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Chapter 24 : Molecular Typing of the Species Complex

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Abstract:

A large number of molecular typing techniques have been applied over the years to discriminate between individual isolates that had been indistinguishable using conventional techniques and to obtain further insights into the epidemiology and population structure of this species complex. This chapter aims to summarize the diverse typing techniques applied to the / species complex, to correlate the obtained results, and to describe the global distribution of the major genotypes. The enzymes malate dehydrogenase, alcohol dehydrogenase, phosphoglyceromutase, and glutamate dehydrogenase could separate from . Electrophoretic karyotyping was for the first time applied to the / species complex to study the genetic diversity between seven cryptococcal strains representing all four serotypes. PCR fingerprinting using the primer (GACA) was applied to 110 cryptococcal isolates obtained mainly from Germany and Africa as well as additional globally collected reference strains. The restriction fragment length polymorphism (RFLP) patterns result from the presence of a restriction enzyme cleavage site at one place in the genome in one individual and the absence of that specific site in another individual. Intergenic spacer (IGS) sequence analysis is a powerful tool to delineate the two varieties of and separate the four major molecular types of .

Citation: Meyer W, Gilgado F, Ngamskulrungroj P, Trilles L, Hagen F, Castañeda E, Boekhout T. 2011. Molecular Typing of the Species Complex, p 327-357. In Heitman J, Kozel T, Kwon-Chung K, Perfect J, Casadevall A (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555816858.ch24

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Restriction Fragment Length Polymorphism
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DNA Fingerprinting
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Image of FIGURE 1
FIGURE 1

The taxonomic, molecular, epidemiological, and clinical correlation within the and species complex. (Names given in bold are currently accepted taxonomic species names.)

Citation: Meyer W, Gilgado F, Ngamskulrungroj P, Trilles L, Hagen F, Castañeda E, Boekhout T. 2011. Molecular Typing of the Species Complex, p 327-357. In Heitman J, Kozel T, Kwon-Chung K, Perfect J, Casadevall A (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555816858.ch24
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Image of FIGURE 2
FIGURE 2

Assessment of the major molecular types within the species complex. (A) PCR fingerprints generated with the primer M13. (B) AFLP profiles generated with the FAM label AC+G kit. (C) gene RFLP profiles identified via double digestion with au96I and HhaI. (D) gene RFLP profiles identified via digestion with AvaI obtained from the reference strains of each major genotype.

Citation: Meyer W, Gilgado F, Ngamskulrungroj P, Trilles L, Hagen F, Castañeda E, Boekhout T. 2011. Molecular Typing of the Species Complex, p 327-357. In Heitman J, Kozel T, Kwon-Chung K, Perfect J, Casadevall A (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555816858.ch24
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Image of FIGURE 3
FIGURE 3

Phenogram of the combined PCR-fingerprinting data obtained with the primer M13 and (GACA) from a selection of global cryptococcal isolates. All isolates fall into eight major molecular types, which correspond to var. , serotype A, with two molecular types, VNI and VNII; var. , serotype D, with the molecular type VNIV; and , serotypes B and C, with the molecular types VGI, VGII, VGIII, and VGIV. In addition to the three major clusters, we can see the intermediate molecular type VNIII, representing the AD hybrids ( ; Meyer et al., unpublished data).

Citation: Meyer W, Gilgado F, Ngamskulrungroj P, Trilles L, Hagen F, Castañeda E, Boekhout T. 2011. Molecular Typing of the Species Complex, p 327-357. In Heitman J, Kozel T, Kwon-Chung K, Perfect J, Casadevall A (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555816858.ch24
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Image of FIGURE 4
FIGURE 4

AFLP analysis of cryptococcal strains. (A) AFLP patterns representing all AFLP types obtained within the species complex. (B) AFLP patterns (AFLP3/VNIII) showing that the AD hybrid strains are a combination of the patterns obtained for serotype A (AFLP1/VNI) and serotype D (AFLP2/VNIV). (C) AFLP patterns indicating AB hybrids. (D) AFLP patterns indicating BD hybrids.

Citation: Meyer W, Gilgado F, Ngamskulrungroj P, Trilles L, Hagen F, Castañeda E, Boekhout T. 2011. Molecular Typing of the Species Complex, p 327-357. In Heitman J, Kozel T, Kwon-Chung K, Perfect J, Casadevall A (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555816858.ch24
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Image of FIGURE 5
FIGURE 5

Geographical distribution of the 86 var. strains studied with their MLMT type, made up of the combination of the three polymorphic microsatellite alleles. The number of strains given per MLMT locus is in parentheses, showing that the largest genetic variation is present in the Americas, especially in South and Latin America (reproduction from reference ).

Citation: Meyer W, Gilgado F, Ngamskulrungroj P, Trilles L, Hagen F, Castañeda E, Boekhout T. 2011. Molecular Typing of the Species Complex, p 327-357. In Heitman J, Kozel T, Kwon-Chung K, Perfect J, Casadevall A (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555816858.ch24
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Image of FIGURE 6
FIGURE 6

(A) Distribution of the eight major molecular types identified among 2,755 globally collected isolates ( ). (B) Data for 2,046 clinical and 68 veterinary cryptococcal strains obtained from 48 countries. (C) Data for 604 environmental isolates obtained from 21 countries. Thirty-seven isolates were excluded from the analysis due to missing source information.

Citation: Meyer W, Gilgado F, Ngamskulrungroj P, Trilles L, Hagen F, Castañeda E, Boekhout T. 2011. Molecular Typing of the Species Complex, p 327-357. In Heitman J, Kozel T, Kwon-Chung K, Perfect J, Casadevall A (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555816858.ch24
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Image of FIGURE 7
FIGURE 7

Global distribution of the eight major molecular types of the species complex. Numbers in italics indicate that clinical and environmental samples have been reported for this molecular type. Nonitalic numbers indicate that only clinical strains have been reported. Data have been combined from the following references: .

Citation: Meyer W, Gilgado F, Ngamskulrungroj P, Trilles L, Hagen F, Castañeda E, Boekhout T. 2011. Molecular Typing of the Species Complex, p 327-357. In Heitman J, Kozel T, Kwon-Chung K, Perfect J, Casadevall A (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555816858.ch24
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Image of FIGURE 8
FIGURE 8

(A) Numbers of clinical isolates obtained from immunocompetent and immunocom-promised patients (HIV-positive) and patients with other risk factors per major molecular type from a total of 1,250 clinical strains from a total of 2,046 strains investigated by PCR fingerprinting with the primer M13 and AFLP analysis for which the clinical data were available ( ). Other risk factors are alcoholism, corticosteroid use, disorder T immunity, diabetes, leukemia, systemic lupus erythematosus, transplant, and tumor. (B) Distribution of the major molecular types including all clinical isolates obtained from immunocompetent patients. (C) Distribution of the major molecular types obtained from immunocompetent patients excluding the Chinese data ( ).

Citation: Meyer W, Gilgado F, Ngamskulrungroj P, Trilles L, Hagen F, Castañeda E, Boekhout T. 2011. Molecular Typing of the Species Complex, p 327-357. In Heitman J, Kozel T, Kwon-Chung K, Perfect J, Casadevall A (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555816858.ch24
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Image of FIGURE 9
FIGURE 9

Distribution of the major molecular types among 586 environmental isolates, excluding the 5 African and 13 Asian environmental isolates ( ). (A) Data for Oceania ( = 51). (B) Data for Europe ( = 37). (C) Data for North America ( = 111). (D) Data for South America ( = 387), indicating a different prevalence between VGI and VGII in the Americas as compared to the rest of the world.

Citation: Meyer W, Gilgado F, Ngamskulrungroj P, Trilles L, Hagen F, Castañeda E, Boekhout T. 2011. Molecular Typing of the Species Complex, p 327-357. In Heitman J, Kozel T, Kwon-Chung K, Perfect J, Casadevall A (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555816858.ch24
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Image of FIGURE 10
FIGURE 10

One of the two most parsimonious trees obtained from heuristic searches based on analysis produced from 10 combined genes (, ITS1/2, IGS, α, and ). Bootstrap support values above 70% are indicated at the nodes. The tree was rooted at the midpoint.

Citation: Meyer W, Gilgado F, Ngamskulrungroj P, Trilles L, Hagen F, Castañeda E, Boekhout T. 2011. Molecular Typing of the Species Complex, p 327-357. In Heitman J, Kozel T, Kwon-Chung K, Perfect J, Casadevall A (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555816858.ch24
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Image of FIGURE 11
FIGURE 11

Molecular divergence between the major haploid molecular types of the species complex in million of years (A) based on the and genes ( ), (B) based on the , and genes ( ), and (C) based on the analysis of the whole genomes ( ).

Citation: Meyer W, Gilgado F, Ngamskulrungroj P, Trilles L, Hagen F, Castañeda E, Boekhout T. 2011. Molecular Typing of the Species Complex, p 327-357. In Heitman J, Kozel T, Kwon-Chung K, Perfect J, Casadevall A (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555816858.ch24
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Tables

Generic image for table
TABLE 1

Concordance of different molecular typing methods used for and

Citation: Meyer W, Gilgado F, Ngamskulrungroj P, Trilles L, Hagen F, Castañeda E, Boekhout T. 2011. Molecular Typing of the Species Complex, p 327-357. In Heitman J, Kozel T, Kwon-Chung K, Perfect J, Casadevall A (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555816858.ch24
Generic image for table
TABLE 2

Characterization of 11 microsatellite loci for var. , the AD hybrid, and var. ( )

Citation: Meyer W, Gilgado F, Ngamskulrungroj P, Trilles L, Hagen F, Castañeda E, Boekhout T. 2011. Molecular Typing of the Species Complex, p 327-357. In Heitman J, Kozel T, Kwon-Chung K, Perfect J, Casadevall A (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555816858.ch24
Generic image for table
TABLE 3

Consensus MLST loci to be used for strain typing within the C. species complex ( )

Citation: Meyer W, Gilgado F, Ngamskulrungroj P, Trilles L, Hagen F, Castañeda E, Boekhout T. 2011. Molecular Typing of the Species Complex, p 327-357. In Heitman J, Kozel T, Kwon-Chung K, Perfect J, Casadevall A (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555816858.ch24
Generic image for table
TABLE 4

Standard/reference strains for C. and C. strain typing

Citation: Meyer W, Gilgado F, Ngamskulrungroj P, Trilles L, Hagen F, Castañeda E, Boekhout T. 2011. Molecular Typing of the Species Complex, p 327-357. In Heitman J, Kozel T, Kwon-Chung K, Perfect J, Casadevall A (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555816858.ch24

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