Cryptococcal meningitis has been described as an opportunistic infection in HIVIP, mainly caused by the Cn/Cg species complex [15, 16]. However, non-neoformans and non-gattii Cryptococcus species have recently been isolated in cryptococcosis both in immunosuppressed and immunocompetent patients. C. curvatus and C. laurentii are among these species [3, 6, 17]. While C. laurentii has already been associated with bloodstream, neuromeningeal, pulmonary, cutaneous, and peritoneal infections [3, 4, 18,19,20], C. curvatus has very rarely been implicated in human infections, mainly in peritoneal and myeloradicular infections [5,6,7].
Of the 29 Cryptococcus spp. meningitis patients with positive culture included in the present study, six (20.7%, 95% CI 6.9–34.5%) had non-neoformans and non-gattii Cryptococcus species, including five cases of C. curvatus and one case of C. laurentii. To our best knowledge, this study is the first to compare the biological and clinical characteristics of Cryptococcus spp. meningitis induced by these two species groups in the sub-Saharan African region. Globally, 79.3% were identified as Cn and 20.7% as Cc/Cl. A similar prevalence of non-neoformans and non-gattii Cryptococcus infections was reported in three states of the United States of America (USA) in 2020 and during the same study, C. laurentii, C. liquefaciens, and C. magnus were considered as pathogenic .
Patients with Cn were most susceptible to headache on admission than patients with Cc/Cl and most neuromeningeal signs were also found preferentially in Cn patients. The distribution of these signs could suggest a more virulent trait of Cn strains compared to Cc/Cl strains. The main virulence factors of the Cryptococcus genus i.e. presence of the polysaccharide capsule, resistance to high temperatures (> 37 °C), and the activity of the laccase enzyme, have been identified for the Cn complex as well as for the non-neoformans and non-gattii Cryptococcus species. However, the laccase enzyme activity was described as lower for non-neoformans and non-gattii Cryptococcus species compared to that of the Cn . This could explain the more severe clinical presentation of the Cn group patients compared to Cc/Cl group, as described also by Cano et al. .
As mentioned above, most species of the genus Cryptococcus possess a polysaccharide capsule and share the same antigenic determinants with minor differences, and are therefore likely to be detected by India ink staining and/or have antigenic activity detectable by diagnostic tests . Nevertheless, identification of Cryptococcus species still requires strain culture and PCR assays. As described in the literature , the positivity rate of India ink detection in the present study was higher in the Cn group than in the Cc/Cl group, similarly for CSF detection of cryptococcal antigens which had a much higher positivity rate in the Cn group. The partial expression of some virulence enzymatic factors in non-neoformans and non-gattii Cryptococcus species as described by K. Ferreira-Paim et al., and mentioned earlier in this manuscript could explain the low reactivity of Cc/Cl capsular antigens to the CrAg assay, which was developed for the detection of Cn/Cg species complex antigens. Given the increase in meningitis cases due to non-neoformans and non-gattii species worldwide, it is useful that the reactivity of these species to commercially available CrAg assays be established in subsequent larger studies .
For culture-positive samples with negative CrAg results, the antigen excess zone (prozone phenomenon) may be the explanation. Dilution of the tested sample could have resolved this discordance.
While Cn strains were easily identified by MALDI-TOF MS, ITS sequencing, and multiplex PCR serotyping used during the study, only four Cc/Cl strains (out of six) were identified by MALDI-TOF MS and all of them had a different profile from the reference agarose gel profile after multiplex PCR. The results of the MALDI-TOF MS identification were only 80% conclusive for the Cc/Cl strains. In the remaining cases, it was either an identification with a bad score or an outright wrong identification. As the spectrum generated during sample analysis is compared with the spectra in the manufacturer’s database to establish a match, a limited panel of spectra or the absence of spectra of a microbial species in the database could result in a failed species identification. As both species are present in the database used, BD 8326 Bruker, an extraction process using ethanol, formic acid, and acetonitrile might have been necessary to improve the results because of the “big shell” of Cryptococcus spp. The non-neoformans/non-gattii shell could be more refractory than that of Cn. For its part, the determination of serotypes of Cc/Cl strains by classical multiplex PCR targeting LAC1 and CAP64 genes was not possible. Based on its initial application, this PCR was designed to characterize strains of the Cn/Cg species complex. Given the superiority of the laccase enzyme activity of the Cn/Cg complex over that of non-neoformans and non-gattii species as described above, these results could be partially explained .
Although some peritonitis due to non-neoformans and non-gattii Cryptococcus species has been cured by early removal of the catheter without antifungal treatment, others require more intensive treatment because of the fluconazole and 5-flucytosine resistance associated with these isolates [3, 25].
In the present study, 33.3% of the Cc/Cl strains had moderately high MICs to fluconazole (16–32 mg/L) compared to 8.7% in the Cn group. The proportion of Cc/Cl strains resistant to 5-flucytosine and fluconazole is evaluated between 50 and 100% in other studies and higher than in Cn/Cg complex, which is more marked than what we could observe [4, 7, 20]. One strain (16.7%) of the Cc/Cl group, C. laurentii, was resistant to amphotericin B which is known as the most effective antifungal agent in the management of Cryptococcus infections by all species [3, 26]. This same strain was also resistant to 5-flucytosine (16.7%).
All patients with Cn meningitis received antifungal agents according to WHO recommendations and guidelines, MSF protocol . In contrast, one patient in the Cc/Cl group was treated with fluconazole alone although the strain was susceptible in vitro. Despite this, the patients’ therapeutic outcome was not significantly different in the two groups. Patients were selected in MSF-supported clinics where the whole management process is codified. Thus, patients for whom the Cryptococcus identification was provided by the study a few weeks after sample collection were not treated based on this identification. Consequently, 4.3% of patients in the Cn group versus 33.3% of patients in the Cc/Cl group were not treated with antifungals. The mean length of hospital stay for patients was 12.4 versus 9.9 days with the usual tri-antifungals, Cc/Cl, and Cn groups respectively. Longer hospital stays (60 days) have been described for C. albicans meningitis on amphotericin B .