Molecular typing of IberoAmerican Cryptococcus neoformans isolates

Abstract

A network was established to acquire basic knowledge of Cryptococcus neoformans in IberoAmerican countries. To this effect, 340 clinical, veterinary, and environmental isolates from Argentina, Brazil, Chile, Colombia, Mexico, Peru, Venezuela, Guatemala, and Spain were typed by using M13 polymerase chain reaction-fingerprinting and orotidine monophosphate pyrophosphorylase (URA5) gene restriction fragment length polymorphism analysis with HhaI and Sau96I in a double digest. Both techniques grouped all isolates into eight previously established molecular types. The majority of the isolates, 68.2% (n=232), were VNI (var. grubii, serotype A), which accords with the fact that this variety causes most human cryptococcal infections worldwide. A smaller proportion, 5.6% (n=19), were VNII (var. grubii, serotype A); 4.1% (n=14), VNIII (AD hybrid), with 9 isolates having a polymorphism in the URA5 gene; 1.8% (n=6), VNIV (var. neoformans, serotype D); 3.5% (n=12), VGI; 6.2% (n=21), VGII; 9.1% (n=31), VGIII, and 1.5% (n=5) VGIV, with all four VG types containing var. gatii serotypes B and C isolates.

Figure 1

Geographic distribution of the molecular types obtained from IberoAmerican Crptococcus neoformans isolates by polymerase chain reaction fingerprinting and URA5 gene restriction fragment length polymorphis analysis (total numbers studied per country given in parentheses).

Figure 2

Dendrogram of the polymerase chain reaction-fingerprinting patterns obtained with the primer M13 from a selection of the IberoAmerican isolates studied. All the isolates fall into eight major molecular types, which fall into three major groups corresponding to Cryptococcus neoformans var. grubii, serotype A, with two molecular types VNI and VNII; C. neoformans var. neoformans, serotype D, with the molecular type VNIV; and C. neoformans var. gattii 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.

Figure 3

Polymerase chain reaction (PCR) fingerprints generated with the primer M13 (2A), and URA5 gene restriction fragment length polymorphism (RFLP) profiles identified by double digest of the gene with Sau96I and HhaI (2B) obtained from a selection of Mexican Cryptococcus neoformans isolates, given as a representative example for the patterns obtained from clinical and environmental isolates from Latin America. Standard patterns obtained from the reference strains of the major molecular types by PCR-fingerprinting patterns with the microsatellite specific primer M13 as a single primer in the PCR (right-hand side of 2A) and URA5 gene RFLP profiles generated after double digest with Sau96I and HhaI (right-hand side of 2B) (VNIII correspond to the seven-band URA5 RFLP pattern and VNIII* correspond to the six-band URA5 RFLP pattern).

Figure 4

Polymerase chain reaction fingerprints generated with the primer M13 (3A) and URA5 gene restriction frgement length polymorphism (RFLP) profiles identified via double digest of the gene with Sau96I and HhaI (3B) obtained from the Spanish clinical, veterinary, and environmental Cryptococcus neoformans isolates (VNIII correspond to the seven-band URA5 RFLP pattern and VNIII* correspond to the six-band URA5 RFLP pattern).