P349 Relationship between genotypes and in vitro pathogenic behavior of Uruguayan strains of Cryptococcus spp.

Abstract   Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM Objective Contribute to the knowledge of the relationship between genetic and pathogenic characteristics of meningeal cryptococcosis-producing strains from Uruguay. Methods We have included a total of 21 isolates of Cryptococcus spp from human cases of meningoencephalitis. We start from the general strain collection of the mycology laboratory of the department of parasitology and mycology, medical school, University of the Republic. The strain was sectioned taking into account the phenotypic diversity and genetic diversity through a previous analysis by PCR fingerprinting, for study by MLST. MLST was performed according to the protocol available at: http://mlst.mycologylab.org/defaultinfo.aspx?Page=CryptoHome. Infection, intracellular survival, and cell proliferation assays were performed. The murine macrophage cell line J774 (ATCC TIB 67) was used. Results Of the 21 strains studied, in 13 we obtained an ST that had been previously reported (9 C. neoformans complex and 4 C. gattii complex). For the remaining 8 strains, we obtained new allelic combinations to which new STs were assigned, which were deposited in the MLST database for Cryptococcus spp. (http://mlst.mycologylab.org) (Table 1). All strains were phagocytosed and all were able to persist within macrophages at 24 h post-infection, although there was great variability between strains (Table 1). In most of the strains, the control of macrophages prevailed through cell lysis mechanisms over Cryptococcus spp, with one proliferation index (PI) ˂1.0. In the proliferation assay, only 4 strains managed to proliferate within the macrophages, 3 of them correspond to C. gattii complex, genotypes VGI and VGII. The 4th strain that achieved proliferation was corresponding to the VNI genotype. Conclusion We obtained a clear differentiation of in vitro behavior between the strains corresponding to C. neoformans complex and C. gattii complex. The strains belonging to C. gattii complex presented less phagocytosis and greater persistence and intracellular proliferation.

Objectives: Candida species are known to colonize human skins and mucous membranes, and cause candidemia under various immunosuppressive conditions. Among these Candida species, C. albicans is reported to be the most frequently isolated species, and could colonize on skins, vaginas, gastrointestinal tracts, and medical devices. Environmental factors including oxygen concentration is thought to affect the capability of colonization and virulence of Candida species; however, most previous research was performed under aerobic condition, and few research focused on hypoxic conditions imitating inside the human bodies. Given these backgrounds, we performed murine experiments by culturing C. albicans under various oxygen conditions to evaluate the effect of environmental oxygen concentration on virulence. Through our studies, we aimed to clarify the actual behavior of C. albicans in the human body.
Methods: In this study, female C57BL/6 mice, 7-8 weeks old, were used and injected via lateral tail vein to cause C. albicans dissemination. Mice were divided into 4 groups according to the pre-culture conditions: aerobic, microaerobic ( 5% oxygen concentration ) , microaerobic ( 1% oxygen concentration ) , and anaerobic . Under each oxygen condition, C. albicans was grown at 37°C for 2 days on yeast extract peptone dextrose ( YPD ) agar and then incubated in YPD broth for 16-24 h. After incubation, C. albicans was collected, washed, resuspended in sterile PBS, and injected into each mouse at ∼2.5 × 105 colony-forming units. In this study, one reference strain ( SC5314 ) and one clinically isolated strain ( from bloodstream infection ) were used. These infected mice were euthanatized 2 or 4 days after injection, and organ ( kidneys and brain ) fungal burdens were evaluated.
Results: The kidneys' fungal burdens were significantly higher in the microaerobic groups than those in the aerobic or anaerobic groups 2 and 4 days after injection. There were no significant differences between 5% oxygen concentration precultured group and 1% oxygen concentration pre-cultured group. On gross examination, disseminated lesion formations were visible in the kidneys of the microaerobic groups. Similarly, the fungal burdens of brain were significantly higher in the microaerobically pre-cultured groups than in the aerobically or anaerobically pre-cultured groups. This tendency was similar for both the reference and clinically isolated strain.
Conclusion: Our results indicated that C. albicans could become more virulent under hypoxia, especially under microaerobic conditions. We assumed that some virulence factors of C. albicans were elevated under microaerobic conditions. These results implicated that C. albicans could become more virulent under hypoxic conditions in human bodies. In the future, we will continue to evaluate factors related to this change in virulence and pathological analysis of infected organs.

P346 Pulmonary fungal infection in Sudan, a retrospective study from the Mycology Reference Laboratory
Mawahib Ismail 1,2 , Aida Gabr 1,2 , Shaoqin Zhou 3 , Elsheikh Mahgoub 1 , Sarah Ahmed 3,4 1 Mycology Reference Laboratory, Khartoum, Sudan, Khartoum, Sudan 2 National Laboratory for Public Health, Sudan 3 RadboudUMC, Netherlands 4 Foundation Atlas of Clinical Fungi, Netherlands Poster session 3, September 23, 2022, 12:30 PM -1:30 PM Objectives: Pulmonary fungal infections are life-threatening diseases, if not diagnosed and properly treated can lead to serious complications. In chronic cases, the condition might mimic tuberculosis and may be misdiagnosed. The aim of this retrospective study is to determine the frequency of fungi among the respiratory samples received at the mycology reference laboratory over 5 years period and to provide a view of the burden of pulmonary fungal diseases in the country.
Methods: A total of 713 sputum samples received at the Mycology Reference Laboratory, Khartoum, Sudan, between 2015-2019 were analyzed. These samples were collected from different health care centers in Khartoum state. For every sample, direct microscopy using 20% KOH and methylene blue stain was performed. In addition, cultures were made by inoculating every sample in three tubes of Sabouraud dextrose gar ( SDA ) containing chloramphenicol. Tubes were incubated at 37°C for 2 and up to 7 days. Isolated fungi were identified phenotypically using the Atlas of Clinical Fungi guidelines.
Results: Out of the 713 samples, 235 ( 33% ) were positive for fungi; both in the direct microscopy and culture. Cultures were identified as Aspergillus species 38 ( 16% ) , while 197 ( 84% ) were found to represent Candida species.
Conclusion: Our study showed a high number of fungi is associated with pulmonary conditions in Sudan. Risk factors might include post tuberculosis, Asthma, HIV, and COPD.

P348 tRNA modifications in Candida virulence
Bettina Böttcher 2,1 , Sebastian Leidel 3 , Sascha Brunke 1 1 Hans Knoell Institute, Jena, Germany 2 Septomics Research Center, Jena, Germany 3 University of Bern, Bern, Switzerland Poster session 3, September 23, 2022, 12:30 PM -1:30 PM Candida albicans is a normal commensal of the gastrointestinal tract, but also an important opportunistic pathogen that can cause life-threatening infections. An important pathogenicity factor of C. albicans is its ability to form hyphae, which are associated with the expression of many different virulence factors. The regulation of hypha formation has been analyzed extensively on the transcriptional and also post-translational level in the past.
Candida albicans has a closely related sister species, C. dubliniensis , which has a lower propensity to form hyphae and only very rarely causes more than superficial infections. We investigated the differences in the genetic setup of these two species and found a gene whose Saccharomyces cerevisiae homolog is involved in tRNA modifications.
We here show that this gene, Hma1, acts as a tRNA-threonyl carbamoyl adenosine dehydratase in C. albicans , and that its activity differs between C. albicans and C. dubliniensis . A deletion mutant shows specific defects in hyphae formation, invasion of host cells, and in virulence in C. albicans . Furthermore, we find that the loss of this tRNA-modifying enzyme leads to changes in ribosome occupancy at 37°C, the temperature encountered in the host.
With these data we link tRNA modifications to host-induced processes of one of the most important fungal pathogens of humans. We thereby add another layer of regulation, between transcription and post-translational modifications, to the virulence program of Candida yeasts.

Relationship between genotypes and in vitro pathogenic behavior of Uruguayan strains of Cryptococcus spp.
Elisa Cabeza 1,2 , Zaida Arteta 1,2 , Laura Betancor 3  Methods: We have included a total of 21 isolates of Cryptococcus spp from human cases of meningoencephalitis. We start from the general strain collection of the mycology laboratory of the department of parasitology and mycology, medical school, University of the Republic . The strain was sectioned taking into account the phenotypic diversity and genetic diversity through a previous analysis by PCR fingerprinting, for study by MLST.
Infection, intracellular survival, and cell proliferation assays were performed. The murine macrophage cell line J774 ( ATCC TIB 67 ) was used.
Results: Of the 21 strains studied, in 13 we obtained an ST that had been previously reported ( 9 C. neoformans complex and 4 C. gattii complex ) . For the remaining 8 strains, we obtained new allelic combinations to which new STs were assigned, which were deposited in the MLST database for Cryptococcus spp. ( http://mlst.mycologylab.org ) ( Table 1 ) .
All strains were phagocytosed and all were able to persist within macrophages at 24 h post-infection, although there was great variability between strains ( Table 1 ) . In most of the strains, the control of macrophages prevailed through cell lysis mechanisms over Cryptococcus spp, with one proliferation index ( PI ) ˂1.0. In the proliferation assay, only 4 strains managed to proliferate within the macrophages, 3 of them correspond to C. gattii complex, genotypes VGI and VGII. The 4th strain that achieved proliferation was corresponding to the VNI genotype.
Conclusion: We obtained a clear differentiation of in vitro behavior between the strains corresponding to C. neoformans complex and C. gattii complex. The strains belonging to C. gattii complex presented less phagocytosis and greater persistence and intracellular proliferation. Methodology: A hospital-based cross-sectional study was conducted for 6 months in the Department of Microbiology of Sukraraj Tropical and Infectious Disease Hospital, Kathmandu, Teku, Nepal. A total of 408 HIV-positive patients attending the ART center of the hospital of different age and sex were included in the study. Study variables like CD4 + count, tobacco consumption, diabetes, URTI, and recent antibiotic consumption were noted.
Standard microbiological guidelines were followed for the oral swab collection and transportation. Specimen was seeded on SDA agar plates ( SDA with chloramphenicol and cycloheximide ) . Preliminary identification of the Candida was done on the basis of colony characteristics, gram staining, and germ tube test. Further for the speciation of Candida species, HiCrome TM Candida Differential Agar ( HImedia, India ) was applied where on the basis of coloration and colony morphology speciation was done. Similarly, Cornmeal Agar ( Dalmau plate culture technique ) and sugar Fermentation test were applied to confirm the Candida species.
Muller-Hinton agar supplemented with 2% glucose and 0.5 μg/ml methylene blue dye ( GMB ) medium was used to perform the antifungal susceptibility testing of Candida isolates. Antifungal susceptibility testing ( AST ) of isolates was performed and interpreted according to Clinical and Laboratory Standards Institute ( CLSI ) M44-A document. Candida albicans ATCC 90028 was taken as the reference strain. All the data were entered in Microsoft excel and Chi-square was used to find out the association between the frequency of oral candidiasis and study variables.
Conclusion: Candida albicans was the common species isolated in our study. Candida Chrome Agar can be used as a routine media for rapid identification and Candida speciation. AST needs to be done routinely to know the susceptibility pattern of isolates and initiate proper treatment of patients. Since oral yeast colonization was associated with low CD4 + count ( < 200 cells/mm 3 ) . Thus, oral lesions can serve as early markers for HIV infection.