Horse: a potential source of Cryptococcus neoformans and Cryptococcus gattii in Egypt.

BACKGROUND: Cryptococcosis is an opportunistic mycozoonosis of global significance in a wide variety of host species. In equines, cryptococcosis is uncommon, and sporadic cases have been reported with rhinitis, sinusitis, pneumonia, and meningitis. Cryptococcus spp. represents a potential risk for immunosuppressed and healthy persons. In Egypt, epidemiological data on cryptococcal infection in horses are limited. The current study was carried out to investigate the occurrence of Cryptococcus spp. in horses and its possible role in the epidemiology of such disease in Egypt. A total of 223 samples was collected from different localities in Egypt included 183 nasal swabs from horses, 28 nasal swabs from humans, and 12 soil samples. Bacteriological examination and the identification of Cryptococcus spp. were performed. Molecular serotyping of Cryptococcus spp. was determined by multiplex PCR using CNa-70S/A-CNb-49S/A. The virulence genes (LAC1, CAP59, and PLB1) of the identified isolates were detected by PCR. Moreover, sequencing and phylogenetic analysis of the C. gattii gene from horses, humans, and soil isolates found nearby were performed. RESULT: The overall occurrence of Cryptococcus spp. in horses were 9.3, 25, and 10.7% in horses, the soil, and humans, respectively. Molecular serotyping of the Cryptococcus spp. isolates recovered from the nasal passages of horses proved that C. gattii (B), C. neoformans, and two hybrids between C. neoformans (A) and C. gattii (B) were identified. Meanwhile, in case of soil samples, the isolates were identified as C. gattii (B). The human isolates were serotyped as C. gattii in two isolates and C. neoformans in only one isolate. Molecular detection of some virulence genes (LAC1), (CAP59), and (PLB1) were identified in both C. gattii and C. neoformans isolates. The C. gattii gene amplicons of the isolates from horses, humans, and the soil were closely related. CONCLUSION: This study provides the first insights into the Egyptian horse ecology of Cryptococcus species and highlights the role of horses as asymptomatic carriers in disseminating the potentially pathogenic Cryptococcus spp. It also presents the possible risk of cryptococcosis infection in humans.

Cryptococcosis.

Cryptococcosis is an invasive fungal infection of global significance caused by yeasts of the genus Cryptococcus. The prevalence of HIV in certain areas of the world and the expanding population of immunocompromised patients contribute to the ongoing global disease burden. Point-of-care serologic testing has allowed for more rapid diagnosis and implementation of screening programs in resource-limited settings. Management involves therapy aimed at reduction in fungal burden, maintenance of intracranial pressure, and optimization of host immunity. Despite diagnostic and therapeutic advances, cryptococcosis continues to be a disease with unacceptably high incidence and mortality, particularly in resource-limited settings.

Hearing impairment as an unusual presenting sign of Cryptococcus gattii meningoencephalitis.

Hearing loss is an unusual presenting feature of Cryptococcus gattii meningoencephalitis. Two cases of HIV-negative patients who presented with hearing loss are discussed and a literature review of published cases was conducted. Possible mechanisms for hearing loss with C. gattii infection are explored. This case series aims to raise awareness among clinicians that hearing loss can be a concerning feature in patients with persistent headache necessitating further investigation.

Development and Application of Rapid Clinical Visualization Molecular Diagnostic Technology for Cryptococcus neoformans/C. gattii Based on Recombinase Polymerase Amplification Combined With a Lateral Flow Strip.

Cryptococcus neoformans (C. neoformans)/C. gattii can easily invade the human central nervous system and cause cryptococcal meningitis (CM). The clinical fatality rate of these fungi is extremely high and causes more than 180,000 deaths worldwide every year. At present, the common clinical identification methods of these fungi are traditional culture methods and Indian ink staining. In addition, enzyme-linked immunosorbent assay (ELISAs), polymerase chain reaction (PCR), real-time quantitative PCR detecting system (qPCR), mass spectrometry, and metagenomic next-generation sequencing (mNGS) have also been applied to detect these fungus. Due to the rapid progress of meningitis caused by C. neoformans/C. gattii infection, there is a desperate need for fast, sensitive, and on-site detection methods to meet the clinical diagnosis. Recombinase polymerase amplification (RPA) is a promising isothermal amplification technique that can compensate for the shortcomings of the above techniques, featuring short reaction time, high specificity, and high sensitivity, thus meeting the demand for in-field detection of C.neoformans/C. gattii. In our study, RPA- lateral flow strip (LFS) was used to amplify the capsule-associated gene, CAP64, of C. neoformans/C. gattii, and the primer-probe design was optimized by introducing base mismatches to obtain a specific and sensitive primer-probe combination for clinical testing, and specificity of the detection system was determined for 26 common clinical pathogens. This system was developed to obtain results in 20 min at an isothermal temperature of 37°C with a lower limit of detection as low as 10 CFU/µL or 1 fg/µL. A total of 487 clinical samples collected from multicenter multiplexes were tested to evaluate the detection performance of the RPA-LFS system, which revealed that the system could specifically detect C. neoformans/C. gattii, meeting the need for rapid, specific, and sensitive detection.

Sequential infection of Epstein-Barr virus and cryptococcal encephalitis after umbilical cord blood transplantation in a child with X-linked adrenoleukodystrophy.

Dual infection with two pathogens can be found in few cases of encephalitis. Cases of sequential infection with EBV and cryptococcal encephalitis in post-transplant patients are rare. We describe a 5-year-old boy with X-linked adrenoleukodystrophy who presented sequential infection with EBV and cryptococcal encephalitis after umbilical cord blood transplant. The patient showed fever, vomiting and emotional agitation with EBV DNA detected in CSF on day 100. The child underwent 3 doses of intravenous rituximab with a good response. However, the child presented with right facial paralysis, headache, and fever on day 130 after 2 weeks of clinical stability. Brain MRI demonstrated chronic granuloma formed with ring enhancement. FilmArray ME PCR confirmed the existence of Cryptococcus neoformans/gattii in the CSF. The child underwent sequential treatment with amphotericin liposome B and flucytosine. Maintenance treatment with fluconazole was administered for 1 year. Facial paralysis was on longer present on day 260. Cryptococcus neoformans/gattii was not detected on day 310. The biochemistry and cell count of the CSF were completely normal on day 520. Follow-up 2.5 years after presentation, brain MRI changes showed near complete resolution of the lesions. The child survived for 3 years to the last following-up. Invasive cryptococcal encephalitis is rare and life-threatening complication of transplantation. It is important to recognize dual infections, and perform treatment quickly to improve the prognosis of encephalitis after transplantation.

Two cases of cryptococcus gattii infection in the rhesus macaque (Macaca Mulatta).

Cryptococcus gattii was diagnosed in two female indoor-housed rhesus macaques. Gross and histopathologic findings included an isolated pulmonary cryptococcoma in a non-SIV-infected macaque and disseminated disease centered on the lungs of an SIV-infected macaque. Fungal yeast were positive with special stains, and the diagnoses were confirmed with a lateral flow assay and PCR.

A Rare Case of Cryptococcus gattii Meningitis in Advanced HIV Disease, Sagittal Thrombosis, and Immune Reconstitution Syndrome, Resolved With Isavuconazonium.

Cryptococcus gattii is a species that has received more recognition in the recent past as distinct from Cryptococcus neoformans. C gattii is known to cause meningeal disease in both immunocompetent and immunosuppressed hosts. Patients may be clinically asymptomatic until immunosuppressive conditions occur such as corticosteroid treatment or an HIV infection. HIV-associated cryptococcal infections are most often due to C neoformans. C gattii is found in a minority. Speciation and subtyping of Cryptococcus are not always accomplished. In many parts of the world, there is no availability for speciation of Cryptococcus. Travel history may provide a clue to the most probable species. This case demonstrates a case of C gattii meningitis with a multiplicity of complications. These include advanced HIV disease secondary to nonadherence, immune reconstitution inflammatory syndrome, and superior sagittal sinus thrombosis. The patient represented diagnostic and therapeutic dilemmas over time. Headache was the primary symptom in cryptococcal meningitis, immune reconstitution inflammatory syndrome, and superior sagittal sinus thrombosis. All are discussed in detail as potential etiologies for the primary disease. Isavuconazonium is a relatively new broad-spectrum antifungal azole that was used as salvage therapy.

Cryptococcal infection in haematologic malignancies and haematopoietic stem cell transplantation.

This review summarises both the recent and relevant studies about cryptococcal infections in haematologic malignancies and haematopoietic stem cell transplantation. Although uncommon in this patient population, this infection carries a high mortality, especially if left untreated. Given the limited data, we draw some conclusions with respect to management from the solid organ transplantation and HIV-infected literature. Herein, we discuss cryptococcosis with a particular attention to its background, epidemiology, risk factors, clinical presentation, diagnosis, treatment and prevention in this group.

A novel method for identifying and distinguishing Cryptococcus neoformans and Cryptococcus gattii by surface-enhanced Raman scattering using positively charged silver nanoparticles.

There are approximately 1 million cryptococcal infections per year among HIV+ individuals, resulting in nearly 625,000 deaths. Cryptococcus neoformans and Cryptococcus gattii are the two most common species that cause human cryptococcosis. These two species of Cryptococcus have differences in pathogenicity, diagnosis, and treatment. Cryptococcal infections are usually difficult to identify because of their slow growth in vitro. In addition, the long detection cycle of Cryptococcus in clinical specimens makes the diagnosis of Cryptococcal infections difficult. Here, we used positively charged silver nanoparticles (AgNPs+) as a substrate to distinguish between C. neoformans and C. gattii in clinical specimens directly via surface-enhanced Raman scattering (SERS) and spectral analysis. The AgNPs+ self-assembled on the surface of the fungal cell wall via electrostatic aggregation, leading to enhanced SERS signals that were better than the standard substrate negatively charged silver nanoparticles (AgNPs). The SERS spectra could also be used as a sample database in the multivariate analysis via orthogonal partial least-squares discriminant analysis. This novel SERS detection method can clearly distinguish between the two Cryptococcus species using principal component analysis. The accuracy of the training data and test data was 100% after a tenfold crossover validation.

Landmark clinical observations and immunopathogenesis pathways linked to HIV and Cryptococcus fatal central nervous system co-infection.

Cryptococcal meningitis remains one of the leading causes of death among HIV-infected adults in the fourth decade of HIV era in sub-Saharan Africa, contributing to 10%-20% of global HIV-related deaths. Despite widespread use and early induction of ART among HIV-infected adults, incidence of cryptococcosis remains significant in those with advanced HIV disease. Cryptococcus species that causes fatal infection follows systemic spread from initial environmental acquired infection in lungs to antigenaemia and fungaemia in circulation prior to establishment of often fatal disease, cryptococcal meningitis in the CNS. Cryptococcus person-to-person transmission is uncommon, and deaths related to blood infection without CNS involvement are rare. Keen to the persistent high mortality associated with HIV-cryptococcal meningitis, seizures are common among a third of the patients, altered mental status is frequent, anaemia is prevalent with ensuing brain hypoxia and at autopsy, brain fibrosis and infarction are evident. In addition, fungal burden is 3-to-4-fold higher in those with seizures. And high immune activation together with exacerbated inflammation and elevated PD-1/PD-L immune checkpoint expression is immunomodulated phenotypes elevated in CSF relative to blood. Lastly, though multiple Cryptococcus species cause disease in this setting, observations are mostly generalised to cryptococcal infection/meningitis or regional dominant species (C neoformans or gattii complex) that may limit our understanding of interspecies differences in infection, progression, treatment or recovery outcome. Together, these factors and underlying mechanisms are hypotheses generating for research to find targets to prevent infection or adequate therapy to prevent persistent high mortality with current optimal therapy.

High genetic variability of clinical and environmental Cryptococcus gattii isolates from Brazil.

Among Cryptococcus gattii genotypes, VGII has gained pivotal relevance in epidemiological, clinical and genetic contexts due to its association with several outbreaks in temperate regions and due to the high variability of this genotype. The aim of this study was to compare 25 isolates of C. gattii from the Southeast region of Brazil with previously described isolates from other regions of the country and around the world. Among the 25 isolates, 24 were VGII and one was VGI. All of them were newly identified. Three new allele types (AT) (AT47 for the URA5 locus, AT56 for the LAC1 locus, and AT96 for the IGS1 region) were also described. Compared with other Brazilian isolates, those from the Southeast region presented the greatest haplotype diversity. In general, the regions presented different sequence types (STs), and only nine STs were found in more than one location. GoeBURST analysis showed two large groups among the Brazilian isolates. The largest group consists of 59 STs predominantly from the North and Northeast regions; the other large group includes 57 STs from the Southeast and Midwest regions. In a global context the South American isolates presented the highest genetic diversity (STs = 145, haplotype diversity (Hd) = 0.999 and π = 0.00464), while the African populations showed the lowest genetic diversity (STs = 3, Hd = 0.667 and π = 0.00225). These results confirm that the Brazilian C. gattii VGII population is highly diverse and reinforce the hypothesis of dispersion of this genotype from South America.

Clinical and microbiological characteristics of Cryptococcus gattii isolated from 7 hospitals in China.

BACKGROUND: Infection, even outbreak, caused by Cryptococcus gattii (C. gattii) has been reported in Canada and the United States, but there were sparsely-reported cases of C. gattii in China. Our interest in occurrence, clinical manifestation, laboratory identification and molecular characterization of Chinese C. gattii strains leads us to this research. RESULTS: Out of 254 clinical isolates, initially identified as Cryptococcus neoformans (C. neoformans), eight strains were re-identified as C. gattii. Multi-locus sequence typing (MLST) showed genotype VGI accounted for the most (6 / 8), the other two strains were genotype VGII (VGIIa and VGIIb respectively) with 3 specific spectra of molecular weight about 4342, 8686, 9611 Da by MALDI-TOF MS. The minimal inhibitory concentrations (MICs) of Fluconazole with Yeast one was 2~4 times higher than that with ATB fungus 3 and MICs of antifungal agents against VGII strains were higher than against VGI strains. Comparative proteome analysis showed that 329 and 180 proteins were highly expressed by C. gattii VGI and VGII respectively. The enrichment of differentially expressed proteins was directed to Golgi complex. CONCLUSIONS: Infection by C. gattii in China occurred sparsely. Genotype VGI was predominant but VGII was more resistant to antifungal agents. There was significant difference in protein expression profile between isolates of VGI and VGII C. gattii.

Genotypic and Phenotypic Stability of Mixed Primary Isolates of Cryptococcus gattii and Cryptococcus neoformans: A Comparative Analysis of Four Preservation Methods.

The choice of a suitable preservation method is critical for long-term microorganisms' viability. The strains should be preserved for long periods using reliable and reproducible methods that minimize genotypic and phenotypic variations and viability losses. The methodologies are usually designed for a better performance in isolated microorganisms. However, atypical primary isolates of Cryptococcus neoformans or Cryptococcus gattii, such as mixed species or even different species of a species complex, are a challenge for long-term preservation and taxonomic review studies. The aim of this study was to evaluate which of the four preservation methods tested presented better performance in the preservation of simulated coexistence strains of C. neoformans and C. gattii. Two environmental strains, one C. gattii and one C. neoformans, were mixed in vitro to test four different preservation methods (freezing at -20°C, -80°C, -196°C, and freeze-drying). The colony-forming units from each preservation method were evaluated, and colonies were randomly selected and cultivated in canavanine glycine bromothymol blue (CGB) agar to evaluate the amounts of CGB-positive (C. gattii) and CGB-negative (C. neoformans) colonies resulting from each preservation method after 1 week, 15 days, 1 month, 6 months, and 1 year. According to our results, cryopreservation at -20°C demonstrated was preferable for C. neoformans species, and further studies after long-term storage are necessary. Recovery of yeast cells after freeze-dried preservation in skim milk is better for both species. Ultrafreezing methods evaluated (-80°C and -196°C) also showed better results in the maintenance of C. gattii. Freeze-drying should be preferred for the maintenance of multilineage isolates from the C. neoformans and C. gattii species complexes.

Clinical and microbiological characteristics of cryptococcosis at an university hospital in China from 2013 to 2017

ABSTRACT Background: This study aims to explore the epidemiology, clinical profile and strain characteristics of cryptococcosis from 2013 to 2017 in a major teaching hospital in China. Methods: Trends in antifungal drug susceptibility of 217 consecutive non-repetitive cryptococcal isolates collected from patients of an university hospital in China were analyzed between 2013 and 2017. Of those, 98 isolates were conserved for identification by internal transcribed spacer (ITS) sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) system. Multilocus sequence typing (MLST) was used to designate molecular types. Clinical characteristics of the 98 patients with cryptococcosis during the period of 2013-2017 were retrospectively evaluated. Results: There was a trend for gradual increase in the MIC range of fluconazole was from 2013 to 2017. The conserved 98 clinical cryptococcal isolates included 97 C. neoformans and one C. gattii, and 90 (91.8%) isolates belonged to ST5 genotype VNI. Out of the 98 patients with cryptococcosis, 28 (28.6%) were HIV-infected and 32 (32.7%) had no underlying diseases. HIV-infected patients had higher mortality than HIV-uninfected patients (28.6% vs 14.3%, p = 0.147). Conclusions: Most of the patients with cryptococcosis were not HIV-infected in this study, while patients with HIV had a higher mortality. Reduced susceptibility to fluconazole was observed among C. neoformans isolates, most of them belonged to ST5 genotype VNI having an impact on the effective dose of fluconazole.

Evaluation of the Biofire FilmArray meningitis/encephalitis assay for the detection of Cryptococcus neoformans/gattii.

OBJECTIVES: Cryptococcal meningitis (CM) remains an important cause of morbidity and mortality among immunocompromised patients. Laboratory diagnostics for CM includes antigen detection, staining and culture. Data on the performance of the Biofire® FilmArray® meningitis/encephalitis (ME) panel for detecting Cryptococcus neoformans/gattii is limited, with several reports describing false negativity for this target. METHODS: A retrospective analysis of 1384 physician-ordered ME panel tests ordered between January 2017 to October 2018 was performed. ME panel results were compared to cerebrospinal fluid (CSF) cryptococcal antigen (CrAg) and CSF culture testing and clinical significance of cryptococcal detection was determined. RESULTS: There were 34 patients positive for cryptococcal detection by either ME panel, CSF CrAg or CSF culture in 2.7% of CSF specimens tested (38/1384). Of the 34 patients positive for cryptococcal detection, 85.3% were human immunodeficiency virus positive (29/34). The ME panel detected 32/38 (84.2%) cryptococcal-positive specimens, culture detected 28/38 (73.7%) and CSF CrAg was positive in 37/38 specimens (97.4%). The ME panel had a sensitivity and specificity of 96.4% (95% CI 81.7-99.9%) and 99.6% (95% CI 99.2-99.9%) compared with culture, and 83.8% (95% CI 68.0-93.8%) and 99.9% (95% CI 99.6-100.0%) compared to CSF CrAg testing, respectively. CrAg titres were lower among ME panel-negative, culture-negative specimens compared with ME panel-positive, culture-negative specimens (reciprocal median end-point titres of 128 ± 60 vs. 1920 ± 1730, p 0.04). All five CrAg-positive, ME panel- and culture-negative specimens were obtained from previously treated CM patients. DISCUSSION: The ME panel had high correlation with CSF culture and a somewhat lower correlation with CSF CrAg testing. The potential utility of using negative ME panel test results to predict culture sterility among patients undergoing treatment for CM warrants further study.

Unusual Presentation of Severe Endobronchial Obstruction Caused by Cryptococcus gattii in a Child.

Disease caused by Cryptococcus gattii typically manifests as meningoencephalitis or pulmonary nodules. Endobronchial lesions are rare, and most cases are caused by Cryptococcus neoformans. We describe here a case of endobronchial disease in a child caused by C gattii. The disease spectrum in this patient was notable for the discovery of anti-granulocyte macrophage colony-stimulating factor autoantibodies.

Clinical insights and epidemiology of central nervous system infection due to Cryptococcus neoformans/gattii species complexes: A prospective study from South India.

In the last two decades, central nervous system (CNS) cryptococcosis (CNSc) has emerged as a major opportunistic infection in the immunocompromised population of India. We have analyzed the clinical features of CNSc and epidemiology of Cryptococcus neoformans and Cryptococcus gattii. A total of 160 clinical isolates of C. neoformans/gattii recovered from CNSc patients were analyzed. The origin, clinical parameters, and imaging features of the patients were recorded, and clinical parameters were analyzed based on their human immunodeficiency virus (HIV) status and infecting species, namely, C. neoformans or C. gattii. Serotypes and mating types of the isolates were determined. Molecular typing was performed by polymerase chain reaction (PCR) fingerprinting using M13 microsatellite primer (GTG)5, and multilocus sequence typing (MLST). Majority of the patients were from Bangalore Urban, Karnataka. Among 160 cases 128 (80%) were HIV seropositive, and 32 (20%) were HIV negative. Middle-aged males (36-55 years) were highly affected. There were statistically significant differences in the clinical manifestations, imaging and CSF parameters of HIV coinfected and noninfected cases, whereas limited differences were observed in these parameters in the cases infected with C. neoformans and C. gattii. We identified 80% C. neoformans VNI, 8.75% VNII and 22.5% C. gattii (VGI), 8.75% C. tetragattii (VGIV) among clinical strains. This comprehensive study will contribute toward a better prognosis of CNS cryptococcosis patients during the hospital stay, treatment strategies for HIV coinfected and noninfected cases and will provide the molecular epidemiology of these two pathogenic fungal species in south India, which was unclear in this part of the country.

Microbiological and clinical characteristics of cryptococcemia: a retrospective analysis of 85 cases in a Chinese hospital.

Cryptococcemia is a life-threatening fungal infection. Sometimes, it is hard to diagnose. The studies to describe the characteristics of cryptococcemia specifically were limited. We performed this retrospective analysis in a Chinese hospital during 2002-2015, including 85 cryptococcemia cases and 52 Cryptococcus spp. isolates. The species, mating type, antifungal susceptibility and multilocus sequence typing of Cryptococcus spp. were determined. C. neoformans var. grubii MATα of sequence type (ST) 5 is the representative strain of cryptococcemia, accounting for 51 isolates. The MIC50/90 values were 0.5/0.5, 1.0/1.0, 2.0/4.0, ≤0.06/0.25, and ≤0.06/≤0.06 µg/ml for amphotericin B, flucytosine, fluconazole, itraconazole, and voriconazole, respectively. Cryptococcemia was the first diagnostic proof of cryptococcosis in 37 patients (43.5%, 37/85). Compared with the patients initially diagnosed of cryptococcosis in other sites (mainly cerebrospinal fluid), the patients firstly diagnosed by blood culture had prolonged time from admission to diagnosis of cryptococcosis (9 days vs. 2 days, P < .001) and higher 30-day mortality (54.1% vs. 20.8%, P = .003), while fewer symptoms of meningitis (45.9% vs. 100%, P < .001). For the patients receiving lumbar puncture, the occurrence of meningitis was similar between the patients firstly diagnosed by blood culture and those firstly diagnosed in other sites (94.1% vs. 100%, P = .26). However, the patients first diagnosed by blood culture had lower baseline intracranial pressure (250 mm H2O vs. 342.5 mm H2O, P = .001). In conclusion, patients with cryptococcemia as the first diagnostic proof of cryptococcosis usually had neglected subtle symptoms of meningitis, which may result in delayed diagnosis and catastrophic outcome.