Molecular Typing and Susceptibility Profile of Cryptococcus neoformans and Cryptococcus gattii species Complex: An updated Review

| Cryptococcosis is a sub-acute or chronic fungal infection of human and animals that is induced by opportunistic capsulated basidiomycetous yeasts of genus Cryptococcus , primarily C. neoformans and C. gattii with a worldwide distribution. The genetic variability of C. neoformans/C. gattii species complex has been investigated by several molecular techniques mainly hybridization and nested and multiplex PCR assays. Other PCR-based methods were employed for identification as PCR fingerprinting, PCR Restriction fragment length polymorphism (PCR-RFLP), Amplified fragment length polymorphism (AFLP), and multi-locus sequence typing (MLST). Moreover, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been recently introduced for Cryptococcus identification and subtyping. Cryptococcus neoformans/C. gattii species complex is recently divided into seven species: C. neoformans , C, deneoformans , C. gattii , C. deuterogattii , C. bacillisporus , C. tetragattii , and C. decagattii in addition to four interspecies hybrids with differences in pathogenicity, epidemiology, and antifungal susceptibility. Long-term usage of antifungal drugs led to the emergence of resistance in C. neoformans and C. gattii species. Thus, antifungal susceptibility is of great importance in the epidemiological investigation for tracking the susceptibility profiles and drug resistance. Moreover, efficient antifungal therapies selection for cryptococcosis treatment is based on minimum inhibitory concentration (MIC) values of the gold standard drugs for cryptococcosis therapy: amphotericin B, fluconazole, 5-flucytosine, voriconazole, ketoconazole, and itraconazole. This review article highlights the main molecular methods used for identification and genotyping of C. neoformans and C. gattii and presents the global prevalence and antifungal susceptibility profiles of these environmental isolates against the commonly used antifungals.

Cryptococcus neoformans and C. gattii have a different and overlapping ecological niche. C. neoformans is frequently isolated from birds dropping, especially in pigeon nests, while C. gattii, occurring principally in tropical or subtropical areas, is found frequently in soil debris and decaying trees like oaks, pink showers, and Eucalyptus trees (Alves et al., 2016). Almost all of the cryptococcal infections are relevant to the species C. neoformans var. grubii, particularly in immunocompromised patients and also apparently healthy hosts (Bandalizadeh et al., 2020). One million cryptococcal meningitis cases are reported annually in patients with human immunodeficiency virus infection and acquired immune deficiency syndrome with a mortality peak at approximately 625,000 deaths (Harris et al., 2011). Meanwhile, C. gattii causes a smaller fraction of cases affecting commonly immunocompetent individuals in temperate regions (Firacative et al., 2012).
Cryptococcus infection is also very important in a wide range of animals worldwide. It may affect various mammals like cats, dogs, horses, cattle, sheep, goats and birds, with two basic forms: pulmonary and cerebral cryptococcosis. The cutaneous, ocular, osseous, and visceral form may be found due to disseminated infection. Cryptococcosis is mostly associated with mastitis in cattle, sheep, and goats besides endometritis and placentitis in mares (Refai et al., 2017).
Concerning Cryptococcus complex epidemiological spreading, C. neoformans var. grubii (VNI, II, and VNB) is more predominant and distributed globally than C. neoformans var. neoformans (VNIV), which is observed in Europe (Cogliati, 2013). The VGI and VGII are the most widely distributed among C. gattii members and they are responsible mainly for the Vancouver outbreak, which is still ongoing in Canada and USA resulting in more than 350 human cryptococcosis cases, 3.5% of them were lethal even with powerful antifungal therapy. Meanwhile, VGIV is the least distributing genotype associated with cryptococcosis infection in central Africa (Byrnes and Marr, 2011;D'Souza et al., 2011). The results from molecular identification and genotyping of C. neoformans and C. gattii species complex can positively impact the monitoring of resistant strains and treating cryptococcosis (Sidrim et al., 2010). This updated review sheds a light on the main molecular techniques for accurate identification, and typing of cryptococcosis primary agents from different environmental sources. Moreover, the susceptibility profiles of environmental isolates of various genotypes were also presented.

environMenTal prevalence
Cryptococcus neoformans and C. gattii are saprophytes and their infections occur by the exogenous route via inhalation of infectious dispersed yeast cells. Hence, awareness by C. neoformans and C. gattii environmental prevalence (Table  1) is critically important for laying out the possible control measurements against cryptococcosis (Chowdhary et al., 2011).

idenTificaTion of C. neoformans/C. gattii species coMplex
The microbiology laboratory must differentiate C. gattii and C. neoformans as they differ in the susceptibility patterns to antifungal drugs. Only 5% of New York State surveyed laboratories had the ability of proper identification and differentiation of C. gattii and C. neoformans; while in developing countries, this issue is more unsatisfactory (Chowdhary et al., 2011). Cryptococcus neoformans/C. gattii species complex has been classically identified by phenotypic methods based on the species characteristics. Various molecular methods have been further applied for confirmation of the identification and also for genotyping purposes. The MALDI-TOF MS technique has been successfully used for identification and discrimination of this species complex (Perfect and Bicanic, 2015;Hagen et al., 2015).

phenoTYpic MeThods
Phenotypically, C. neoformans and C. gattii were identified by different tests such as polysaccharide capsule production, which is detected with India ink staining, urease and phenoloxidase tests, and growth at 37 o C. Cryptococcal species are maintained in the culture of solid media as Sabouraud dextrose agar medium with or without the addition of antibiotics after isolation. The opaque, and creamy to tan or brown colored colonies are fast-growing and observed after up to 3 days of incubation at 25°C and 37°C. Carotenoid pigments may be produced after a long incubation period. The colony slimy appearance is related to the capsule size (Doering, 2009). For C. gattii differentiation from C. neoformans, many phenotypic methods have been used including Canavanine Glycine Bromothymol blue agar (CGB), which may give falsepositive results, Glycine Cycloheximide Phenol red agar, Creatinine-Dextrose Bromothymol blue agar, and Creatinine Dextrose Bromothymol blue thymine agar (Byrnes and Marr, 2011). Available commercial approaches
Limitations of conventional methods of identification and diagnosis led to many molecular methods development that were used for identification and genotyping of C. neoformans and C. gattii species complex (Cogliati, 2013).

Molecular MeThods for idenTificaTion and genoTYping of C. neoformans and C. gattii species coMplex
Molecular approaches are of great sensitivity and specificity, and could overcome the restrictions of traditional methods. It is employed for identification and typing of C. neoformans/C. gattii complex (Table 2) besides the molecular epidemiology research (Meyer, 2015).

dna-dna hYbridiZaTion MeThods and elecTro-karYoTYping
DNA-DNA hybridization techniques and electrokaryotyping were used in a wide range in 1990 for cryptococcosis research with a special reference to

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Journal of Animal Health and Production 2020 | Volume 9 | Special Issue 1 | Page 20 its main causative agents. They were more useful and accurate when combined with PCR in the researches that are concerned with the most rapid and economical progressions of alternative technologies in Cryptococcus species identification (Hu et al., 2008). These methods are costly and complex as they need previous electrophoresis reliability, denaturing buffer preparation, nitrocellulose or nylon membrane acquiring for DNA impregnation, uniquely marked probes, and suitable detection equipment (Sidrim et al., 2010).

pcr assaYs
These techniques are widely used in laboratories to detect Cryptococcus DNA and to genotype C. neoformans complex from clinical and also environmental samples as it is specific, fast, easily performed, and sensitive (Feng et al., 2013). Moreover, PCR assays are entirely automatic and able to discriminate yeasts from either clinical samples or contaminated cultures. PCR is used with more other techniques to be valuable for molecular epidemiology researches (Cogliati, 2013). The most commonly used PCR methods in identifying C. neoformans and C. gattii are nested, multiplex, and real-time targeting the orotidine monophosphate pyrophosphorylase (URA5) and the capsule synthesis (CAP59) genes, minisatellite-specific core sequence (M13), and ITS regions of rDNA (18S, 5.8S, and 28S) target sequences (Hagen et al., 2012) as following.

nesTed pcr
Nested PCR is a greatly sensitive, rapid, and reliable approach for identifying Cryptococcus species and diagnosing cryptococcosis, in which the DNA template is a product of the first-round PCR. It is also a useful technique used for patients monitoring throughout the therapy and for confirmation of the fungal pathogen clearance in the follow-up examinations (Rivera et al., 2015). In 2002, C. neoformans was directly detected from laboratory animal tissue samples by 18S region of rDNA amplification (Bialek, 2005). Hyper-branched rolling circle amplification (HRCA) is a semi-nested PCR, which is based on PLB1, the padlock probes locus. This practice was highly sensitive and more specific with the ability of distinct nucleotide polymorphisms identification and it is used in the direct cryptococcosis diagnosis (Trilles et al., 2014).

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Journal of Animal Health and Production 2020 | Volume 9 | Special Issue 1 | Page 21

MulTiplex pcr
Multiplex PCR is another desirable approach that enables the amplification of more loci in just one reaction. This technique is rapid, done with less amount of DNA, and species-specific. Moreover, it has been used to check the fungal isolates mating-type profile and it was applied in conjunction with other methods; for instance, the realtime PCR assay (Ito-Kuwa et al., 2007). Leal and his coworkers established a protocol of multiplex PCR using species-specific primers for ITS region. The obtained findings indicated a specific and rapid differentiation between C. neoformans and C. gattii from different isolates (Leal et al., 2008).

real-TiMe pcr
Real-time PCR, a recent method applied for rapid and accurate C. neoformans and C. gattii identification, allows the evaluation of gene expression associated with the microorganism virulence. This technique achieves higher levels of sensitivity, but it still of high cost, where expensive materials and special equipment are needed (Feng et al., 2013).

pcr fingerprinTing
The major typing approach in C. neoformans comprehensive molecular epidemiological studies is PCR fingerprinting. It allowed the determination of molecular types in sporadic diseases and it was used in molecular epidemiology studies (Posteraro et al., 2012). This assay relies on DNA sequences amplification lined by single primers in PCR including primers for the minisatellite-specific core sequence of the wild-type phage M13 as well as microsatellitespecific single-primers (GACA) 4 (Meyer et al., 2009). PCR fingerprinting classified C. neoformans complex into eight main types depending on the polymorphic DNA sequences as following: C. neoformans var. grubii serotype A1 (VNI), C. neoformans var. grubii serotype A2 (VNII), C. neoformans serotype AD (VNIII), C. neoformans var. neoformans serotype D (VNIV), and C. gattii B and C serotypes (VGI, VGII, VGIII, and VGIV) (Perfect and Bicanic, 2015).

pcr-rflp
PCR-RFLP using the URA5 and PLB1 genes was previously used in C. neoformans complex molecular types confirmation (Brito-Santos et al., 2015). This assay was applied in molecular epidemiological surveys not only for evaluation of the possible relationships between clinical and environmental C. neoformans complex molecular types, but also for cryptococcosis diagnosis as it commonly targets the URA5 gene (Kwon-Chung et al., 2017). This practice is also suggested when it is desired to get more information about a particular strain. Moreover, other targets rather than URA5 were used for molecular typing and accurate serotypes differentiation such as the capsular gene, CAP59 (Feng et al., 2013). rapd assaY RAPD analysis had been used for investigation of the genetic variability of Cryptococcus spp. from different sources and also for determination of serotypes and molecular types. It is rapid, simple, and highly discriminatory requiring strict quality control measures, but it is recently replaced by more recent techniques for C. neoformans and C. gattii genotyping (Sidrim et al., 2010).

aflp assaY
AFLP is another useful technique in C. neoformans complex genotyping. It is reliable, more specific than RAPD due to using of longer primers in the PCR cycles that prevent the existence of conflict during the PCR reaction of the RAPD technique (Meyer, 2015). The AFLP technique stages are DNA cleavage using EcoRI restriction enzyme and rarely MseI, then ligation of particular adaptors to the ends of DNA fragments, PCR cycles using MseI-G and EcoRI-AC primers, and finally high-resolution gel electrophoresis. The large numbers of stages, reagents, and devices as well as DNA quality are considered limitations of this technique (Sidrim et al., 2010). The AFLP assay exposed C. neoformans and C. gattii strains to subdivision into 7 -9 genetically diverse monophyletic clades (Kwon-Chung et al., 2017). The AFLP assay has helped to clarify the Vancouver island outbreak causative agent due to its high discriminatory power besides the ability to give a particular distinctive profile to each strain; the outbreak agent was two AFLP6 subtypes (AFLP6A and AFLP6B) (Byrnes and Marr, 2011). Some studies in the Netherlands applied the AFLP typing technique to Cryptococcus strains and discovered novel hybrids among C. neoformans and C. gattii, AFLP9 (AFLP1×AFLP4) and AFLP8 (AFLP3×AFLP4) (Hagen et al., 2012).

MulTi-locus sequence TYping
Multi-locus sequence typing has significant importance for the global epidemiological characterization of Cryptococcus species complex genotypes around the world (Firacative et al., 2016). It exploits the unique characteristics of nucleotide sequences of multiple genes such as capsular, urease, and phospholipase encoding genes for allowing Cryptococcus spp typing (Beale et al., 2015). MLST analysis is fully automated after the target regions amplification and sequencing and it can be interconnected between laboratories (Meyer, 2015). A limitation of this technique is that it provides incomplete or inaccurate measures of the species relationships as a relatively small portion of sequence diversity is detected by MLST and also it yields weak recombination interpretations (Beale et al., 2015).

MulTi-locus MicrosaTelliTe TYping
Multi-locus microsatellite typing (MLMT) has been commonly used for typing of different fungi owing to its strong discriminatory power, which might be an effective and provable approach for wide-range epidemiology studies (Feng et al., 2013). The MLMT with MLST were applied for studying the genotypic diversity and genetic relationships between environmental and clinical isolates of C. neoformans in an Indian survey, where environmental isolates showed more genetic diversity than clinical ones (Prakash et al., 2020).

Whole genoMe sequencing
The whole fungal genome sequencing technique is an accurate and modern technique for typing of Cryptococcus strains (D' Souza et al., 2011). Genes` contents and structure characterization indicated variations in the Cryptococcus genes in comparison with other fungi as Cryptococcus genes are intron-rich with predicted highly alternative splicing and antisense transcription ( Janbon et al., 2014). The whole genome structure is designed to be significantly heterogeneous within Cryptococcus strains with few alterations of either species or molecular types (Farrer et al., 2015). Cryptococcus neoformans var. grubii and C. neoformans var. neoformans genomes showed extensive rearrangements ( Janbon et al., 2014).

TiMe of flighT Mass specTroMeTrY
The MALDI-TOF MS is a simple, fast, and accurate technique based on mass spectrometry of different microorganisms. It was used as an alternative for phenotypic and genotypic methods for C. neoformans and C. gattii differentiation (Firacative et al., 2012;Hagen et al., 2015). This technique allows the discrimination between species by evaluation of the determined spectrum of peptides and proteins of integral microbial cells either from biological samples or uncontaminated cultures within few minutes and it also separates C. neoformans and C. gattii into eight molecular patterns (Firacative et al., 2012).

anTifungal suscepTibiliTY TesTing
The best antifungal drug should target either a component vital for the viability of fungal cell or a virulence factor, but not the host to avoid cell toxicity. That drug should have a fungicidal effect when used alone or in combination with another. It also should be of good bioavailability and capable of reaching cryptococcal receptors within the host (May et al., 2016). The World Health Organization and the Infectious Diseases Society of America recommended the gold standard cryptococcosis therapy guidelines including three drugs: amphotericin B (AmB), flucytosine, and fluconazole (Maziarz and Perfect, 2016).
Amphotericin B deoxycholate, which was released in 1960, acts as fungicidal by binding to the ergosterol of fungal cell membrane and also by cell death induction as a result of oxidative damage (Gray et al., 2012). Using AmB prevailing formulations requires the intravenous route and its usage is difficult in oral administration because of low bioavailability (Kwon-Chung et al., 2014).
Flucytosine is converted inside the fungal cells principally by the cytosine deaminase enzyme to 5-fluorouracil (5-FU). The mammalian cells are lacking this enzyme (Loyse et al., 2013). Fluconazole has a good bioavailability, essentially in the initial stage therapy and it may be used during the maintenance stage. It acts as a fungistatic (rather than fungicidal) as it inhibits the ergosterol synthesis leading to accumulation of destructive steroidal substances in the cell membrane (Gray et al., 2012).
Using the standard antifungal susceptibility testing methods has facilitated the detection of antifungal resistance; for example, clinical breakpoints and epidemiologic cut off values for Candida and Aspergillus spp., the Clinical and

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Journal of Animal Health and Production 2020 | Volume 9 | Special Issue 1 | Page 23  Laboratory Standards Institute (CLSI) and Antifungal Susceptibility Testing of the European Committee on Antibiotic Susceptibility Testing (Pfaller, et al., 2011).
The sensitivity of C. neoformans and C. gattii to antifungal agents have been determined using broth micro-dilution and macro-dilution methods according to the CLSI guidelines (Moraes et al., 2003) in addition to E-test (Khan et al., 2007).
Antifungal susceptibility testing helps in preference of the efficient antifungal therapies for immunocompromised patients with disseminated mycosis (Andrade- Silva et al., 2013). Studying the antifungal susceptibility patterns of C. neoformans and C. gattii environmental isolates (Table 3) is limited (Pedroso et al., 2006). Several studies revealed relatively low minimum inhibitory concentrations (MICs) of typical antifungals to C. gattii in contrast to C. neoformans with no increase over time (Thompson et al., 2008). In the past two decades, the reports of fluconazole-resistant strains increased globally. Geographical data indicates that increased fluconazole resistance was reported in Asia, Africa, and Latin America; while in North America, Europe, and Spain, low resistance rates were reported (May et al., 2016).
Many published studies stated that C. gattii and C. neoformans origins and genotypes had a great impact on the susceptibility to antifungal agents (Chong et al., 2010). In contrast, other investigators observed that susceptibility to antifungal agents was not influenced by the environmental or clinical origins of Cryptococcus species (Moraes et al., 2003).
The high prevalence and severity of cryptococcal infections are being considered as significant public health issues as a result of the high expansion in immunocompromised patients. Accordingly, using antifungals, principally in long-term therapies led to the resistance of C. neoformans and C. gattii species (Yang et al., 2010).

CONCLUSIONS AND RECOMMENDATIONS
In conclusion, introducing molecular methods for accurate and rapid identification of C. neoformans species complex is warranted in the mycology laboratories for efficient monitoring of resistant strains and treating cryptococcosis. This updated review threw more light on the global prevalence, genetic diversity, ecology, and susceptibility profiles of the environmental isolates of C. neoformans species complex to investigate the severity of health hazards generated by Cryptococcus species and to design possible control measures against cryptococcosis.

AUTHOR'S CONTRIbUTION
MT and YHT contributed to the conception and design of the article and participated with SZI and RMA in interpreting the relevant literature. SZI and RMA drafted the manuscript. YHT and MT revised it critically for important intellectual content.