Medical Mycology, 2018, 0, 1–3

doi: 10.1093/mmy/myy022
Advance Access Publication Date: 0 2018
Brief Report

Brief Report
Molecular-Type Specific Multiplex PCR produces a distinct VNII PCR
pattern among Cryptococcus neoformans species complex
Massimo Cogliati1,∗ , Mala Rakoto Andrianarivelo2 , Mohamed Ellabib3 ,
Emmanuel N. Nnadi4 and Muriel Cornet5
1
Lab. Medical Mycology, Dip. Scienze Biomediche per la Salute, Università degli Studi di Milano, Milano, Italy,
2
Centre d’Infectiologie Charles Mérieux (CICM), University Ankatso, Antananarivo, Madagascar, 3 Medical College
University of Tripoli, Tripoli, Libya, 4 Department of Microbiology, Plateau State University, Bokkos, Plateau State,
Nigeria and 5 Université Grenoble Alpes, CNRS, Grenoble INP, CHU Grenoble Alpes, TIMC-IMAG, Grenoble, France
∗
To whom correspondence should be addressed. Massimo Cogliati. Lab. Medical Mycology, Dip. Scienze Biomediche per la Salute,
Università degli Studi di Milano, Via pascal 36 - 20133 Milano –Italy. Tel: +39 0250315144; Fax: +39 0250315146;
E-mail: massimo.cogliati@unimi.it
Received 17 November 2017; Revised 26 February 2018; Accepted 4 April 2018; Editorial Decision 7 March 2018

Abstract
Prevalence of molecular type VNII among Cryptococcus neoformans species complex is probably underes-
timated since it can be distinguished from VNI only using molecular typing methods such as URA5-RFLP,
AFLP, MLST, or whole genome sequencing. Previously, we described a multiplex polymerase chain reaction
(PCR) method able to identify VNI, VNIV, and VNIII hybrids, but, at that time, VNII molecular type was not
described yet. In this study, 16 VNII global isolates were analyzed by our multiplex PCR method, and results
showed that it was able to produce a specific pattern for all the studied VNII isolates, which was different
from those of VNI, VNIV, and VNIII.
Key words: Cryptococcus neoformans species complex, molecular typing, molecular type VNII, MLST, multiplex PCR.

Cryptococcus neoformans species complex includes encapsu-
lated yeasts, which are the major cause of cryptococcosis world-
wide.1 Based on molecular typing methods, five molecular types
can be distinguished inside the species complex: VNI, VNII,
and VNB (C. neoformans var. grubii), VNIV (C. neoformans
var. neoformans), and the intervarietal hybrids identified by
molecular type VNIII.2 A recent taxonomic revision proposes
to define C. neoformans var. grubii and C. neoformans var.
neoformans as two separate species: C. neoformans and C. de-
neoformans, respectively.3 However, both molecular types VNII
and VNB was shown to be phylogenetically very distant from
VNI4 ; therefore, their taxonomic position in the same species
represents a further issue for discussion in the scientific commu-
nity.
Molecular type VNII has been isolated from all the conti-
nents although less frequently than VNI.5 However, the preva-
lence of this molecular type is probably underestimated because
it can be distinguished from VNI only using molecular typ-
ing methods such as URA5-restriction fragment length poly-
morphism (URA5-RFLP),6 amplified fragment length polymor-
phism (AFLP),7 multilocus sequence typing (MLST),2 and whole
genome sequencing (WGS).4 Previously, we described a multi-
plex polymerase chain reaction (PCR) method able to identify
VNI, VNIV, and VNIII hybrids, but at that time, VNII molecular
type was not described yet.8 In the present study, we analyzed
a set of VNII isolates to verify if our multiplex PCR method
is suitable to distinguish molecular type VNII from molecular
types VNI, VNIV, and VNIII.
The study included 16 VNII isolates from different geo-
graphical areas of the world and with different MLST profiles
(Table 1). Five strains were from Italy,9 10 were from three
different countries of Africa,10,11 and one from Australia.6 In


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Table 1. List of the 16 isolates belonging to Cryptococcus neoformans species complex, molecular type VNII, analyzed in this study.

MLST
Date of Mating Molecular Sequence
Strain Geographical origin Type of isolate Source isolation type type type References

LYTIP1HO1-1 Africa, Libya Environmental Eucalyptus 2013 αA VNII 40 10

LYTIP3SO1-1 Africa, Libya Environmental Eucalyptus 2013 αA VNII 490 10

IUM 15.0045 Africa, Madagascar Clinical CSF 2015 αA VNII na Unpublished

IUM 15–0110 Africa, Madagascar Clinical CSF 2015 αA VNII na Unpublished
IUM 15–0189 Africa, Madagascar Clinical CSF 2015 αA VNII na Unpublished
IUM 16–0061 Africa, Madagascar Clinical CSF 2016 αA VNII na Unpublished
IUM 16–0062 Africa, Madagascar Clinical CSF 2016 αA VNII na Unpublished
IUM 14–0304 Africa, Nigeria Environmental Pigeon droppings 2014 αA VNII 43 9

IUM 14–0305 Africa, Nigeria Environmental Pigeon droppings 2014 αA VNII 43 9

IUM 14–0306 Africa, Nigeria Environmental Pigeon droppings 2014 αA VNII 43 9

IUM 97–4879 Europe, Italy Clinical CSF 1997 αA VNII 96 8

IUM 99 -4427 Europe, Italy Clinical CSF 1999 αA VNII 107 8

IUM 01–4726 Europe, Italy Clinical CSF 2001 αA VNII 96 8

IUM 08–0068 Europe, Italy Clinical CSF 2008 αA VNII na Unpublished

IUM 13–0224 Europe, Italy Clinical Human 2013 αA VNII 43 Unpublished
WM626 Oceania, Australia Clinical CSF 1993 αA VNII 97 5

CSF = cerebrospinal fluid; na = not available.

Figure 1. Multiplex PCR banding pattern of the 10 VNII isolates confirmed by MLST compared to those of the three reference strains. M = 100 bp DNA Ladder
(Promega Italy, Milano, Italy). MLST, multilocus sequence typing; PCR, polymerase chain reaction.

addition, isolates were both from clinical (nine isolates) and
environmental (seven isolates) sources. The molecular type of all
the isolates was previously determined by URA5-RFLP method,
and for 10 isolates, it was confirmed by MLST profile (Table 1).
Multiplex PCR was performed as previously described 7 using
four pairs of primers able to amplify four specific genomic DNA
regions12 whose combinations allow identifying C. neoformans
species complex molecular types. Strains H99 (VNI), JEC21
(VNIV), and CBS132 (VNIII) were used as reference strains to
compare the banding profile of VNII isolates.
Results of the multiplex PCR confirmed the expected band-
ing profile for the three reference strains. Strain H99 showed
the characteristic VNI profile with three bands (670, 430, and
420 bp), strain JEC21 showed the VNIV profile with two bands
(420 and 230 bp), and CBS132 the VNIII pattern with all four
bands (670, 430, 420, and 230 bp). In contrast, all VNII iso-
lates showed a different PCR pattern with three bands at 430,
420, and 230 bp. Although among VNII isolates six different
MLST profiles (ST40, ST43, ST96, ST97, ST107, and ST490)
were identified, they showed all the same PCR pattern (Fig. 1).
The present study shows that our molecular typing method
using specific multiplex PCR is able to identify VNII isolates
of Cryptococcus neoformans species complex producing a dis-
tinct PCR banding pattern. At present, molecular type VNII
can be distinguished from VNI only using molecular typing
methods such as URA5-RFLP,6 AFLP,7 MLST,2 and WGS.4

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 Cogliati et al.
However, URA5-RFLP requires two steps, PCR of URA5 gene
and subsequently a double restriction reaction of the ampli-
con, whereas AFLP, MLST, and WGS are laborious and require
expensive instruments. In contrast, the method reported here
requires only a one-step multiplex PCR, and it is easily inter-
pretable on the basis of the combination of four specific bands.
These characteristics also make it useful for molecular type iden-
tification in epidemiological studies in laboratories, which oper-
ate with low resources. The fact that our method is also able
to identify molecular type VNII represents an additional tool
for discovering new isolates belonging to this genotype that, at
present, is underestimated. The presence of molecular type VNII
was reported in all five continents,5 but little is known about its
pathobiology, ecology, and antifungal susceptibility. Therefore,
tools that are able to identify it are important to better under-
stand how it is distributed in the environment, how it interacts
with the host, and finally if it needs a different and more specific
therapeutic treatment to improve the patient outcome. A higher
number of VNII isolates will also contribute to understanding
the phylogenetic relationship of this genotype compared to the
other ones, revealing if it represents a separate species in the C.
neoformans species complex.
Acknowledgments
Isolates from Madagascar were obtained thanks to a study funded by
European & Developing Countries Clinical Trials Partnership.
Declaration of interest
The authors report no conflicts of interest. The authors alone are respon-
sible for the content and the writing of the paper.
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