novel treatments for various patient populations. Future research must assess the potential advantages of early therapy for individuals at risk for invasive *Candida* infection based on real-time polymerase chain reaction.

### **Conflict of interest**

There is no conflict of interest.

#### **Author details**

Muataz Mohammed Al-Taee Department of Medical Laboratory Technology, AL-Nisour University College, Baghdad, Iraq

Address all correspondence to: muataz.m.path@nuc.edu.iq

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Molecular Detection and Identification of* Candida *DOI: http://dx.doi.org/10.5772/intechopen.107899*

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*Molecular Detection and Identification of* Candida *DOI: http://dx.doi.org/10.5772/intechopen.107899*

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#### **Chapter 4**

## Laboratory Diagnosis of Candidiasis

*Benson Musinguzi, Obondo J. Sande, Gerald Mboowa, Andrew Baguma, Herbert Itabangi and Beatrice Achan*

#### **Abstract**

The burden of Candidiasis continues to increase and so does the *Candida* species. Although *Candida* species are closely similar phenotypically, they differ from each other in terms of epidemiology, genetic characteristics, antifungal susceptibility and virulence profile. Therefore, reliable and accurate laboratory methods for identification of *Candida* species can determine the Candidiasis burden and enable the administration of the most appropriate antifungal drug therapy to reduce fungal mortality rates. Conventional and biochemical methods are often used in identification of *Candida* species. However, these techniques are specific and sensitive enough in detecting the non albicans candida (NAC) species. Molecular techniques have improved the laboratory diagnosis and management of Candidiasis due to improved sensitivity and specificity threshold. This chapter provides an overview of different laboratory methods for diagnosis of Candidiasis.

**Keywords:** *Candida*, identification, candidiasis, laboratory, diagnosis, non-C. *albicans*

#### **1. Introduction**

There is a global raise in the burden of Candidiasis among immunocompromised individuals and this has to an increase in *Candida* species [1]. These species include both *C. albicans* and non *C. albicans* (NAC); *C. glabrata, C. tropicalis, Candida krusei, C. dubliniensis, Candida parapsilosis, Candida guilliermondii, Candida famata, C. kefyr, Candida norvegensis, Candida sake, Candida lusitaniae, C. pintolopesii, C. pseudotropicalis, C. globosa, C. dattila, C. inconspicua, Cobitis hellenica, Calamagrostis holmii, C. pulcherrima, C. valida, Candida fabianii, C. cacaoi, Candida zeylanoides* among [2, 3].

The phenotypic appearance of *Candida* species are relatively similar, however, different species differ from each other in terms of antifungal sensitivity, epidemiological distribution, genetic makeup and virulence attributes [4]. The diagnosis of Candidiasis is often clinical and empirical management is no longer adequate. This is partly due to misdiagnosis and varied antifungal susceptibility profile of the different *Candida* species [5]. This has worsened with the ever-increasing taxonomical shift in the etiology of Candidiasis towards resistant non albicans candida (NAC) [6]. This is partly caused by laboratory diagnosis which is frequently based on the conventional phenotypic and biochemical methods that are often not specific and sensitive in detecting NAC species [7]. However, diagnostic approaches have improved over the

years with the invention of advanced molecular techniques [8]. This chapter provides an overview of the laboratory methods for diagnosis of Candidiasis.

#### **2. Laboratory diagnosis of candidiasis**

The laboratory diagnosis of the Candidiasis involves the use of both Conventional (phenotypic) and molecular (genotypic) methods to detect visible and genetic characteristics of Candida respectively.

#### **2.1 Conventional methods**

Conventional methods are still commonly used for diagnosis and identification of fungi. These techniques are based on microscopic examination and fungal culture. Oral swab is collected, followed by microscopy and culture on selective media [4]. Microscopy can be done directly either from fresh samples or from fungal cultures. However, microscopy is non-specific, as different species can show the same morphological patterns and it is not possible to identify the *Candid*a species causing the Candidiasis [9]. Swab culture is normally the first test that is commonly done for identification of *Candida* species causing Candidiasis. However, It takes 1 to 3 days to have results [4]. Once positive cultures are available, other methods can be used to identify species of *Candida*. For instance, CHROM agar is a selective and differential medium for the identification of *Candida* species and can be used to identify *C. albicans, C. parapsilisis*, *C. dubliniensis*, *C. tropicalis* and *C. krusei.* It is widely used in mycology and it is found to be an effective primary identification test, where each species gives different colors of the colony forming units when species-specific enzymes split the chromogenic substrates [10]. Discrepancies may occur due to variations in the enzymatic reactions within the same *Candia* species [11]. *C. albicans* can be presumptively identified using the germ tube test; *C. albicans* shows a distinctive, tube-like structure when incubated in serum for 2–4 hours at 37°C. However, a possible limitation about the germ tube test is that some other *Candida* species such as *C. dubliniensis* also show a positive test result [12]. However, an easy and rapid commercialized latex agglutination test, Bichro-Dubli Fumouze® (Fumouze Diagnostics, France) has been evaluated to differentiate *C. albicans* from *C. dubliniensis* by detecting specific antigens located on the surface of *C. dubliniensis* blastoconidia [13]. In addition, automated biochemical and assimilation tests such as API and VITEK (BioMerieux Vitek, Inc., Hazelwood, USA) have been developed for *Candida* species identification. The API 20C system (Analytab Products, Plainview, USA) was one of the first available commercial kits used for the identification of yeast [12]. The ID 32C system (bioMérieux, France) has 12 substrates more than API which can enable identification of a diverse set of clinically important yeasts and can also differentiate between *C. albicans* and *C. dubliniensis* [14]. The Vitek 2 system is able to identify and detect *Candida* species and their antifungal susceptibility profile [15]. The main concern of these tests is that they require isolated fungal colonies and an incubation time of 2 to 3 days and misidentification of *Corynebacterium auris* may occur [16].

Indirect nonculture-based methods are available such as *C. albicans* germ tube antibody (CAGTA), circulating (1,3)-ß-D-glucan (BDG) antigen detection, mannan and anti-mannan antibody tests [17]. Much as, BDG Fungitell assay (Associates of Cape Cod, Inc) has been approved by the Food and Drug Administration (FDA) for the diagnosis of candidiasis, it associated with high false-positive, low sensitivity and


*a Peptide nucleic acid fluorescent in situ hybridization.*

*b Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.*

#### **Table 1.**

*Sensitivity and specificity of some methods used in diagnosis of invasive candidiasis as compared to conventional methods.*

specificity results as shown in the **Table 1** above. This has limited its use for screening purposes [16, 22].

Enzyme-linked immunosorbent assay (ELISA) kits can be used to identify both mannan and anti-mannan antibodies however, this test is not recommended for identification of *Candida* species due poor specificity and sensitivity [23]. *C. albicans* germ tube antibody is an indirect immunofluorescence assay that detects antibodies against *C. albicans* germ tube and commercial kits for CAGTA assay include VirClia IgG Monotest and Vircell kit (Vircell, Spain). However, FDA has not yet approved CAGTA for use in clinical settings [24].

#### **2.2 Molecular method**

Molecular methods are more accurate and rapid in detecting *Candida* species*.* They have higher sensitivity and specificity as shown in **Table 1** above. Most molecular methods have the power to rapidly detect both primary and secondary antifungal resistance alleles, which may necessitate these methods to progressively replace conventional techniques which have reduced sensitivity and specificity as shown in **Table 1** above [15]. The D1/D2 region located in the larger ribosomal deoxyribonucleic (DNA) subunit and intervening transcribed spacers (ITS) 1 and 2 located between 18S, 5.8S, and 28S ribosomal ribonucleic acid (RNA) genes as shown in **Figure 1** below are useful markers for *Candida* species identification and phylogenetic studies [11, 15].

**Figure 1.**

*Adopted from Chen et al., 2000, showing the non-coding internal transcribed spacer between the coding regions of 18S, 5.8S and 28S ribosomal RNA [25].*

These regions contain sufficient sequence heterogeneity to provide differences at the species level [26].

Molecular techniques are categorized into two methods, i.e., polymerase chain reaction-based methods and non-polymerase chain reaction (PCR) based methods.

#### *2.2.1 Polymerase chain reaction-based techniques*

Polymerase chain reaction (PCR) is one of the most important molecular techniques used to detect *Candida* species, as it is fast and easy use [15].

#### *2.2.1.1 Polymerase chain reaction (PCR)*

PCR is based on the amplification of a small specific DNA target through multiple repeated cycles of temperature changes into multiple copies. The main PCR steps are denaturation of the template DNA into single strands (94–98°C), annealing of the primers to the target sequence (50–65°C), and elongation whereby DNA polymerase elongates a DNA complementary to each strand of the target (72°C) [27].

Various PCR techniques have been developed, such as real-time PCR, Restriction Fragment Length Polymorphism (RFLP) PCR technique, multiplex PCR, reverse transcriptase PCR and nested PCR [27, 28].

Real-time PCR can be used to quantify the PCR product during amplification. Moreover, it has advantages over the conventional PCR in that it does not require agarose gel electrophoresis to visualize the amplified products. In real-time PCR, the amplified product can be measured automatically after each cycle by a fluorometer [29].

Nested PCR is based on the amplification of DNA by using two sets of primers to improve its specificity and sensitivity. *Candida* DNA topoisomerase II genes have been used to adjust Nested PCR for identification of specific *Candida* species [30]. Reverse transcriptase PCR (RT-PCR) is based on the reverse transcription of ribonucleic acid (RNA) into complementary DNA (cDNA) using a reverse transcriptase enzyme. The cDNA can then be amplified by regular PCR [31].

Restriction Fragment Length Polymorphism (RFLP) PCR is an important technique to detect and identify strains of *Candida* species using portions of ribosomal DNA, such as the intervening transcribed spacers (ITS) region that are located in between the small and large ribosomal subunits, and the D1/D2 region of the large (26S) ribosomal subunit [12, 22, 32].

Multiplex PCR requires multiple different primers and specific probes labeled with different fluorophores in a single PCR tube to allow the identification of many different *Candida* species from the same sample. For instance, amplification of two DNA fragments from the ITS1 and ITS2 regions in combination with specific primers in a single PCR reaction is very accurate in *Candida* species speciation [33]. It is worth noting that multiplex PCR has the following advantages, has a high specificity and sensitivity of approximately 2 cells per, is rapid and easy to use, whole yeast cells may be employed directly in the PCR mixture, has the potential to discriminate specific *Candida* species in polyfungal infections to a maximum ratio of 1:10, and has a good reproducibility among different PCR thermal cyclers and within different laboratories [34]. In addition, commercial Multiplex qPCR kits for *Candida* detection including *Cand*ID/Plus (OLM diagnostics, UK) and Fungiplex *Candida* IVD (Bruker, Germany) are now available [17, 33].

#### *2.2.1.2 Sequencing*

Sanger sequencing is a first-generation sequencing technique developed by Sanger Frederick and it is based on chain-termination (Sanger *et al.*, 1977). Sanger sequencing has been used extensively for identification of many fungal pathogens [35]. The most commonly conserved regions in fungi are the ribosomal RNA genes including 5.8S, 18S and 28S and in between these are the ITS1 and ITS2 regions, non-coding regions, which vary in different species and sequencing of these regions supports rapid identification of different *Candida* species [15, 22]. Limitations of Sanger sequencing include high cost for whole genome sequencing and reduced accuracy when using only one copy for each strand [35].

Next generation sequencing (NGS) is accurate and rapid high throughput sequencing technique and is very vital in genome sequencing, fungal research, diagnostic purposes, outbreak monitoring [36]. Most of NGS platforms including the Ion Torrent PGM (Life Technologies), HiSeq, MiSeq and NextSeq (Illumina), 454 GS (Roche) and SOLiD System (Applied Biosystems) are based on sequencing by synthesis and have three main steps: template preparation, sequencing and imaging and data analysis [37, 38]. In addition to *Candida* species identification, NGS can be used for detecting genetic mutations associated with antifungal resistance [15]. As compared to Sanger sequencing, NGS is accurate and faster as massive DNA strands can be sequenced in parallel on a single run and a lesser amount of DNA is required. However, NGS reagents are expensive and the software requires technical expertise [38]. Nanopore sequencing is the fourth-generation DNA sequencing technology which is fairly cheap and uses short sequencing time with long sequencing reads [39]. Nanopore platforms like GridION™, PromethION™ and MinION™ are the latest portable and affordable NGS technologies with high genotyping accuracy [40].

Pyrosequencing is another PCR based technique which depends on the release of pyrophosphate when nucleotides are incorporated into the nucleic acid chain by DNA polymerase and produced pyrophosphate is then subsequently converted to Adenosine-5'triphosphate (ATP) by ATP sulfurylase, and that provides energy for luciferin oxidation by luciferase, which produces light that can be detected as a peak on the pyrogram [37]. Any unincorporated nucleotides are degraded by apyrase to allow iterative nucleotide addition into the nucleic acid chain and peak heights are associated with the number of the same nucleotides added to the nascent strand [41].

Pyrosequencing is a rapid and accurate molecular method for the detection of point mutations in any selected gene within short DNA fragments. It has been used widely for the identification and detection of antifungal drug resistance [42].

#### *2.2.2 Non-polymerase chain reaction-based methods*

These methods can facilitate rapid identification of *Candida* directly from candida culture broth without the need for DNA amplification. Non-PCR methods include peptide nucleic acid fluorescent *in situ* hybridisation (PNA-FISH) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). These methods have sensitivity and sensitivity of up 100% in detecting *Candida* species causing invasive diagnosis as shown in **Table 1** above. PNA-FISH is based on the rapid hybridization between synthetic oligonucleotide fluorescence-labeled probes and species-specific ribosomal RNA that can be detected via fluorescent microscopy [28]. PNA-FISH has been used to effectively identify *Candida* species with high

sensitivity and specificity directly from positive cultures, with final identification provided within 2.5 hours [12]. However, this test is very expensive and needs technical expertise [11].

MALDI-TOF MS is a method that uses mass spectrometry to identify the protein fingerprints of microorganisms that are compared with databases of reference spectra [43]. MALDI-TOF MS is able to accurately detect and identify *Candida* species in a timely manner with up 100% sensitivity and specificity as shown in **Table 1** above. However, high set up cost is the main limitation of this test include the high setup [44].

#### *2.2.3 The internal transcribed spacer marker for Candida species identification and phylogenetics*

The ITS region of ribosomal DNA (rDNA) is the most useful genetic marker for rapid and accurate molecular identification of *Candida* species and phylogenetic studies due to its region sequence variability among different species [15, 45–47]. The ITS 1 and ITS 2 are two vital non-coding regions composed of conservative and variable subregions outside and inside respectively [45]. The ITS1 fragment is positioned between the 18S and 5.8S ribosomal RNA genes while ITS2 fragment is positioned between 5.8S and 28S ribosomal RNA genes [48]. Furthermore, the amplicon sizes differ according to the target ITS1 region based on specific *Candida* species of interest [33, 49]. It is worth noting that ITS primer design, PCR amplification and sequencing has been made easy due to availability of several conserved sequences, frequent copies of the ribosomal operon and moderately limited length of ITS region [48].

#### **3. Conclusions**

Emergence of non albicans *Candida* species causing Candidiasis has highlighted importance of accurate *Candida* species identification. Laboratory diagnosis of Candidiasis is often based on conventional and biochemical identification of *Candida* species. However, these methods are labor intensive, time consuming and often do not permit sufficient specificity and sensitivity. Furthermore, conventional based identification of *Candida* species is affected by the variable nature of phenotypic characteristics. Molecular based methods are more proficient, rapid and easier diagnostic technologies for Candidiasis due to their increased sensitivity, specificity and accurate early detection of different *Candida* species. Early diagnosis allows clinicians to combat Candidiasis at an early stage through choice-specific and effective antifungal therapy, avoiding empirical management and development of resistance to antifungal drugs. From this review, it is expected that progress in use of molecular approaches will continue to have a positive impact on exploration of molecular epidemiology of *Candida* species and subsequently improve diagnosis and management of candidiasis.

#### **Author details**

Benson Musinguzi1,2\*, Obondo J. Sande1 , Gerald Mboowa1 , Andrew Baguma<sup>3</sup> , Herbert Itabangi4 and Beatrice Achan<sup>5</sup>

1 Department of Immunology and Molecular Biology, School of Biomedical Sciences, Makerere University, Uganda

2 Faculty of Health Sciences, Department of Medical Laboratory Science, Muni University, Arua, Uganda

3 Department of Microbiology, School of Medicine, Kabale University, Kabale, Uganda

4 Faculty of Health Sciences, Department of Microbiology and Immunology, Busitema University, Mbale, Uganda

5 Department of Medical Microbiology, School of Biomedical Sciences, Makerere University, Uganda

\*Address all correspondence to: b.musinguzi@muni.ac.ug

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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## Section 3
