Review Article

 

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

 

Mohammed Taha1, Yasmine Hasanine Tartor1*, Sara Zaki Ibrahim2, Rana Mohamed Abd El-Aziz3

1Department of Microbiology, Faculty of Veterinary Medicine, Zagazig University, 44511-Zagazig, Sharkia, Egypt; 2Veterinarian, Faculty of Veterinary Medicine, Zagazig University; 3Cairo International Airport Veterinary Quarantine, General Organization for Veterinary Services, Ministry of Agriculture, Cairo, Egypt

 

Editor | Asghar Ali Kamboh, Sindh Agriculture University, Tandojam, Pakistan.

Received | November 02, 2020; Accepted | November 25, 2020; Published | December 27, 2020

*Correspondence | Yasmine Hasanine Tartor, Department of Microbiology, Faculty of Veterinary Medicine, Zagazig University 44511-Zagazig, Sharkia, Egypt; Email: [email protected]; [email protected]

Citation | Taha M, Tartor YH, Ibrahim SZ, Abd El-Aziz RM (2020). Molecular Typing and Susceptibility Profile of Cryptococcus neoformans and Cryptococcus gattii species Complex: An updated Review. J. Anim. Health Prod. 9(s1): 17-26.

DOI | http://dx.doi.org/10.17582/journal.jahp/2020/9.s1.17.26

ISSN | 2308-2801

Copyright © 2020 Taha et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

 

INTRODUCTION

 

Cryptococcus neoformans/C. gattii species complex comprises capsulated basidiomycetous yeasts, the primary causative agent of cryptococcosis, which is a worldwide distributing life-threatening fungal disease producing tissue infections, pneumonia, and commonly meningoencephalitis mainly in immunosuppressed patients (Kwon-Chung et al., 2014).

 

Biological, epidemiological, clinical, pathogenicity, and drug susceptibility differences were observed between the species complex. Biochemically, C. neoformans is divided into two varieties (C. neoformans var. grubii and C. neoformans var. neoformans), while the capsule polysaccharides difference subdivided it into three serotypes: A, D, and AD in addition to serotypes B and C for C. gattii (Silva and de Albuquerque Maranhão, 2015). Recently, after various molecular studies, the complex C. neoformans/C. gattii was rearranged into 7 haploid species: C. neoformans var. grubii, C. deneoformans var. neoformans, along with C. gattii, C. bacillisporus, C. deuterogattii, C. tetragattii, and C. decagattii as well as four interspecies hybrids (Hagen et al., 2015).

 

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 37oC. 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 false-positive 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

 

Table 1: Environmental prevalence of Cryptococcus neoformans and Cryptococcus gattii species complex globally.

 

Type of samples (No.)	Prevalence (%)		Country	Molecular type	Method of typing	Reference
	C. neoformans	C. gattii
Pigeon droppings (191)	4.7	-	Brazilian amazon	VNI	PCR-RFLP	Alves et al. (2016)
Trees hollows (pottery tree) (255)	-	0.4		VGII
Pigeon droppings (50)	10	-	Mexico	C. neoformans var grubii	PCR and ITS1,ITS2 sequencing analysis	Canónico-González et al. (2013)
Cocktail and love bird excreta (200)	7.5	-	Egypt	serotype A	Multiplex PCR, ITS sequencing	Elhariri et al. (2015)
Eucalyptus trees sample (311)	4.2	-	Egypt	C. neoformans var grubii serotype A	Multiplex PCR	Elhariri et al. (2016)
Olive Trees (Olea europaea) (388)	22.4	-	coastal line of Anatolia, Turkey	Serotype A	Multiplex PCR, ITS sequencing	Ergin et al. (2019)
Passerine and Psittacine excreta (141)	25.5	-	Brazil	VNI	Multiplex PCR, PCR fingerprinting	Lugarini et al. (2008)
Pigeon dropping (100)	32	-	Libya	VNI	Multiplex PCR, URA5 RFLP	Ellabib et al. (2016)
Eucalyptus trees (210)	1.4	-		VNI
Hollows of trees and soil around trees (272)	-	5.9	California, USA	VGI	MLST	Hurst et al. (2019)
Olive tree hollows (172)	12.2	1.1	Italy	VNI	Multiplex PCR, MLST	Trovato et al. (2019)
Castanopsis argyrophylla trees hollows (48)	-	2.08	Thailand	VGI/ AFLP4	AFLP genotyping and MLST	Khayhan et al. (2017)
Olea europea and Pinus sylvestris trees hollows (472)	0.8	-	Croatia	VNI/ VNIV	Multiplex PCR	Pllana-Hajdari et al. (2019)

PCR-RFLP: PCR-restriction fragment length polymorphism, ITS: internal transcribed spacer, MLST: multi-locus sequence typing, AFLP: amplified fragment length polymorphism.

 

were used for yeast identification; for instance, API 20 AUX (bioMeriex, Paris, France) and VITEK 2 system (bioMérieux, Inc., Hazelwood, MO), which couldn’t differentiate C. gattii from C. neoformans, while MALDI-TOF MS was successfully used for this differentiation (Mctaggart et al., 2011).

 

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).

 

Numerous molecular typing methodologies were already applied in the determination of subgroups of each species of C. neoformans/ C. gattii complex as multilocus enzyme electrophoresis (MLEE), random amplification of polymorphic DNA (RAPD), PCR fingerprinting (Cogliati, 2013), restriction fragment length polymorphism (RFLP) of phospholipase (PLB1), orotidine monophosphate pyrophosphorylase (URA5), and GEF1 genes (Brito-Santos et al., 2015), and sequencing of internal transcribed spacer regions (ITS1 and ITS2) or intergenic spacer region (IGS), amplified fragment length polymorphism (AFLP) (Meyer et al., 2009). Recently, multilocus sequence typing (MLST) (Beale et al., 2015), multilocus microsatellite typing (MLMT), genes sequence analysis, whole-genome analyses, whole-genome sequencing in addition to MALDI-TOF MS analysis (D’Souza et al., 2011; Hagen et al., 2015) were introduced for genotyping.

 

DNA–DNA hybridization methods and electro-karyotyping

DNA–DNA hybridization techniques and electro-karyotyping were used in a wide range in 1990 for cryptococcosis research with a special reference to 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).

 

Table 2: Molecular techniques used for identification and genotyping of Cryptococcus neoformans/C. gattii species complex.

 

Technique	Target	Advantage	Disadvantage	References
Identification
Hybridization	Repeatable and polymorphic DNA	High specificity and sensitivity	Costly and laborious	Hu et al., (2008)
Nested PCR	ITS ribosomal DNA	High sensitivity and specificity	Presence of reaction contaminants Presence of polymorphism	Rivera et al. (2015)
Multiplex PCR	Serotype specific	Amplification of two or more loci in just one reaction. Small amounts of DNA extracted	Reagent competition Non-specific products	Leal et al. (2008)
Real-time PCR	18S/28 ribosomal RNA	High sensitivity, specificity and speed Determining levels of gene expression, Fast	Contamination with genomic DNA Requires technical ability and support Expensive	Feng et al. (2013)
Genotyping:
PCR fingerprinting	Microsatellite (GACA)4	Previous knowledge of target sequences is not required Using of short primers Detection of polymorphism	Standardization of the technique under the conditions of each laboratory	Meyer et al. (2009)
RAPD	Minisatellite (M13)	Previous knowledge of target sequences is not required Using of short primers Detection of polymorphism	Standardization of the technique under the conditions of each laboratory	Sidrim et al. (2010)
PCR-RFLP	Urease	Specificity	Decreased sensitivity in case of mutation	Cogliati (2013)
AFLP	Capsule	High sensitivity and specificity Detection of genetic variability.	Large number of phases and reagents. Expensive	Meyer et al. (2009)
MLST	IGS, capsule, laccase, urease, phospholipase	Reproducible and accurate. Completely automated analysis. Analysis of multiple loci	Restrictions in strains differentiation when genes are conserved	Beale et al. (2015)

ITS: internal transcribed spacer, RAPD: random amplification of polymorphic DNA, PCR-RFLP: PCR-estriction fragment length polymorphism, AFLP: amplified fragment length polymorphism, MLST: multi-locus sequence typing, IGS: intergenic spacer region.

 

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 real-time PCR assay (Ito-Kuwa et al., 2007). Leal and his co-workers 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 microsatellite-specific 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).

 

In 2007, International Society of Human and Animal Mycoses developed and sponsored a workgroup that declared MLST as a standard method for C. neoformans - C. gattii genotyping (Firacative et al., 2016). Typical MLST involves 7 loci sequences: capsular-associated protein (CAP59), glyceraldehyde-3-phosphate dehydrogenase (GPD1), URA5, PLB1, laccase (LAC1), Cu, Zn superoxide dismutase (SOD1), and intergenic spacer region (IGS1). These sequences together represent the minimal genes numbers that give the maximum power of discrimination (Farrer et al., 2015).

 

The recent proposed designation for C. neoformans/C. gattii species complex depending on AFLP and MLST results is as follows: I. C. neoformans serotype A (VNI) AFLP1, VNII AFLP1A, AFLP1B, VNB, II. C. deneoformans serotype D (VNIV) AFLP2, and III. C. neoformans - C. deneoformans hybrid or AD hybrids (VNIII) AFLP3. Cryptococcus gattii has been categorized as separate species named C. gattii (VGI) AFLP4, C. deuterogattii (VGII) AFLP6, C. bacillisporus (VGIII) AFLP5, C. tetragattii (VGIV) AFLP7, and C. decagattii (VGIV) and (VGIIIc) AFLP10. The hybrids between C. neoformans and C. gattii complexes isolates are named C. deneoformans × C. gattii hybrid (AFLP8), C. neoformans × C. gattii hybrid (AFLP9), and C. neoformans × C. deuterogattii hybrid (AFLP11) (Hagen et al., 2015). The expression “neoformans” was used for long time to represent species and variety. C. neoformans var. neoformans in that new system “C. neoformans” represents only serotype A strains with VNI and VNII/VNB molecular types (Kwon-Chung et al., 2017).

 

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).

 

Matrix-assisted laser desorption ionization-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

 

Table 3: Antifungal susceptibility profiles of Cryptococcus neoformans and C. gattii from environmental samples according to Clinical Laboratory Standards Institute guidelines.

 

Species (No.)	Geographic region	MIC range (µg/mL)									Reference
		AMB	FLU	5-FC	VRC	KET	ITC	POS	ISA	Nys
C. neoformans (10)	Cameroon	4-8	64- >256	ND	ND	16-64	ND	ND	ND	0.125-0.5	Dongmo et al. (2016)
C. gattii (10)		64-128	16- >256	ND	ND	8-64	ND	ND	ND	0.5-1
C. neoformans (86)	India	0.062-0.5	2-8	1-16	0.031-0.250	ND	0.031 -0.5	ND	ND	ND	Chowdhary et al. (2011)
C. gattii (60)		0.125-1	2-16	1-4	0.062-0.250	ND	0.125-0.5	ND	ND	ND
C. neoformans (117)	India	0.004-0.25	0.032-12	ND	0.006-0.125	0.002- 0.19	0.004 -0.75	ND	ND	ND	Khan et al. (2007)*
C. gattii (65)		0.023-0.5	0.032-16	ND	0.004-0.125	0.003- 0.19	0.006-2	ND	ND	ND
C. neoformans (81)	Brazil	0.3-1	0.12-64	ND	0.3-2	0.3-2	0.3-1	ND	ND	ND	Andrade-Silva et al. (2013)
C. neoformans (40)	Brazil	0.15-0.125	0.25-2	ND	0.003-0.25	ND	0.007 -0.125	ND	ND	ND	Souza et al. (2005)
C. neoformans (40)	Goiania, Brazil	0.03-0.5	0.5-4	ND	0.03-0.25	ND	0.03 -0.125	ND	ND	ND	Souza et al. (2010)
C. neoformans (16)	Sao Paulo, Brazil	0.25-2	0.5- >64	1- >64	ND	ND	0.06-4	ND	ND	ND	Pedroso et al. (2006)
C. gattii (60)	North western India	0.6-1	0.25-16	0.125-64	0.3-0.25	0.016 >-0.5	ND	0.016 >-0.5	0.016 >-0.25	ND	Chowdhary et al. (2013)
C. neoformans (50)	India	ND	0.063-64	ND	ND	0.03 -0.25	0.03-1	ND	ND	ND	Gutch et al. (2015)
C. gattii (4)		ND	2-64	ND	ND	0.03 -0.125	0.03-0.5	ND	ND	ND
C. neoformans (8)	Brazil	≤0.031–0.5	≤0.125–8	1-8	ND	ND	≤0.031 –0.125	ND	ND	ND	Moraes et al. (2003)
C. gattii (6)		≤0.031–0.125	0.125–8	≤0.125–8	ND	ND	0.5–2	ND	ND	ND

MIC: minimum inhibitory concentration. No.: number of cryptococcal species, AMB: amphotericin B, FLU: fluconazole, 5-FC: 5-flucytocine, VRC: voriconazole, KET: ketoconazole, ITC: itraconazole, POS: posaconazole, ISA: isavuconazole, Nys: nystatin. ND: not determined, *: Antifungal sensitivity was performed using E-Test method.

 

 

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.

 

Conflict of interest

 

The authors have declared no conflict of interest.

 

REFERENCES

 

• Alves GSB, Freire AKL, Bentes ADS, et al., (2016). Molecular typing of environmental Cryptococcus neoformans/C. gattii species complex isolates from Manaus, Amazonas, Brazil. Mycoses, 59: 509–515. https://doi.org/10.1111/myc.12499

• Andrade-Silva L, Ferreira-Paim K, Mora DJ, et al., (2013). Susceptibility profile of clinical and environmental isolates of Cryptococcus neoformans and Cryptococcus gattii in Uberaba, Minas Gerais, Brazil. Med. Myc., 51: 635–640. https://doi.org/10.3109/13693786.2012.761737

• Bandalizadeh Z, Shokohi T, Badali H, et al., (2020). Molecular epidemiology and antifungal susceptibility profiles of clinical Cryptococcus neoformans/Cryptococcus gattii species complex. J. Med. Microbiol., 69: 72-81. https://doi.org/10.1099/jmm.0.001101

• Beale M, Sabiiti W, Robertson E, et al., (2015). Genotypic diversity is associated with clinical outcome and phenotype in cryptococcal meningitis across southern Africa. PLoS Neglect. Trop. D., 9: e0003847. https://doi.org/10.1371/journal.pntd.0003847

• Bialek R. (2005). PCR based identification and discrimination of agents of mucormycosis and aspergillosis in paraffin wax embedded tissue. J. Clin. Pathol., 58: 1180–1184. https://doi.org/10.1136/jcp.2004.024703

• Brito-Santos F, Barbosa GG, Trilles L, et al., (2015). Environmental isolation of Cryptococcus gattii vgii from indoor dust from typical wooden houses in the deep amazonas of the Rio Negro Basin. PLoS One, 10: e0115866. https://doi.org/10.1371/journal.pone.0115866

• Byrnes E, Marr K (2011). The Outbreak of Cryptococcus gattii in western North America: epidemiology and clinical issues. Curr. Infect. Dis. Rep., 13: 256-261. https://doi.org/10.1007/s11908-011-0181-0

• Canónico-González Y, Adame-Rodríguez JM, Mercado-Hernández R, et al., (2013). Cryptococcus spp. isolation from excreta of pigeons (Columba livia) in and around Monterrey, Mexico. Springer Plus, 2: 632. https://doi.org/10.1186/2193-1801-2-632

• Chong HS, Dagg R, Malik R et al., (2010). In vitro susceptibility of the yeast pathogen Cryptococcus to fluconazole and other azoles varies with molecular genotype. J. Clin. Microbiol., 48: 4115-4120. https://doi.org/10.1128/JCM.01271-10

• Chowdhary A, Prakash A, Randhawa HS, et al., (2013). First environmental isolation of Cryptococcus gattii, genotype AFLP5, from India and a global review. Mycoses, 56: 222–228. https://doi.org/10.1111/myc.12039

• Chowdhary A, Randhawa HS, Prakash A, et al., (2011). Environmental prevalence of Cryptococcus neoformans and Cryptococcus gattii in India: An update. Crit. Rev. Microbiol., 38: 1–16.

• Cogliati M. (2013). Global molecular epidemiology of Cryptococcus neoformans and Cryptococcus gattii: an atlas of the molecular types. Scientifica, 2013: 1-23. https://doi.org/10.1155/2013/675213

• D’Souza C, Kronstad J, Taylor G, et al., (2011). Genome variation in Cryptococcus gattii, an emerging pathogen of immunocompetent hosts. mBio, 2: e00342-10.

• Doering TL. (2009). How sweet it is! Cell wall biogenesis and polysaccharide capsule formation in Cryptococcus neoformans. Annu. Rev. Microbiol., 63: 223–247. https://doi.org/10.1146/annurev.micro.62.081307.162753

• Dongmo W, Kechia F, Tchuenguem R, et al., (2016). In vitro antifungal susceptibility of environmental isolates of Cryptococcus spp. From the west region of Cameroon. Ethiop. J. Health Sci., 26: 555. https://doi.org/10.4314/ejhs.v26i6.8

• Elhariri M, Hamza D, Elhelw R, et al., (2016). Eucalyptus tree: a potential source of Cryptococcus neoformans in Egyptian environment. Int. J. Microbiol., 2016: 1–5. https://doi.org/10.1155/2016/4080725

• Elhariri, M, Hamza D, Elhelw R, et al., (2015). Lovebirds and cockatiels risk reservoir of cryptococcus neoformans, a potential hazard to human health. J. Vet. Sci. Med. Diagn., 04: 4. https://doi.org/10.4172/2325-9590.1000168

• Ellabib MS, Aboshkiwa MA, Husien WM, et al., (2016). Isolation, identification and molecular typing of Cryptococcus neoformans from pigeon droppings and other environmental sources in Tripoli, Libya. Mycopathologia, 181: 603–608. https://doi.org/10.1007/s11046-016-9996-4

• Ergin Ç, Şengül M, Aksoy L, et al., (2019). Cryptococcus neoformans recovered from olive trees (oleo European) in turkey reveal allopatry with African and south American lineages. Front Cell Infect. Microbiol., 9: 384. https://doi.org/10.3389/fcimb.2019.00384

• Farrer RA, Desjardins CA, Sakthikumar S, et al., (2015). Genome evolution and innovation across the four major lineages of Cryptococcus gattii. mBio, 6: e00868-15. https://doi.org/10.1128/mBio.00868-15

• Feng, Xiaobo, Yao, Zhirong, Liao, Wanqing. (2013). Approaches for molecular identification and typing of the Cryptococcus species complex: An update. Rev. Med. Microbiol., 24: 1-6. https://doi.org/10.1097/MRM.0b013e32835736c1

• Firacative C, Roe CC, Malik R, et al., (2016). Plos Ne MLST and whole-genome-based population analysis of Cryptococcus gattii VGIII links clinical, veterinary and environmental strains, and reveals divergent serotype specific sub-populations and distant ancestors glect Trop D, 10: e0004861. https://doi.org/10.1371/journal.pntd.0004861

• Firacative C, Trilles L, Meyer W (2012). MALDI-TOF MS enables the rapid identification of the major molecular types within the Cryptococcus neoformans/C. gattii species complex. PLoS One, 7: e37566. https://doi.org/10.1371/journal.pone.0037566

• Gray KC, Palacios DS, Dailey I, et al., (2012). Amphotericin primarily kills yeast by simply binding ergosterol. Proc. Natl. Acad. Sci. USA, 109: 2234–2239. https://doi.org/10.1073/pnas.1117280109

• Gutch RS, Nawange SR, Singh SM, et al., (2015). Antifungal susceptibility of clinical and environmental Cryptococcus neoformans and Cryptococcus gattii isolates in Jabalpur, a city of Madhya Pradesh in Central India. Braz. J. Microbiol., 46: 1125–1133. https://doi.org/10.1590/S1517-838246420140564

• Hagen F, Colom M, Swinne D, et al., (2012). Autochthonous and dormant Cryptococcus gattii infections in Europe. Emerg. Infect. Dis., 18: 1618-1624. https://doi.org/10.3201/eid1810.120068

• Hagen F, Khayhan K, Theelen B, et al., (2015). Recognition of seven species in the Cryptococcus gattii/Cryptococcus neoformans species complex. Fungal Genet. Biol., 78: 16–48. https://doi.org/10.1016/j.fgb.2015.02.009

• Harris JR, Lockhart SR, Debess E, et al., (2011). Cryptococcus gattii in the United States: Clinical aspects of infection with an emerging pathogen. Clin. Infect. Dis., 53: 1188–1195. https://doi.org/10.1093/cid/cir723

• Hu G, Liu I, Sham A, et al., (2008). Comparative hybridization reveals extensive genome variation in the AIDS-associated pathogen Cryptococcus neoformans. Genome Biol., 9: R41. https://doi.org/10.1186/gb-2008-9-2-r41

• Hurst S, Lysen C, Cooksey G, et al., (2019). Molecular typing of clinical and environmental isolates of Cryptococcus gattii species complex from southern California, United States. Mycoses, 62: 1029–1034. https://doi.org/10.1111/myc.12980

• Ito-Kuwa, S., Nakamura, K., Aoki, S., et al., (2007). Serotype identification of Cryptococcus neoformans by multiplex PCR. Mycoses, 50: 277–281. https://doi.org/10.1111/j.1439-0507.2007.01357.x

• Janbon G, Ormerod KL, Paulet D, et al., (2014). Analysis of the genome and transcriptome of Cryptococcus neoformans var. grubii reveals complex RNA expression and microevolution leading to virulence attenuation. PLoS Genet., 10: e1004261.

• Khan ZU, Randhawa HS, Kowshik T, et al., (2007). Antifungal susceptibility of Cryptococcus neoformans and Cryptococcus gattii isolates from decayed wood of trunk hollows of Ficus religiosa and Syzygium cumini trees in north-western India. J. Antimicrob. Chemother., 60: 312-316. https://doi.org/10.1093/jac/dkm192

• Khayhan K, Hagen F, Norkaew T, et al., (2017). Isolation of Cryptococcus gattii from a Castanopsis Argyrophylla tree hollow (Mai-Kaw), Chiang Mai, Thailand. Mycopathologia, 182: 365–370. https://doi.org/10.1007/s11046-016-0067-7

• Kwon-Chung K, Fraser J, Doering T, et al., (2014). Cryptococcus neoformans and Cryptococcus gattii, the etiologic agents of cryptococcosis. Csh. Perspect. Med., 4: a019760-a019760. https://doi.org/10.1101/cshperspect.a019760

• Kwon-Chung KJ, Bennett JE, Wickes BL, et al., (2017). The Case for adopting the “Species Complex” nomenclature for the etiologic agents of cryptococcosis. mSphere, 2: e00357-16. https://doi.org/10.1128/mSphere.00357-16

• Leal AL, Faganello J, Bassanesi MC, et al., (2008). Cryptococcus species identification by multiplex PCR. Med. Mycolo., 46: 377–383. https://doi.org/10.1080/13693780701824429

• Loyse A, Dromer F, Day J, et al., (2013). Flucytosine and cryptococcosis: time to urgently address the worldwide accessibility of a 50-year-old antifungal. J. Antimicrob. Chemother., 68: 2435-2444. https://doi.org/10.1093/jac/dkt221

• Lugarini C, Goebel CS, Condas LAZ, et al., (2008). Cryptococcus neoformans isolated from passerine and psittacine bird excreta in the state of paraná, Brazil. Mycopathologia, 166: 61–69. https://doi.org/10.1007/s11046-008-9122-3

• May RC, Stone NR, Wiesner DL, et al., (2016). Cryptococcus: from environmental saprophyte to global pathogen. Nat. Rev. Microbiol., 14: 106-117. https://doi.org/10.1038/nrmicro.2015.6

• Maziarz EK, Perfect JR. 2016. Cryptococcosis. Infect. Dis. Clin. North Am., 30: 179-206. https://doi.org/10.1016/j.idc.2015.10.006

• Mctaggart LR, Lei E, Richardson SE, et al., (2011). Rapid Identification of Cryptococcus neoformans and Cryptococcus gattii by matrix-assisted laser desorption ionization-time of flight mass spectrometry. J. Clin. Microbiol., 49: 3050–3053. https://doi.org/10.1128/JCM.00651-11

• Meyer W, Aanensen D, Boekhout T, et al., (2009). Consensus multi-locus sequence typing scheme for Cryptococcus neoformans and Cryptococcus gattii. Med. Mycol., 47: 561-570. https://doi.org/10.1080/13693780902953886

• Meyer W. 2015. Cryptococcus gattii in the age of whole-genome sequencing. mBio, 6: e01761-15. https://doi.org/10.1128/mBio.01761-15

• Moraes EMP, Primola NS, Hamdan JS, et al., (2003). Antifungal susceptibility of clinical and environmental isolates of Cryptococcus neoformans to four antifungal drugs determined by two techniques. Empfindlichkeit von klinischen und Umgebungsisolaten von Cryptococcus neoformans fur vier Antimykotika. Vergleichsstudie zweier Techniken. Mycoses, 46: 164–168. https://doi.org/10.1046/j.1439-0507.2003.00872.x

• Pedroso RDS, Ferreira JC, Candido RC, et al., (2006). In vitro susceptibility to antifungal agents of environmental Cryptococcus spp. isolated in the city of Ribeirão Preto, São Paulo, Brazil. Memórias do Instituto Oswaldo Cruz. 101: 239–243. https://doi.org/10.1590/S0074-02762006000300002

• Perfect J, Bicanic T. (2015). Cryptococcosis diagnosis and treatment: What do we know now? Fungal Genet. Biol., 78: 49-54. https://doi.org/10.1016/j.fgb.2014.10.003

• Pfaller MA, Castanheira M, Diekema DJ, et al., (2011). Wild-type MIC distributions and epidemiologic cutoff values for fluconazole, posaconazole, and voriconazole when testing Cryptococcus neoformans as determined by the CLSI broth microdilution method. Diagn. Microbiol. Infect. Dis., 71: 252–259. https://doi.org/10.1016/j.diagmicrobio.2011.07.007

• Pllana-Hajdari, D, Cogliati, M, Čičmak, L et al., (2019). First isolation, antifungal susceptibility, and molecular characterization of Cryptococcus neoformans from the environment in Croatia. JoF, 5: 99. https://doi.org/10.3390/jof5040099

• Prakash A, Sundar G, Sharma B, et al., (2020). Genotypic diversity in clinical and environmental isolates of Cryptococcus neoformans from India using multilocus microsatellite and multilocus sequence typing. Mycoses, 63: 284–293. https://doi.org/10.1111/myc.13041

• Refai M, Elhariri M, Alarousy R. (2017). Cryptococcosis in animals and birds: A Review. 4: 202-223.

• Rivera V, Gaviria M, Muñoz-Cadavid C, et al., (2015). Validation and clinical application of a molecular method for the identification of Cryptococcus neoformans/Cryptococcus gattii complex DNA in human clinical specimens. Braz. J. Infect. Dis., 19: 563–570. https://doi.org/10.1016/j.bjid.2015.07.006

• Sidrim J, Costa A, Cordeiro R, et al., (2010). Molecular methods for the diagnosis and characterization of Cryptococcus: A review. Can. J. Microbiol., 56: 445-458. https://doi.org/10.1139/W10-030

• Silva DMW and de Albuquerque Maranhão FC (2015). Current Status of the Diagnostic and Genomics of Cryptococcus neoformans/C. gattii Species Complex. Fungal Genom. Biol., 05: 2. https://doi.org/10.4172/2165-8056.1000e118

• Souza LKH, Fernandes OFL, Kobayashi CCBA et al., (2005). Antifungal susceptibilities of clinical and environmental isolates of Cryptococcus neoformans in Goiânia City Goiás Brazil. Rev. Inst. Med. Trop. Sao. Paulo., 47: 253-256. https://doi.org/10.1590/S0036-46652005000500003

• Souza LKH, Junior AHS, Costa CR, et al., (2010). Molecular typing and antifungal susceptibility of clinical and environmental Cryptococcus neoformans species complex isolates in Goiania, Brazil. Mycoses, 53: 62–67. https://doi.org/10.1111/j.1439-0507.2008.01662.x

• Thompson GR, Wiederhold NP, Fothergill AW, et al., (2008). Antifungal susceptibilities among different serotypes of Cryptococcus gattii and Cryptococcus neoformans. Antimicrob. Agents Chemother., 53: 309–311. https://doi.org/10.1128/AAC.01216-08

• Trilles L, Wang B, Firacative C, et al., (2014). Identification of the major molecular types of Cryptococcus neoformans and c. gattii by hyperbranched rolling circle amplification. PLoS One, 9: e94648. https://doi.org/10.1371/journal.pone.0094648

• Trovato L, Oliveri S, Esposto MC, et al., (2019). Cryptococcus neoformans and Cryptococcus gattii species complex isolates on the slopes of Mount Etna, SICILY, Italy. Front Microbiol., 10: 2390. https://doi.org/10.3389/fmicb.2019.02390

• Yang YL, Cheng MF, Wang CW, et al., (2010). The distribution of species and susceptibility of amphotericin B and fluconazole of yeast pathogens isolated from sterile sites in Taiwan. Med. Mycol., 48: 328–334. https://doi.org/10.3109/13693780903154070