Cryptococcus neoformans,a global threat to human health

Background Emerging fungal pathogens pose important threats to global public health. The World Health Organization has responded to the rising threat of traditionally neglected fungal infections by developing a Fungal Priority Pathogens List (FPPL). Taking the highest-ranked fungal pathogen in the FPPL,Cryptococcus neoformans, as a paradigm, we review progress made over the past two decades on its global burden, its clinical manifestation and management of cryptococcal infection, and its antifungal resistance. The purpose of this review is to drive research efforts to improve future diagnoses, therapies, and interventions associated with fungal infections.Methods We first reviewed trends in the global burden of HIV-associated cryptococcal infection, mainly based on a series of systematic studies. We next conducted scoping reviews in accordance with the guidelines described in the Preferred Reporting Items for Systematic Reviews and Meta-analyses extension for Scoping Reviews using PubMed and ScienceDirect with the keywordCryptococcus neoformans to identify case reports of cryptococcal infections published since 2000. We then reviewed recent updates on the diagnosis and antifungal treatment of cryptococcal infections. Finally, we summarized knowledge regarding the resistance and tolerance ofC. neoformans to approved antifungal drugs.Results There has been a general reduction in the estimated global burden of HIV-associated cryptococcal meningitis since 2009, probably due to improvements in highly active antiretroviral therapies. However, cryptococcal meningitis still accounts for 19% of AIDS-related deaths annually. The incidences of CM in Europe and North America and the Latin America region have increased by approximately two-fold since 2009, while other regions showed either reduced or stable numbers of cases. Unfortunately, diagnostic and treatment options for cryptococcal infections are limited, and emerging antifungal resistance exacerbates the public health burden.Conclusions The rising threat ofC. neoformans is compounded by accumulating evidence for its ability to infect immunocompetent individuals and the emergence of antifungal-resistant variants. Emphasis should be placed on further understanding the mechanisms of pathogenicity and of antifungal resistance and tolerance. The development of novel management strategies through the identification of new drug targets and the discovery and optimization of new and existing diagnostics and therapeutics are key to reducing the health burden.

Microcomputer System for Automatic Identification of the Cryptococcus neoformans and Its Clinical Application

In this study,microcomputer image processing and pattern recognition technology,and the knowledge of morphology and optical characteristics of Cryptococcus neoformans were used for identification of Cryptococcus neoformans.Four groups of mice were lethally infected with standard strain,Wuhan strain,American B-2643 strain and Var.Shanghainesis of the Cryptococcus neoformans.The samplescollected included mice brain,lung,kidney,liver,small intestine tissue and were observed under a light microscope.More than 600 images of the fungus were input into a microcomputer.A system of computer for automatic identification of the Cryptoccocus neoformans was developed. The technique involved image preprocessing,imagesegmenting,coding of line-length on the edge,curve fitting,extracting of image feature,building of image library and feature data bank etc..And then,768 images of the clinical samples and other fungus samples whose morphological features tend to be confused with Cryptococcus neoformans were input into microcomputer and subjected to automatic identification.The Cryptococcus neoformans was accurately identified within 15 min,and the consistence rate with results of routine culture was 98%.

Cryptococcus and Cryptococcosis in Human and Animal Model:An Overview

Background: Yeasts from the genus Cryptococcus (species C. neoformans and C. gattii) are opportunistic pathogens that afflict humans with low immunity caused by diseases such as AIDS or by the usage of immunosuppressive drugs. It is a fatal disease that has been studied around the whole world and its most fatal form is the brain cryptococcosis. Purpose: The present review describes the disease from pathogen isolation, the various clinical presentations of the disease, the most important virulence factors of yeast in human and animal model and their clinical issues. Methods: On this review, several published studies about the disease are presented. Results: Numerous researches have been done worldwide in order to find a kind of therapy that is more effective against the disease. Amphotericin B, in all forms is still the drug of choice in the treatment of the cryptococcosis. Fluconazole, as well as voriconazole in combination with amphotericin B, is recommended in the cases of treatment failure. Conclusion: This study presented has elucidated a little more about the disease. Further studies should be conducted to find more diagnoses that are accurate as well as more effective treatments for eradicating the disease. In this study, the bibliographic survey makes reference to the world literature;with regard to ecology, taxonomy, mains factors related to virulence, the clinical manifestations, the action of antifungal drugs and histopathological analysis used in an animal model, were the objectives deleterious aspects of this study, thus informing, in a simple way, the importance of this microorganism for research and researchers working with this global disease, called Cryptococcosis.

Isolated cryptococcal osteomyelitis of the ulna in an immunocompetent patient:A case report

BACKGROUND Cryptococcal osteomyelitis is a bone infection caused by cryptococcus.As an opportunistic infection,bone cryptococcosis usually occurs in patients with immunodeficiency diseases or in those undergoing immunosuppressive therapy and often displays characteristics of disseminated disease.Isolated cryptococcal osteomyelitis is extremely unusual in immunocompetent person.The pathogenic fungus often invades vertebrae,femur,tibia,rib,clavicle,pelvis,and humerus,but the ulna is a rare target.CASE SUMMARY A 79-year-old woman complaining of chronic pain,skin ulceration and a sinus on her right forearm was admitted,and soon after was diagnosed with cryptococcal osteomyelitis in the right ulna.Unexpectedly,she was also found to have apparently normal immunity.After treatment with antifungal therapy combined with surgery debridement,the patient’s osteomyelitis healed with a satisfactory outcome.CONCLUSION Although rare,cryptococcal osteomyelitis should be considered in the differential diagnosis of osteolytic lesions even in immunocompetent patients,and good outcomes can be expected if early definitive diagnosis and etiological treatment are established.

Pleural involvement in cryptococcal infection

Pleural involvement of cryptococcal infection is uncommon and is more commonly observed in immunocompromised hosts than in immunocompetent ones.Pleural involvement in cryptococcal infections can manifest with or without pleural effusion.The presence of Cryptococcus spp.in the effusion or pleura is required for the diagnosis of cryptococcal pleural infection,which is commonly determined by pleural biopsy,fluid culture,and/or detection of cryptococcal antigen in the pleura or pleural fluid.

Transmission of cryptococcosis by liver transplantation:A case report and review of literature

BACKGROUND Cryptococcosis is a fungal infection caused by the yeast-like encapsulated basidiomycetous fungus of the Cryptococcus neoformans(C.neoformans)species complex.These fungi are ubiquitous in soil and bird droppings,and infection by them is an important global health concern,particularly in immunosuppressed patients,such as organ transplant recipients and those infected by the human immunodeficiency virus.The fungus usually enters the body through the respiratory tract,but extremely rare cases of infection acquired by transplantation of solid organs have been reported.CASE SUMMARY We report a case of disseminated cryptococcosis in a liver transplant recipient,diagnosed 2 wk after the procedure.The patient initially presented with fever,hyponatremia and elevated transaminase levels,manifesting intense headache after a few days.Blood cultures were positive for C.neoformans.Liver biopsy showed numerous fungal elements surrounded by gelatinous matrix and sparse granulomatous formations.Magnetic resonance imaging of the brain showed multiple small lesions with low signal in T2,peripheric enhancement and edematous halo,diffuse through the parenchyma but more concentrated in the subcortical regions.Treatment with amphotericin B for 3 wk,followed by maintenance therapy with fluconazole,led to complete resolution of the symptoms.The recipients of both kidneys from the same donor also developed disseminated cryptococcosis,confirming the transplant as the source of infection.The organ donor lived in a rural area,surrounded by tropical rainforest,and had negative blood cultures prior to organ procurement.CONCLUSION This case highlights the risk of transmission of fungal diseases,specifically of C.neoformans,through liver graft during liver transplantation.

Ecological surveys of the Cryptococcus species complex in China

Background Despite recent reports on the molecular epidemiology of cryptococcal infections in China,clinical isolates have been mostly reported from human immunodeficiency virus （HIV）-negative patients,and environmental isolates from China have rarely been included.The aim of this study was to investigate the ecological profile of Cryptococcus （C.）neoformans and C.gattii in China.Methods A survey was performed in 10 cities from 20°N （North latitude） to 50°N and in a Eucalyptus （E.） camaldulensis forestry farm at the Guixi forestry center,China.Results Six hundred and twenty samples of pigeon droppings from 10 cities and 819 E.camaldulensis tree samples were collected and inoculated on caffeic acid cornmeal agar （CACA）.The brown-colored colonies were recultured to observe their morphology,growth on canavanine-glycine-bromothymol-blue （CGB） medium,phenol oxidase and urease activities,serotype and mating type.There were obvious differences in the positive sample rates of C.neoformans in pigeon droppings collected from the different cities,ranging from 50% in the cities located at latitudes from 30°N-40°N,29% at 20°N-30°N and 13% at 40°N-50°N.Conclusions There were no differences in positive bevy rates （approximately 80%） among the three grouped cities.Mycological tests of 101 isolates purified from pigeon droppings revealed that they were C.neoformans var.grubii.We also observed variable capsular size around the C.neoformans cells in colonies with variable melanin production and the bio-adhesion of the natural C.neoformans cells with other microorganisms.One urease-negative C.neoformans isolatewas isolated from pigeon droppings in Jinan city.No C.gattiiwas isolated in this study.

Taxonomic analysis of cryptococcus species complex strain $8012 revealed Cryptococcus gattii with high heterogeneity on the ~enetics

Background Initially, Cryptococcus （C.） neoformans was previously divided into two varieties comprising C. neoformans var. neoformans and C. neoformans var. gattii. Currently, taxonomic studies defined C. neoformans as C. species complex, which contains C. neoformans var. neoformans （serotype D）, the hybrid isolates （serotype AD）, C. neoformans var. grubfi （serotype A） and C. gattii （serotypes B and C）. However, Liao and his team once isolated a unique C. gattii isolate, namely strain S8012 with unique phenotype from cerebrospinal fluid （CSF） of a 43-year-old male patient in the Shanghai Changzheng Hospital and described as C. neoformans var. shanghaiensis in 1980s. The aim of this study was to explore the genetic background and polymorphism of Chinese clinical C. gattii isolates. Methods S8012 was analyzed as representative strain using the M13-polymerase chain reaction （PC R） fingerprinting pattern and multilocus sequence analysis including internal transcribed spacers of rDNA （ITS region）, the intergenic spacer 1 regions （IGS1）, RPB1, RPB2, CNLACl, and TEF1 genes. Results The PCR fingerprinting pattern results showed strain S8012 belonged to molecular types VGI, and phylogenetic analysis suggested strain S8012 was grouped into the cluster of C. gattii environmental isolates originated from Eucalyptus camaldulensis trees in Australia. Conclusion C. gattii isolates from Chinese patients expresses high polymorphism on the phenotype, and molecular type VGI isolates from China have a close genetic relationship with the C. gattii isolates from Australia.

Advanced approach for antifungal susceptibility and characterization of resistance properties in clinical and environmental isolates of Cryptococcus species complex

Background:Meningitis due to Cryptococcus neoformans/gattii is a fatal infection affecting immunocompromised population worldwide.Amphotericin B(AmB),fluconazole(FLC)and 5-flucytosine are the drugs of choice to treat the infection.We studied antifungal susceptibility pattern of clinical and environmental cryptococcal species using newer approach and analyze their resistant characteristics.Methods:Eighty clinical(54 C.neoformans and 26 C.gattii)and 18 environmental(14 C.neoformans and 4 C.gattii)isolates were subjected to antifungal susceptibility testing by automated(VITEK2C)method.Minimum inhibitory concentrations(MIC)were analyzed statistically.Genomic DNA of FLC resistant isolates was extracted and amplified to detect presence of CnAFR1 gene.Results:C.neoformans showed 1.85%and 21.4%AmB resistance,and 1.85%and 28.5%FLC-resistance,whereas C.gattii showed 25%and 50%FLC-resistance among clinical and environmental isolates respectively.MIC values were significantly(p<0.05)different for the isolates from 2 sources.CnAFR1 gene sequence analysis revealed phylogenetic relationship among the resistant isolates.Conclusions:This pioneering study provides an insight into the sensitivity patterns of clinical and environmental cryptococcal isolates from south India.The recent emergence of AmB-resistance may transpire as a challenge for the clinicians.As the clinical and environmental isolates are phylogenetically evolved from CnAFR1 gene of Filobasidiella neoformans,the resistance is most probably an inherent attribute.This study emphasizes the need for speciation and antifungal susceptibility testing of cryptococcal isolates from clinical sources to institute appropriate antifungal therapy and to reduce the mortality and morbidity.

Construction of a capsule associated protein 10 gene eukaryotic expression vector for RNA interference and confirmation of biologic relevance

Background The capsule associated protein 10 gene （caplO） is indispensible for the formation of the polysaccharide capsule, and is important in maintaining virulence of the Cryptococcus （C.） neoformans. In this study, we aimed to construct an short hairpin RNA （shRNA） expression vector targeting C. neoformans caplO gene expression and confirm its biologic relevance. Methods A pair of oligonucleotides targeting the cap10 cDNA sequence was designed and synthesized. It was cloned into the plasmid psilencer4.1-CMV neo to construct an eukaryotic shRNA expression vector. The vector was transfected into C. neoformans cells using the LiAc method. The expression of cap10 was assessed by real-time fluorescence quantitative PCR. Groups of C. neoformans cells were incubated with murine macrophage-like J774A.1 cells, and the phagocytic indexes and ratios were determined by the microscopic observation method. Results The expression of cap10 in C. neoformans cells transfected with ps4.1 nee-cap10 （（175 535.00±47 004.00） copies/μl） was lower than that of cells transfected with the empty vector （（512 698.89±32 318.02） copies/μl） and mock transfected cells （（562 931.66±65 928.41） copies/μl）. The average phagocytic ratio and phagocytic index of J774A.1 cells following incubation with C. neoformans were higher for cells transfected with ps4.1 neo-capl0 （0.21±0.02, （19.06±1.66）%） than for the control experimental group （0.08±0.02, （6.57±1.23）%） and the blank experimental group （（0.07±0.01）, （5.89±1.07）%） （P 〈0.05）. Conclusions The cap10 shRNA vector was successfully prepared and transfected into C. neoformans cells. The effect of RNA interference on the expression of the C. neoformans caplO gene is effective, and it can induce phagocytosis of C. neoformans.

Disseminated cryptococcal lymphadenitis with negative latex agglutination test

We reported an unusual case of disseminated cryptococcal lymphadenitis in an immunocompetent host who presented with fever and lymphadenopathy, which were the only two symptoms and signs. Latex agglutination test of serum and cerebrospinal fluid （CSF） were negative, while lymph node biopsy showed Cryptococcus neoformans. A diagnosis of disseminated cryptococcal lymphadenitis was made. Then the patient was treated with amphotericin B for 15 days as initial therapy and itraconazole for 6 months as maintenance therapy respectively. The patient received re-examination per 6 months and was followed up for 2 years. Swollen lymph nodes diminished gradually, and no fever or other symptoms were found. Latex agglutination test of serum and CSF were negative throughout the follow-up period, and anti-HIV, syphilis and tuberculosis antibody were all negative.

A forkhead transcription factor contributes to the regulatory differences of pathogenicity in closely related fungal pathogens

Cryptococcus neoformans and its sister species Cryptococcus deuterogattii are important human fungal pathogens.Despite their phylogenetically close relationship,these two Cryptococcus pathogens are greatly different in their clinical characteristics.However,the determinants underlying the regulatory differences of their pathogenicity remain largely unknown.Here,we show that the forkhead transcription factor Hcm1 promotes infection in C.neoformans but not in C.deuterogattii.Monitoring in vitro and in vivo fitness outcomes of multiple clinical isolates from the two pathogens indicates that Hcm1 mediates pathogenicity in C.neoformans through its key involvement in oxidative stress defense.By comparison,Hcm1 is not critical for antioxidation in C.deuterogattii.Furthermore,we identified SRX1,which encodes the antioxidant sulfiredoxin,as a conserved target of Hcm1 in two Cryptococcus pathogens.Like HCM1,SRX1 had a greater role in antioxidation in C.neoformans than in C.deuterogattii.Significantly,overexpression of SRX1 can largely rescue the defective pathogenicity caused by the absence of Hcm1 in C.neoformans.Conversely,Srx1 is dispensable for virulence in C.deuterogattii.Overall,our findings demonstrate that the difference in the contribution of the antioxidant sulfiredoxin to oxidative stress defense underlies the Hcm1-mediated regulatory differences of pathogenicity in two closely related pathogens.