Molecular Analysis of Cryptococcus spp. Reveals Species Diversity and Multilocus Sequence Typing Heterogeneity among People Living with HIV in Kinshasa

Objectives: In the Democratic Republic of Congo (DRC), the neuromeningeal cryptococcosis
(NMC) hospital prevalence varies between 8.8 and 11% with a death rate of about
50%. However, the Cryptococcus isolates circulating are poorly and formerly described.
We describe the molecular characterization of Cryptococcus spp. isolated from people
living with HIV (PLWHIV), and the association between NMC severity factors and the
Cryptococcus multilocus sequence typing (MLST) profiles.
Materials & Methods: Cerebral spinal fluid (CSF) was collected from PLWHIV with
neuromeningeal syndrome hospitalized in three Kinshasa public hospitals from 1 February
2019 to 29 February 2020, and cultured on Sabouraud dextrose agar + chloramphenicol
medium afterwards. Serotyping PCR, internal transcribed spacer (ITS) sequencing, and
MLST from next-generation sequencing (NGS) data were performed to genotype the
Cryptococcus isolates. Raw NGS data were processed and the loci alleles were manually
extracted using Geneious software. NMC severity factors such as hypoglycorrhachia
(<50 mg/dL), raised CSF opening pressure (>30 cm water), and the patient pejorative
outcome were compared to the Cryptococcus sequence type (ST)-MLST profile which was
identified, using Pearson chi-square test or Fisher exact test.
Results: Out of 29 isolates included in the present study, 23 (79.3%; 95% IC: 65.5–93.1)
have been identified as serotype A using serotyping PCR, while 6 (20.7%; 95% IC: 6.9–34.5)
had unidentifiable profile. Moreover, all the 29 isolates have been characterized by ITS
sequencing as follow: Cryptococcus neoformans var. grubii (23 of 29 isolates, 79.3%), Cryptococcus
curvatus (5 of 29, 17.2%), and Cryptococcus laurentii (1 of 29, 3.5%). Apart from
this highlighted species diversity, C. neoformans var. grubii isolates were all identified as
molecular type VNI using the MLST ISHAM scheme, with seven different STs including
ST93 (14 out of 23, 60.9%), ST53 (1 out of 23, 4.3%), ST31 (1 out of 23, 4.3%), ST5 (1 out of
23, 4.3%), ST4 (1 out of 23, 4.3%), ST69 (1 out 23, 4.3%), and one novel ST not yet reported
worldwide (2 out of 23, 8.6%). Furthermore, two isolates (8.6%) showed a mixed molecular
profile (under investigation). Phylogenetic analysis carried out by the unweighted pair
group method with arithmetic mean algorithm (UPGMA) of concatenated gene sequences
from the seven MLST loci indicated that the main ST isolated in this study is very close
to the only isolate previously reported from the DRC. The others are distant from it, even
more so for ST69. All the STs identified in the present study have been isolated in the neighbouring
countries of the DRC except for ST53, which has only been reported in Southeast Asia. Among NMC severity factors, only the patient pejorative outcome was associated
with the minority STs infections (87.5%, p = 0.02) (ST53, ST31, ST5, ST4, STA, ST69).
Conclusions: Molecular analysis of Cryptococcus spp. isolates showed wide species
diversity, and ST-MLST heterogenicity within the only one molecular type identified. The
existence of the STs isolated in this study in the DRC neighbouring countries suggests probable
close-to-home spread. Furthermore, infections due to minority STs were associated
with a pejorative outcome than those due to ST93.