Clinical epidemiology and high genetic diversity amongst Cryptococcus spp. isolates infecting people living with HIV in Kinshasa, Democratic Republic of Congo

Abstract

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 (< 50 mg/dL), increased cerebral spinal fluid opening pressure (> 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.

What’s New in Cryptococcus gattii: From Bench to Bedside and Beyond

“…A minority of Cryptococcus spp. (ranging from 2–21%), are acapsular or pauci-capsular, and so untypeable [ 16 , 17 , 18 ].…”

“…MLST has revealed geographical associations with clonal populations with increased heterogeneity possibly related to the origins of the species; for example, C. gattii , may have spread from Australia and Northern Brazil to North America and Canada [ 28 , 29 ] whereas for C. neoformans, ST5 is the predominant serotype in China and Taiwan, ST93 in Brazil, ST41 in Japan and the US, with greater diversity in African countries [ 16 , 28 , 29 , 30 , 31 , 32 ].…”

Correlation of antifungal susceptibility and sequence types within Cryptococcus neoformans VNI from HIV patients, and ERG11 gene polymorphism

“…The long delay in CM diagnosis due to the lack of laboratory tools, the advanced stage of the patients' HIV disease on arrival at the clinics, the genetic diversity of Cryptococcus spp. and the low sensitivity of some species to common antifungal agents as previously described in DRC could partially explain this fatal outcome [17,18].…”

“…The overall data from this survey were included in our previous article. The MLST characterisation of the isolates carried out previously and allowed identification of seven different STs, including one main ST (ST93, n = 15) and six less common STs [ST659 (n = 2), ST5 (n = 2), ST4 (n = 1), ST 53 (n = 1), ST31 (n = 1), and ST69 (n = 1)] [17].…”

Exposure of Cryptococcus neoformans to Seven Commonly Used Agricultural Azole Fungicides Induces Resistance to Fluconazole as Well as Cross-Resistance to Voriconazole, Posaconazole, Itraconazole and Isavuconazole

“…Indirect clues about this possibility have emerged in various studies. Previous studies in Western Africa have shown that strains already resistant to fluconazole or exhibiting an elevated fluconazole MIC were present in patients naïve for this molecule in Cameroon [ 31 , 32 , 33 ], Ivory Coast [ 34 ] and DRC [ 35 ]. From a general point of view, there is increasing FCZ resistance in Cryptococcus strains isolated from Africa [ 36 , 37 , 38 , 39 ].…”