Épidémiologie clinique et grande diversité génétique parmi les isolats de Cryptococcus spp. infectant les personnes vivant avec le VIH à Kinshasa, République démocratique du Congo

Neuromeningeal cryptococcosis (NMC) is a life-threatening opportunistic infection in advanced HIV disease patients (AHDP). It is caused by Cryptococcus spp. complexes and mainly occurs in sub-Saharan Africa. In this study, we performed molecular characterization and antifungal susceptibility profiling of Cryptococcus isolates from AHDP in Kinshasa (DRC). Additionally, we investigated a possible association between NMC severity factors and the Cryptococcus neoformans (Cn) multilocus sequence typing (MLST) profiles. We characterized the isolates using PCR serotyping, MALDI-TOF MS, internal transcribed spacer (ITS) sequencing, and MLST. Susceptibility testing for the major antifungal drugs was performed according to the EUCAST guidelines. Parameters associated with NMC severity, such as hypoglycorrhachia ( 30 cm H2O), and poor therapeutic outcome were compared with the Cn MLST sequences type (ST). Twenty-three out of 29 Cryptococcus isolates were identified as serotype A using PCR serotyping (79.3%; 95% IC: 65.5-93.1), while six (20.7%; 95% IC: 6.9-34.5) were not serotypable. The 29 isolates were identified by ITS sequencing as follows: Cryptococcus neoformans (23/29, 79.3%), Cutaneotrichosporon curvatus (previously called Cryptococcus curvatus) (5/29, 17.2%), and Papiliotrema laurentii (Cryptococcus laurentii) (1/29, 3.5%). Using the ISHAM MLST scheme, all Cn isolates were identified as molecular type VNI. These comprised seven different STs: ST93 (n = 15), ST5 (n = 2), ST53 (n = 1), ST31 (n = 1), ST4 (n = 1), ST69 (n = 1), and one novel ST that has not yet been reported from other parts of the world and was subsequently assigned as ST659 (n = 2). Of the included strains, only Papiliotrema laurentii was resistant to amphoterin B (1/29, 3.5%), 6.8% (2/29) were resistant to 5-flucytosine (the single Papiliotrema laurentii strain and one Cryptococcus neoformans isolate), and 13.8% (4/29) to fluconazole, including two of five (40%) Cutaneotrichosporon curvatus and two of 23 (8.7%) C. neoformans strains. We found a significative association between poor therapeutic outcome and a non-ST93 sequence type of causative strains (these concerned the less common sequence types: ST53, ST31, ST5, ST4, ST659, and ST69) (87.5% versus 40%, p = 0.02). Molecular analysis of Cryptococcus spp. isolates showed a wide species diversity and genetic heterogenicity of Cn within the VNI molecular type. Furthermore, it is worrying that among included strains we found resistances to several of the commonly used antifungals.Cryptococcose chez les personnes vivant avec le VIH à Kinshasa : étude épidémiologique et moléculaire3. Good health and well-bein

L'analyse moléculaire de Cryptococcus spp. révèle une diversité d'espèces et une hétérogénéité du typage des séquences multilocus chez les personnes vivant avec le VIH à 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 (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.Cryptococcose chez les personnes vivant avec le VIH à Kinshasa (RDC) : Etude épidémiologique et moléculair