Genotypic diversity and antifungal susceptibility of Cryptococcus neoformans isolates from paediatric patients in China

Cryptococcosis is a life‐threatening mycosis primarily occurring in adult patients particularly those with immunosuppression such as HIV infection/AIDS. The number of reported cases of paediatric cryptococcosis has increased in the last decade around the world, including China. However, current information on the characteristics of cryptococcosis in children, particularly the genotypic diversity and antifungal susceptibility of the isolates, is limited. In the present study, a total of 25 paediatric isolates of Cryptococcus neoformans were genotyped using the ISHAM‐MLST scheme. In vitro susceptibility to antifungal agents of the 22 isolates was tested using the CLSI M27‐A3 method. Our analyses revealed that the genotypic diversity of C. neoformans isolates from Chinese paediatric patients was low, with ST 5 (80%) and ST 31 (12%) being the two major sequence types. Reduced susceptibility to fluconazole (FLU), 5‐flucytosine (5‐FC) and itraconazole (ITR) was observed among C. neoformans isolates from Chinese paediatric patients, particularly among the ST5 isolates, which was similar to observations made on C. neoformans isolates from Chinese adult patients. In addition, the majority of isolates (3/4, 75%) obtained from deceased patients showed decreased antifungal susceptibility, which indicates that further monitoring of antifungal susceptibility of Cryptococcus isolates is warranted in management of paediatric cryptococcosis.     

Cryptococcal infections over a 15 year period at a tertiary facility & impact of guideline management

Cryptococcosis is an invasive fungal infection caused primarily by Cryptococcus neoformans and Cryptococcus gattii species, presenting predominantly as meningoencephalitis. The aim of this study is to assess all cryptococcal infections managed at our facility from 2001 to 2015 to determine incidence, risk factors, and comparison of outcomes prior to and following introduction of the 2010 Infectious Disease Society of America (IDSA) guidelines. Retrospective analysis of all patients diagnosed and treated for cryptococcal infection occurring between January 2001 and December 2015. Of 102 patients diagnosed with cryptococcal infection, 97 were eligible for study inclusion. There appears to be an overall increased incidence of cryptococcosis in both transplant and non‐transplant cohorts with a peak in 2015 of 6 transplant and 13 non‐transplant cases. In the meningitis cohort, 38/52 (73%) of identified isolates were C. neoformans, and 14/52 (27%) were C. gattii. Notably, 14/14 (100%) of C. gattii isolates were associated with meningitis, as compared to only 38/64 (59%) C. neoformans associated with meningitis (P: .003). It appears that patients presenting with cough are less likely to have meningitis, 17/27 (63%), (P: .005). When stratifying for culture positive meningitis lumbar puncture opening pressure, the median in the culture positive cohort was 31.5 cm H₂O compared with 15.5 cm H₂O (P: .036).Multiple admissions were required prior to diagnosis in the majority of cases with only 18/72 (25%) diagnosed on 1st presentation. Postguideline mortality has improved from 15% to 6.1% (P: .046). Cryptococcal infection remains relatively uncommon, but there appears to be an increasing trend in incidence. Overall mortality is relatively low and has improved since introduction of the 2010 IDSA guidelines.     

Primary cutaneous cryptococcosis in an immunocompetent patient due to Cryptococcus gattii molecular type VGI in Brazil: a case report and review of literature

Primary Cutaneous Cryptococcosis is an uncommon infection caused by the yeast Cryptococcus neoformans and C. gattii. Few case reports are available in the literature describing in detail primary cutaneous cryptococcosis due to C. gattii in immunocompetent patients. Herein, we present a case of a 68‐year‐old immunocompetent male patient with erythematous nodular lesions on the right forearm due to C. gattii mating‐type α and molecular type VGI. The virulence factors test was performed for capsule diameter, melanin production and phospholipase activity. In vitro fluconazole testing showed the sensitivity profile of this clinical isolate. In addition, a review of the literature on this subject was carried out and verified that this is the first reported case of VGI in the south‐east region of Brazil.     

Evaluation of antifungal combination against Cryptococcus spp.

The second cause of death among systemic mycoses, cryptococcosis treatment represents a challenge since that 5‐flucytosine is not currently available in Brazil. Looking for alternatives, this study evaluated antifungal agents, alone and combined, correlating susceptibility to genotypes. Eighty Cryptococcus clinical isolates were genotyped by URA5 gene restriction fragment length polymorphism. Antifungal susceptibility was assessed following CLSI‐M27A3 for amphotericin (AMB), 5‐flucytosine (5FC), fluconazole (FCZ), voriconazole (VRZ), itraconazole (ITZ) and terbinafine (TRB). Drug interaction chequerboard assay evaluated: AMB + 5FC, AMB + FCZ, AMB + TRB and FCZ + TRB. Molecular typing divided isolates into 14 C. deuterogattii (VGII) and C. neoformans isolates were found to belong to genotype VNI (n = 62) and VNII (n = 4). C. neoformans VNII was significantly less susceptible than VNI (P = 0.0407) to AMB; C. deuterogattii was significantly less susceptible than VNI and VNII to VRZ (P < 0.0001). C. deuterogattii was less susceptible than C. neoformans VNI for FCZ (P = 0.0170), ITZ (P < 0.0001) and TRB (P = 0.0090). The combination FCZ + TRB showed 95.16% of synergistic effect against C. neoformans genotype VNI isolates and all combinations showed 100% of synergism against genotype VNII isolates, suggesting the relevance of cryptococcal genotyping as it is widely known that the various genotypes (now species) have significant impact in antifungal susceptibilities and clinical outcome. In difficult‐to‐treat cryptococcosis, terbinafine and different antifungal combinations might be alternatives to 5FC.     

The Use of Intravenous Methotrexate in the Treatment of Ectopic Pregnancy

Abstract

Cryptococcosis is a disseminated fungal disease typically associated with immuno-suppression and characterized by high mortality rates. Cryptococcus neoformans has been reported to be isolated from blood cultures in around 20% of patients with cryptococcosis, and cryptococcemia has been correlated with poor prognosis.

We report a case of fatal C. neoformans fungemia in a neutropenic patient with a history of chronic lymphocytic leukemia treated with alemtuzumab. The patient presented with loss of consciousness and died after 5 days of antifungal therapy with liposomal amphotericin B. The international literature regarding opportunistic infections after immunosuppressive therapy with alemtuzumab with particular attention on fun-gal infections has also been reviewed.     

Infective capacity of Cryptococcus neoformans and Cryptococcus gattii in a human astrocytoma cell line

Pathogenesis of cryptococcosis in the central nervous system (CNS) is a topic of ongoing research, including the mechanisms by which this fungus invades and infects the brain. Astrocytes, the most common CNS cells, play a fundamental role in the local immune response. Astrocytes might participate in cryptococcosis either as a host or by responding to fungal antigens. To determine the infectivity of Cryptococcus neoformans var. grubii and Cryptococcus gattii in a human astrocytoma cell line and the induction of major histocompatibility complex (MHC) molecules. A glioblastoma cell line was infected with C. neoformans var. grubii and C. gattii blastoconidia labelled with FUN‐1 fluorescent stain. The percentage of infection and expression of HLA class I and II molecules were determined by flow cytometry. The interactions between the fungi and cells were observed by fluorescence microscopy. There was no difference between C. neoformans var. grubii and C. gattii in the percentage infection, but C. neoformans var. grubii induced higher expression of HLA class II than C. gattii. More blastoconidia were recovered from C. neoformans‐infected cells than from C. gattii infected cells. Cryptococcus neoformans var. grubii may have different virulence mechanisms that allow its survival in human glia‐derived cells.     

Cryptococcosis and cryptococcal meningitis: New predictors and clinical outcomes at a United States academic medical centre

As the diagnosis of cryptococcosis is challenging in low‐prevalence settings, uncovering predictive factors can improve early diagnosis and timely treatment. The aim of the study was to relate clinical outcomes to predictive variables for the presence of cryptococcosis. A retrospective case‐control study matched by collection date, age and gender at a 1:2 ratio (55 cases and 112 controls) was performed in case patients diagnosed with Cryptococcus infection at the University of Colorado Hospital between 2000 and 2017 (n = 167). A bivariate and a forward, stepwise multivariable logistic regression model were performed to identify predictors of cryptococcosis infection. In an adjusted multivariable model, cryptococcal infection was significantly associated with the presence of respiratory symptoms, hyponatremia, lung disease or corticosteroids. Additionally, cryptococcal meningitis was associated with headaches, corticosteroids or increased CSF protein. Conversely, a reduced risk of cryptococcosis was associated with hypertension or peripheral monocytosis. Cryptococcal meningitis leads to subsequent hearing impairment (16% vs 4% (control), P = .013), muscle weakness (40% vs 20%, P = .021), cognitive deficits (33% vs 6%, P = .0001) or any adverse outcome (84% vs 29%, P = .0001). We uncovered novel clinical predictors for the presence of cryptococcal infection or cryptococcal meningitis. This study in patients at a low‐prevalence US medical centre underscores the importance of early diagnosis in this population.     

Cryptococcus and Cryptococcosis in Cuba. A minireview

Cryptococcosis has emerged as an important public health problem in Africa, Asia and the Americas due to the increasing numbers of persons at risk of this infection and the adaptation of its aetiological agents to new environments. The proper management requires early recognition of Cryptococcus neoformans/C. gattii species complex infection, familiarity with the use and limitations of diagnostic tests and knowledge of the available treatment options. This review will address these issues with the goal of providing sufficient information to suspect, diagnose and treat patients with cryptococcosis based on Cuban data and review of the literature.     

Epidemiology of cryptococcosis in Malaysia

This study describes the isolation of Cryptococcus neoformans and Cryptococcus gattii from patients with chronic meningitis who were admitted to 16 Malaysian hospitals, from 2003 to 2004. Of the 96 cryptococcal cases reported over the 2‐year period, 74 (77.1%) patients were male and 45 (46.9%) patients were between 30 and 39 years old. Cryptococcosis was uncommon in children. A total of 57 (59.4%) and 23 (24.0%) patients were Malay and Chinese respectively. Human immunodeficiency virus infection was the major underlying disease reported in 36 (37.5%) patients. C. neoformans var. grubii (serotype A and molecular type VNI) was the predominant Cryptococcus species isolated from 88.5% of cryptococcal cases in this country. Cryptococcal cases due to C. neoformans var. grubii were reported from all the five regions in Malaysia, with the most number of cases reported from the central and northern regions. Cryptococcus gattii (all were serotype B and molecular types VGI/II) was isolated from all regions except the southern region. Compared with a study conducted prior to the AIDS era, our findings show substantial changes in the demographical characteristics of patients.     

Cryptococcosis‐related deaths and associated medical conditions in the United States, 2000–2010

Cryptococcosis is an invasive mycotic infection primarily affecting immunocompromised individuals. The objective of this study was to describe cryptococcosis mortality and associated medical conditions in the US for the period 2000–2010. Cryptococcosis‐related deaths were identified from the national multiple‐cause‐of‐death dataset. Mortality trends and comparison analyses were performed on overall cases of cryptococcosis and by subset [i.e. clinical manifestations of disease and human immunodeficiency virus (HIV) status]. A matched case–control analysis was also conducted to describe the associations between this disease and comorbid medical conditions. A total of 3210 cryptococcosis‐related deaths were identified. Cerebral cryptococcosis was the most commonly reported clinical manifestation of the disease. Approximately one‐fifth of the decedents (n = 616) had a co‐diagnosis of HIV. Mortality rates were highest among men, blacks, Hispanics, Native Americans and older adults. Poisson regression analysis indicated a 6.52% annual decrease in mortality rates for the study period. HIV (MOR = 35.55, 95% CI 27.95–45.22) and leukaemia (MOR = 16.10, 95% CI 11.24–23.06) were highly associated with cryptococcosis‐related deaths. Cryptococcosis mortality declined significantly during 2000–2010. However, the disease continues to cause appreciable mortality in the US. With the majority of decedents having no HIV co‐diagnosis, there is still much to be learned about the epidemiology of this mycosis.     

Genotypic diversity of environmental Cryptococcus neoformans isolates from Northern Portugal

The Cryptococcus neoformans/C. gattii species complex members are the main agents of systemic cryptococcosis. This disease is believed to be acquired from the environment via fungal cell inhalation. Often, isolates recovered from environmental and clinical sources have proven to be genotypically similar. We assessed the occurrence of C. neoformans and C. gattii in environmental substrates collected in a Portuguese region. Twenty‐eight isolates were identified as C. neoformans – five from decaying Eucalyptus leaves and 23 from domestic pigeon droppings. The isolates were genotyped using a URA5‐RFLP approach. The C. neoformans VNIV (53.6%, n = 15) and VNI (32.1%, n = 9) genotypes were abundantly present among environmental isolates. The hybrid VNIII (14.3%, n = 4) genotype was underrepresented and the VNII was not found. Cryptococcus gattii was also not found although some isolates yielded a positive canavanine–glycine–bromothymol blue test.     

First case of disseminated cryptococcosis in a Gorilla gorilla

In humans, Cryptococcus mainly infects individuals with HIV infection or other types of immunosuppression. Here, we report the first case of disseminated cryptococcosis in a simian immunodeficiency virus‐negative 27‐year‐old female Gorilla gorilla presenting with lethargy, progressive weight loss and productive cough. The diagnosis was confirmed by positive lung biopsy culture, serum cryptococcal antigen, and cerebral histopathology demonstrating encapsulated yeasts. Molecular characterisation of lung culture isolate yielded Cryptococcus neoformans var. grubii. An immune‐deficiency could not be demonstrated.     

Cryptococcosis in patients with diabetes mellitus II in mainland China: 1993‐2015

Diabetes mellitus II (DM II) is a newly defined independent factor contributing to the morbidity and mortality of cryptococcosis. This retrospective case analysis aims to explore the epidemiology, clinical profile and strain characteristics of cryptococcosis in Chinese DM II patients. This study included 30 cases of cryptococcosis with DM II occurring from 1993 to 2015 in mainland China. The hospital‐based prevalence of cryptococcosis in DM II was 0.21%. The mean age of the patients was 56.1 years (95% confidence interval: 51.5, 60.6), and 93% of the patients were older than 40 years. Sixty‐two per cent of the patients experienced untreated or poorly controlled blood glucose before infection. Multilocus sequence typing analysis categorised all cultured strains as Cryptococcus neoformans and sequence type 5. Sixty‐nine per cent of pulmonary cryptococcosis patients experienced misdiagnoses and treatment delays. Sixty per cent of cryptococcal meningitis patients received substandard antifungal therapy. The overall death rate was 33%. Considering the large population size of DM II patients in China, improved attention should be paid to the high prevalence of cryptococcosis as revealed by us. We also emphasised the importance of blood glucose control for infection prevention, especially among the elderly.     

Isolation of Cryptococcus neoformans from pigeon (Columba livia) droppings in Mexico City

To determine the ecological and epidemiological significance of pigeon excrement in cryptococcosis in Mexico City, 251 samples of pigeon droppings were studied. These were collected from houses, public buildings, churches, parks and pigeon nests. Each sample was suspended 1 : 10 in isotonic saline solution and then cultured in Staib medium. Identification of Cryptococcus neoformans was performed based on the development of brown-coloured colonies and the presence of encapsulated yeasts. Of 251 samples, 52 (20.7%) were positive for Cr. neoformans. The highest frequency was observed in droppings from public buildings (31.2%), followed by churches (22.0%) and houses (13.3%). No significant differences in isolation frequency were observed between fresh or dried excrement. All isolates obtained were Cr. neoformans var. neoformans. As in other studies on Cr. neoformans from pigeons, performed in other countries, these frequency data are considered ‘normal’. The results, however, accentuate the potential risk of cryptococcosis acquisition, especially now that cryptococcosis frequency is increasing in Mexico, mainly in AIDS patients.     

Cryptococcal meningitis due to Cryptococcus neoformans genotype AFLP1/VNI in Iran: a review of the literature

Cryptococcal meningitis is the most important opportunistic fungal infection with a high mortality in HIV‐patients in less developed regions. Here, we report a case of cryptococcal meningitis in a 49‐year‐old HIV‐positive female due to Cryptococcus neoformans (serotype A, mating‐type alpha, genotype AFLP1/VNI) in Sari, Iran. In vitro antifungal susceptibility tests showed MICs of isavuconazole (0.016 μg ml^?1), voriconazole (0.031 μg ml^?1), posaconazole (0.031 μg ml^?1), itraconazole (0.063 μg ml^?1), amphotericin B (0.125 μg ml^?1) and fluconazole (8 μg ml^?1). Despite immediate antifungal therapy, the patient died 4 days later due to respiratory failure. Cryptococcal infections have been infrequently reported from Iran and therefore we analysed all published cases of cryptococcosis in Iran since the first reported case from 1969.     

In vitro susceptibility testing of amphotericin B for Cryptococcus neoformans variety grubii AFLP1/VNI and Cryptococcus gattii AFLP6/VGII by CLSI and flow cytometry

Cryptococcus neoformans var. grubii AFLP1/VNI is the main causative agent of cryptococcosis associated with AIDS in the world. Cryptococcus gattii AFLP6/VGII causes mainly endemic primary infection in immunocompetent hosts. To determine the minimum inhibitory concentrations (MICs) of C. neoformans var. grubii AFLP1/VNI and C. gattii AFLP6/VGII against amphotericin B (AMB) in a short period of time, flow cytometry (FCM) with FUN‐1 fluorochrome was used to compare with broth microdilution method (CLSI M27‐A3). The minimum incubation period was evaluated by minimum fungicidal concentration procedure. Seventeen clinical isolates of C. neoformans var. grubii AFLP1/VNI and 18 of C. gattii AFLP6/VGII were analysed. The time for the determination of MICs by FCM was 2 h against 72 h by CLSI M27‐A3 and the comparison of MIC showed a positive significant correlation (P = 0.048). It is important to highlight the role of the FCM as an alternative method to determine the MICs for AMB in within a day, with positive cost‐benefit.     

Fulminant Cryptococcus neoformans infection with fatal pericardial tamponade in a patient with chronic myelomonocytic leukaemia who was treated with ruxolitinib: Case report and review of fungal pericarditis

Cryptococcus neoformans is a saprophytic fungal pathogen that can cause serious illness in immune‐compromised hosts and it presents with a wide variety of clinical symptoms. We present a fatal case of fulminant C. neoformans infection presenting as pericardial tamponade in a 71‐year‐old male with chronic myelomonocytic leukaemia undergoing chemotherapy with the JAK‐STAT inhibitor ruxolitinib. We also review the published cases of fungal pericarditis/tamponade. In addition to illustrating an atypical presentation of C. neoformans, this case highlights the risk for opportunistic fungal infections in patients with haematological malignancies, especially the ones treated with small molecule kinase inhibitors.     

An analysis of the utilisation of chemoprophylaxis against Pneumocystis jirovecii pneumonia in patients with malignancy receiving corticosteroid therapy at a cancer hospital

Pneumocystis jirovecii pneumonia (PCP) is associated with high mortality in immunocompromised patients without human immunodeficiency virus infection. However, chemoprophylaxis is highly effective. In patients with solid tumours or haematologic malignancy, several risk factors for developing PCP have been identified, predominantly corticosteroid therapy. The aims of this study were to identify the potentially preventable cases of PCP in patients receiving corticosteroid therapy at a tertiary care cancer centre and to estimate the frequency of utilisation of chemoprophylaxis in these patients. Two retrospective reviews were performed. Over a 10-year period, 14 cases of PCP were identified: no cases were attributable to failed chemoprophylaxis, drug allergy or intolerance. During a 6-month period, 73 patients received high-dose corticosteroid therapy (> or =25 mg prednisolone or > or =4 mg dexamethasone daily) for > or =4 weeks. Of these, 22 (30%) had haematologic malignancy, and 51 (70%) had solid tumours. Fewer patients with solid tumours received prophylaxis compared to patients with haematologic malignancy (3.9 vs 63.6%, P<0.0001). Guidelines for PCP chemoprophylaxis in patients with haematologic malignancy or solid tumours who receive corticosteroid therapy are proposed. Successful primary prevention of PCP in this population will require a multifaceted approach targeting the suboptimal prescribing patterns for chemoprophylaxis.     

Epidemiology of Cryptococcus and cryptococcosis in Western Africa

Cryptococcosis is a serious and sometimes fatal fungal disease caused by Cryptococcus species. Worldwide, it is estimated to kill over 180 000 annually, with 75% of deaths occurring in sub‐Saharan Africa. Though cryptococcal infections are rare in otherwise healthy individuals, there have been reported cases in immunocompetent persons. Most cases occur in individuals who have weakened immune systems, particularly those with advanced HIV/AIDS, thus making West Africa a potential hotspot of the disease. Despite this, there is no recent review article with a focus on published findings on cryptococcosis in Western Africa. Common clinical symptoms include chest pain, dry cough, headache, nausea, confusion, fever, fatigue and stiffness of the neck/neurological impairment. The CNS and the lung remain its preferred target even though rare cases of attack on other parts of the body were reported in this review. Cryptococcal antigen screening and India ink preparation were the most commonly used diagnostic methods. Repeated isolation from environmental samples was observed. Overall, data on the clinical prevalence of Cryptococcus are scarce and variable in the region. The environmental prevalence ranges from 2.3% to 22%. This review covers all published research findings on cryptococcosis in West Africa till date. The epidemiological data will likely be of interest to clinicians within and outside the continent. The nations covered in this review include the following: Benin Republic, Burkina Faso, Cote d'ivoire, Ghana, Guinea, Guinea‐ Bissau, Mali, Nigeria, Senegal and Sierra Leone. More studies are warranted to fill the observed gaps on the epidemiology of Cryptococcus in the region.     

Mycological-diagnostic assessment of the efficacy of amphotericin B + flucytosine to control Cryptococcus neoformans in AIDS patients

Abstract: Optimal diagnostic data (microscopy, culture and serology) on 15 cases of Cr. neoformans infection in AIDS patients served as a basis for the preliminary subdivision of the stages of cryptococcosis: Primary (minor involvement of the lungs only) and secondary (hematogenous dissemination with involvement of various organs) stages. Cr. neoformans counts in body fluids and antigen titres in serum and CSF proved to be useful criteria to assess the stage of infection. The efficacy of the combination therapy with amphotericin B + flucytosine seems to be related to the stage of infection. From the mycological point of view, the standard combination of 0.3–0.5 mg/kg BW/d amphotericin B and 150 mg/kg BW/d flucytosine proved to be effective. Data on sensitivity of the agent to amphotericin B and flucytosine are presented in a summarized form. The subjects of duration of therapy, relapse and resistance to flucytosine are commented by means of examples. It should be the aim of an optimal therapy of cryptococcosis to diagnose this mycotic disease in its primary stage. Zusammenfassung: Anhand von 15 Fällen wird versucht, die Cr. neoformans-Infektion AIDS-Kranker in das Primärstadium (alleiniger und geringer Befall der Lunge) und das Sekundärstadium (hämatogene Dissemination mit Befall verschiedener Organe) einzuteilen. Die Keimzahl von Cr. neoformans in Körperflüssigkeiten und der Cr. neoformans-Antigentiter in Serum und Liquor dienten hierbei als die beiden wesentlichen Kriterien zur Einschätzung des Stadiums der Infektion und seiner Therapierbarkeit. Aus mykologischer Sicht erwies sich die Standardkombination von Amphotericin B (0,3–0,5 mg/kg KG täglich) + Flucytosin (150 mg/kg KG täglich) als wirksam. Anhand von Beispielen wird zu den Themen: Dauer der Therapie, Rezidiv und Flucytosin-Resistenz Stellung genommen. über die Empfindlichkeit der Cr. neoformans-Stämme gegen Amphotericin B und Flucytosin (Ancotil ) wird zusammenfassend berichtet. Voraussetzung für eine optimale Therapierbarkeit der Kryptokokkose scheint die Diagnostik dieser Mykose in ihrem Primärstadium zu sein.