Cryptococcosis in Patients with Nephrotic Syndrome: A Pooled Analysis of Cases

Cryptococcosis is an infection that may be lethal in patients with nephrotic syndrome (NS). However, there is relatively limited epidemiological and clinical data about cryptococcosis in NS patients. We performed a pooled analysis to systemically summarize the epidemiology, risk factors, clinical and laboratory characteristics, treatments and outcomes of cryptococcosis in NS patients. Using data pooled from our hospital and studies identified via searches of three literature databases, 17 cases were identified for inclusion in this analysis. The prevalence of cryptococcosis in NS was 0.3%, with a higher rate in more recent years. Most patients were Asian (94%) and from upper-middle to high-income countries (76%). The median time interval from NS diagnosis to cryptococcosis diagnosis among the cryptococcosis patients was 16 months, and 46% of the identified cryptococcal infections were diagnosed within the first year of NS diagnosis. Cutaneous cryptococcosis was frequently diagnosed among the included patients (35%), 58% received an erroneous diagnosis and inappropriate treatment, 90% of whom had a cryptococcal infection mistaken for a bacterial infection. The mortality rate was 35%. Standard therapeutic strategies should be emphasized for both antifungal treatment and renal disease control. Further studies conducted in various medical centers are warranted to confirm our conclusions.     

A proteomic‐based approach for the identification of immunodominant Cryptococcus neoformans proteins

Cryptococcus neoformans is an opportunistic fungal pathogen that can cause life‐threatening meningoencephalitis in immune compromised patients. Previous, studies in our laboratory have shown that prior exposure to an IFN‐γ‐producing C. neoformans strain (H99γ) elicits protective immunity against a second pulmonary C. neoformans challenge. Here, we characterized the antibody response produced in mice protected against experimental pulmonary C. neoformans infection compared to nonprotected mice. Moreover, we evaluated the efficacy of using serum antibody from protected mice to detect immunodominant C. neoformans proteins. Protected mice were shown to produce significantly more C. neoformans‐specific antibodies following a second experimental pulmonary cryptococcal challenge compared to nonprotected mice. Immunoblot analysis of C. neoformans proteins resolved by 2‐DE using serum from nonprotected mice failed to show any reactivity. In contrast, serum from protected mice was reactive with several cryptococcal protein spots. Analysis of these spots by capillary HPLC‐ESI‐MS/MS identified several cryptococcal proteins shown to be associated with the pathogenesis of cryptococcosis. Our studies demonstrate that mice immunized with C. neoformans strain H99γ produce antibodies that are immune reactive against specific cryptococcal proteins that may provide a basis for the development of immune based therapies that induce protective anticryptococcal immune responses.     

Pulmonary cryptococcosis: A rare but emerging disease

Pulmonary cryptococcosis occurs primarily in immunocompromised hosts as an isolated infection or as a precursor to disseminated disease. Recent studies indicate that patients with isolated pulmonary cryptococcosis may be asymptomatic or, if symptomatic, will often have very low serum cryptococcal antigen, distinguishing them from patients with disseminated disease. Amphotericin B plus flucytosine is the initial treatment for disseminated cryptococcosis or severe symptomatic pulmonary cryptococcosis, followed by fluconazole once symptoms have improved. Fluconazole is recommended for asymptomatic or mild disease. Targeted immunotherapies and vaccines are being studied and offer exciting new prevention and treatment strategies.     

Identification and characterization of Cryptococcus neoformans protein fractions that induce protective immune responses

Cryptococcus neoformans, the main causative agent of cryptococcosis, is a fungal pathogen that causes life‐threatening meningoencephalitis in immunocompromised patients. To date, there is no vaccine or immunotherapy approved to treat cryptococcosis. Cell‐ and antibody‐mediated immune responses collaborate to mediate optimal protection against C. neoformans infections. Accordingly, we identified cryptococcal protein fractions capable of stimulating cell‐ and antibody‐mediated immune responses and determined their efficacy to elicit protection against cryptococcosis. Proteins were extracted from C. neoformans and fractionated based on molecular mass. The fractions were then evaluated by immunoblot analysis for reactivity to serum extracted from protectively immunized mice and in cytokine recall assays for their efficacy to induce pro‐inflammatory and Th1‐type cytokine responses associated with protection. MS analysis revealed a number of proteins with roles in stress response, signal transduction, carbohydrate metabolism, amino acid synthesis, and protein synthesis. Immunization with select protein fractions containing immunodominant antigens induced significantly prolonged survival against experimental pulmonary cryptococcosis. Our studies support using the combination of immunological and proteomic approaches to identify proteins that elicit antigen‐specific antibody and Th1‐type cytokine responses. The immunodominant antigens that were discovered represent attractive candidates for the development of novel subunit vaccines for treatment and/or prevention of cryptococcosis.     

Characterization of<Emphasis Type="Italic">Cryptococcus neoformans</Emphasis> var.<Emphasis Type="Italic">neoformans</Emphasis> serotype A and A/D in samples from Egypt

The cryptococcal polysaccharide antigen was detected in 10 cerebrospinal fluid (CSF) and 23 serum samples from cryptococcal meningitis and intestinal cryptococcosis by the cryptococcal antigen latex agglutination system (CALAS). CALAS titers in CSF and serum samples of cryptococcal meningitis ranged over 8-2048 and 32-2048, respectively, while in cases of intestinal cryptococcosis, serum titers ranged over 8-2048. The isolates of yeast Cryptococcus neoformans were determined to be of serotype A or of the A/D pair. The total leukocyte count and biochemical parameters in CSF were significantly increased as indicators of microbial infection. Furthermore, the in vitro change of the teleomorph (sexual state) to the anamorph (asexual state) was also detected and the teleomorph state changed in vivo to the encapsulated anamoph state which is more virulent during infection in vivo than the yeast-like noncapsulated form. Two primers for internal transcribed spacer (ITS) regions of ribosomal DNA were used for molecular detection of C. neoformans. After PCR amplification, a DNA band of 415 bp, visualized on agarose gel, indicated the presence of C. neoformans cells in the tested CSF and serum samples. The primer sensitivity was also characterized using purified yeast chromosomal DNA as template; it was about 20 pg or more chromosomal DNA which represents about 10 cells of C. neoformans. The primers were also specific for ITS regions of C. neoformans and gave negative results with Candida albicans and E. coli chromosomal DNA templates.     

Value and Interpretation of Serological Tests for the Diagnosis of Cryptococcosis

MAXIMAL SEROLOGICAL DIAGNOSIS OF CRYPTOCOCCOSIS MAY BE ACCOMPLISHED THROUGH THE CONCURRENT USE OF THREE TESTS: the latex agglutination (LA) test for cryptococcal antigen, and the indirect fluorescent antibody (IFA) and tube agglutination (TA) tests for Cryptococcus neoformans antibodies. These tests were applied to 141 serum and cerebral spinal fluid specimens from 66 culturally proven cases of cryptococcosis and to 42 sera from normal subjects and from patients with other systemic mycotic diseases. The LA test was sensitive and completely specific; of the sera from proven cases, 55% were positive. With the TA test, 37% of the specimens were positive and the test was highly specific. With the IFA test, 38% of the specimens were positive and the test appears to be the least specific of the three. Cross-reactions were most evident with blastomycosis and histoplasmosis case sera. When the three tests were used concurrently, 87% of the cryptococcosis case specimens were positive and permitted a presumptive diagnosis of C. neoformans infections in 61 (92%) of the 66 patients whose specimens were examined.     

Detection of Antibody against Fungal Glucosylceramide in Immunocompromised Patients: A Potential New Diagnostic Approach for Cryptococcosis

We have developed an ELISA to determine the value of anti-glucosylceramide antibody for the prediction of disseminated cryptococcosis in immunocompromised subjects and performed a clinical prospective study at the Medical University of South Carolina. The study enrolled a total of 53 patients who were free of active fungal diseases at the time of enrollment but at risk of developing one because they were all immunocompromised, e.g., (1) patients positive for HIV and (2) patients post- or awaiting solid organ transplantation. Among 53 patients enrolled, two patients developed invasive cryptococcosis, and in both patients, IgM anti-GlcCer was detected in sera using the ELISA at least 6 weeks prior to the clinical presentation of the brain disease. These results were corroborated by a cryptococcal antigen lateral flow assay, which was also positive in serum prior to the development of meningoencephalitis. However, a high number of positive results were also detected in patients with no evidence of cryptococcosis. This study highlights the potential utility of this new assay in early diagnostic testing algorithms for patients at risk for cryptococcosis, but further investigations are needed to validate the sensitivity and specificity of the glucosylceramide ELISA as a predictor of cryptococcosis.     

Evaluation of a New Cryptococcal Antigen Lateral Flow Immunoassay in Serum,Cerebrospinal Fluid and Urine for the Diagnosis of Cryptococcosis: A Meta-Analysis and Systematic Review

BackgroundA new lateral flow immunoassay (LFA) for the detection of cryptococcal antigen was developed.ObjectiveWe aimed to systematically review all relevant studies to evaluate the diagnostic accuracy of the cryptococcal antigen LFA on serum, CSF and urine specimens.MethodsWe searched public databases including PubMed, Web of Science, Elsevier Science Direct and Cochrane Library for the English-language literature published up to September 2014. We conducted meta-analyses of sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR) and diagnostic odds ratios (DOR) and SROC of LFA in serum and CSF, respectively. The sensitivity of LFA in urine was also analyzed. Subgroup analyses were carried out to analyze the potential heterogeneity.Results12 studies were included in this study. The pooled sensitivity and specificity values of LFA in serum were 97.6% (95% CI, 95.6% to 98.9%) and 98.1% (95% CI, 97.4% to 98.6%), respectively. The average PLR of LFA in serum was 43.787 (95% CI, 22.60–84.81) and the NLR was 0.03 (95% CI, 0.01–0.09). The pooled DOR was 2180.30 (95% CI, 868.92–5471.00) and the AUC was 0.9968. The pooled sensitivity and specificity values of LFA in CSF were 98.9% (95% CI, 97.9% to 99.5%) and 98.9% (95% CI, 98.0% to 99.5%), respectively. The average PLR of LFA in serum was 48.83 (95% CI, 21.59–110.40) and the NLR was 0.02 (95% CI, 0.01–0.04). The pooled DOR was 2931.10 (95% CI, 1149.20–7475.90) and the AUC was 0.9974. The pooled sensitivity value of LFA in urine was 85.0% (95% CI, 78.7% to 90.1%)ConclusionsThe study demonstrates a very high accuracy of LFA in serum and CSF for the diagnosis of cryptococcosis in patients at risk. LFA in urine can be a promising sample screening tool for early diagnosis of cryptococcosis.     

The Eiken Latex test for detection of a cryptococcal antigen in cryptococcosis

A latex agglutination test for cryptococcal antigen, the Eiken Latex test (Eiken, Tokyo, Japan), was compared with a monoclonal antibody-based agglutination assay, Pastorex^® Cryptococcus (Diagnostics Pasteur, Marneur-la-Coquette, France). In a murine model of disseminated cryptococcosis, the kinetics of the antigen titers by the Eiken Latex were similar to those by the Pastorex^® Cryptococcus, but sensitivity was much higher. In HIV-negative patients with pulmonary cryptococcosis, a cryptococcal antigen was detected in 6 of 10 patients by the Eiken Latex test and in only 3 of those patients by the Pastorex^® Cryptococcus. The results indicate that the Eiken Latex is more sensitive for the detection of the cryptococcal antigen, even in non-disseminated cryptococcosis. The sensitivity and specificity of the Eiken Latex were examined using 195 sera from 25 patients with pulmonary cryptococcosis and 170 patients with non-cryptococcosis. The cutoff value of 1:8 showed a sensitivity of 76% (19/25) and a specificity of 98.9% (168/170).     

Performance of Cryptococcal Antigen Lateral Flow Assay Using Saliva in Ugandans with CD4 <100

Background

Cryptococcal meningitis can best be diagnosed by cerebrospinal fluid India ink microscopy, cryptococcal antigen detection, or culture. These require invasive lumbar punctures. The utility of cryptococcal antigen detection in saliva is unknown. We evaluated the diagnostic performance of the point-of-care cryptococcal antigen lateral flow assay (CrAg LFA) in saliva.

Methods

We screened HIV-infected, antiretroviral therapy naïve persons with symptomatic meningitis (n = 130) and asymptomatic persons with CD4+<100 cells/µL entering into HIV care (n = 399) in Kampala, Uganda. The diagnostic performance of testing saliva was compared to serum/plasma cryptococcal antigen as the reference standard.

Results

The saliva lateral flow assay performance was overall more sensitive in symptomatic patients (88%) than in asymptomatic patients (27%). The specificity of saliva lateral flow assay was excellent at 97.8% in the symptomatic patients and 100% in asymptomatic patients. The degree of accuracy of saliva in diagnosing cryptococcosis and the level of agreement between the two sample types was better in symptomatic patients (C-statistic 92.9, κ-0.82) than in asymptomatic patients (C-statistic 63.5, κ-0.41). Persons with false negative salvia CrAg tests had lower levels of peripheral blood CrAg titers (P<0.001).

Conclusion

There was poor diagnostic performance in testing saliva for cryptococcal antigen, particularly among asymptomatic persons screened for preemptive treatment of cryptococcosis.     

Pulmonary cryptococcosis in late pregnancy and review of published literature

Naturally occurring maternal immunosuppression increases the risk of infection by a variety of pathogens during pregnancy and the postpartum period. Pulmonary cryptococcosis during pregnancy is relatively rare. Here, we report on two cases of pulmonary cryptococcosis during pregnancy and puerperium. Both cases were successfully treated using oral fluconazole after parturition to avoid fetal toxicity. For the two patients, the placentas were checked and found to be pathologically normal, and the cryptococcal serum antigen in both infants was negative. Pulmonary cryptococcosis should be considered during differential diagnosis as a possible cause of abnormal chest shadow in pregnant patients.     

Cavitary pneumonia in an AIDS patient with cryptococcosis

Pulmonary cryptococcosis is an unusual fungal infection that is most often found in AIDS or in organ transplant recipients. Although in immunocompromised patients, cryptococcal infection often causes pulmonary infections, the diagnosis of lung involvement is generally difficult. The presentation of pulmonary cryptoccosis in HIV-infected patients appears to be more acute and severe than in other immunocompromised patients, probably related with the severe immunosuppression. Diffuse infiltrates, mediastinal and hilar lymph nodes enlargement are the most common radiological findings in AIDS-associated pulmonary cryptococcosis. Cavitation is a rare form of and includes only 10% to 15% of all cases. Only a few case reports or studies with small number of patients of pulmonary cryptococcosis have been published over the past two decades. We report a case of an AIDS patient who developed cavitary pneumonia as the only clinical expression of cryptococcosis.     

Clinical Utility of the Cryptococcal Antigen Lateral Flow Assay in a Diagnostic Mycology Laboratory

Background

Cryptococcus neoformans causes life-threatening meningitis. A recently introduced lateral flow immunoassay (LFA) to detect cryptococcal antigen (CRAG) is reportedly more rapid and convenient than standard latex agglutination (LA), but has not yet been evaluated in a diagnostic laboratory setting.

Methods

One hundred and six serum, 42 cerebrospinal fluid (CSF), and 20 urine samples from 92 patients with known or suspected cryptococcosis were tested by LA and LFA, and titres were compared. Results were correlated with laboratory-confirmed cryptococcosis. Serial samples were tested in nine treated patients.

Results

Twenty-five of 92 patients had confirmed cryptococcosis; all sera (n = 56) from these patients were positive by LFA (sensitivity 100%, 95% confidence interval (CI) 93.6–100%) compared with 51/56 positive by LA (sensitivity 91.1%, 95% CI 80.7–96.1%). Fifty sera from 67 patients without cryptococcosis tested negative in both assays. While LA yielded more false negative results (5/56) this did not reach statistical significance (p = 0.063). Nine CSF samples from patients with cryptococcal meningitis yielded positive results using both assays while 17/18 urine samples from patients with cryptococcosis were positive by the LFA. The LFA detected CRAG in C. gattii infection (n = 4 patients). Agreement between titres obtained by both methods (n = 38 samples) was imperfect; correlation between log-transformed titres (r) was 0.84. Turn-around-time was 20 minutes for the LFA and 2 h for LA. The cost per qualitative sample was 18USD and 91 USD, respectively and per quantitative sample was 38USD and 144USD, respectively.

Conclusions

Qualitative agreement between the LFA and LA assays performed on serum and CSF was good but agreement between titres was imperfect. Ease of performance of the LFA and the capacity for testing urine suggest it has a role in the routine laboratory as a rapid diagnostic test or point-of-care test.     

Cryptococcal phospholipase B antigen is not detected in serum of patients infected with Cryptococcus neoformans using a sandwich enzyme-linked immunosorbent assay

Extracellular phospholipase B (PLB) is a virulence determinant of Cryptococcus neoformans and Cryptococcus gattii. In this study, we developed a sensitive enzyme-linked immunosorbent assay (ELISA) for PLB antigen with a detection limit of 3.9 ng mL(-1). PLB was detected in culture supernatants of C. neoformans and C. gattii. PLB, however, was not detected in sera of seven human patients and 10 feline patients with active cryptococcosis. Furthermore, none of five rats with extensive pulmonary C. gattii infection had a positive ELISA test result. In conclusion, cryptococcal PLB could not be detected in serum using a PLB antigen-based ELISA. Despite its sensitivity, this ELISA is of limited diagnostic value. Exploration of further extracellular molecules suitable for serodiagnosis of active cryptococcal infection is warranted.     

Evidence for Interference by Immune Complexes in the Serodiagnosis of Cryptococcosis

The latex agglutination test was used to compare cryptococcal antigen titers before and after protease treatment in 19 patients with pulmonary cryptococcosis. Antigen was detected by the LA test in 13 of 33 serum samples before protease treatment, and in an additional 13 samples following treatment. Of 26 antigen-positive serum samples, 22 (84.6%) showed an increased antigen titer after protease treatment. Using the cell slide agglutination test, antibody was detected in 3 of 19 cases. In one of these 3, antigen could only be detected after protease treatment. Soluble immune complex was prepared in vitro using anti-C. neoformans rabbit antiserum and polysaccharide antigen of C. neoformans, and the effects of immune complexes on the LA test were examined. In this experimental model, soluble immune complexes seemed to be observed in antibody excess region, because the antigen titers were increased after the protease treatment. We concluded that C. neoformans capsular polysaccharide antigen and anti-C. neoformans antibody formed soluble immune complexes in patients' sera, which interfered with antigen detection by the latex agglutination test without protease treatment.     

A synthetic peptide as a novel anticryptococcal agent

An engineered, killer decapeptide (KP) has been synthesized based on the sequence of a recombinant, single-chain anti-idiotypic antibody (KT-scFv) acting as a functional internal image of a yeast killer toxin. Killer decapeptide exerted a strong fungicidal activity against Candida albicans, which was attributed to peptide interaction with beta-glucan. As this polysaccharide is also a critical component of the cryptococcal cell wall, we wondered whether KP was also active against Cryptococcus neoformans, a human pathogen of increasing medical importance. We found that KP was able to kill both capsular and acapsular C. neoformans cells in vitro. Furthermore, KP impaired the production of specific C. neoformans virulence factors including protease and urease activity and capsule formation, rendering the fungus more susceptible to natural effector cells. In vivo treatment with KP significantly reduced fungal burden in mice with cryptococcosis and, importantly, protected the majority of immunosuppressed animals from an otherwise lethal infection. Given the relevance of cryptococcosis in immunocompromised individuals and the inability of conventional drugs to completely resolve the infection, the results of the present study indicate KP as an ideal candidate for further studies on novel anticryptococcal agents.     

Cryptococcosis in China (1985–2010): Review of Cases from Chinese Database

Background

Cryptococcosis is a potential fatal disease, especially in immunocompromised patients. In China, the profile of cryptococcosis is unclear. Therefore, we summarize the epidemiology and therapy of cryptococcosis in china.

Methods

All cases reports about cryptococcosis in China were collected from CBMdisk database (China Biology and Medicine data disc) with key words of cryptococcosis, or cryptococcal infection, or cryptococcus, and case. The features of the cryptococcosis were retrospectively analyzed.

Results

There were 1,032 reports about cryptococcosis, including 8,769 cases. Among them, there were 16% patient with AIDS/HIV, and 17% ones without underlying diseases. There were 2,371 cases of CNS infection. Among them of 2,068 cases, the treatment protocols and outcome were clearly described. The percentages of patients who received intrathecal treatment of amphotericin B(AmB), AmB?+?5-FC(5-fluorocytosine), AmB?+?FCZ(fluconazole), and AmB?+?5-FU?+?FCZ in each medication group were 10, 43, 53, and 33%, respectively. The mortalities were significantly lower in the AmB, Amb?+?5-FC, AmB?+?FCZ intrathecal treatment groups compared with their non-intrathecal treatment controls (6% vs. 23%, 25% vs. 35%, 20% vs. 30%, respectively, P?P?>?0.05).

Conclusion

The Chinese cryptococcosis had its own special clinical features, such as more patients without identifiable underlying diseases. Intrathecal injection of amphotericin B was effective treatment method for cryptococcal CNS infection in China.     

Induction of Protective Immunity Against Cryptococcosis

Cryptococcus neoformans, the predominant etiological agent of cryptococcosis, is an encapsulated fungal pathogen that can cause life-threatening infections of the central nervous system in immune compromised individuals resulting in high morbidity and mortality. Consequently, several studies have endeavored to understand those mechanisms that mediate resistance and susceptibility to Cryptococcus infection. In this review, we will examine the contributions of various components of the innate and adaptive immune response toward protection against cryptococcosis. We will focus our discussion on studies presented at the 8th International Conference on Cryptococcus and Cryptococcosis (ICCC). Remarkable progress has been made toward our understanding of host immunity and susceptibility to cryptococcal infection and the potential for vaccine development.     

Cell-mediated immunity in Cryptococcosis

Cell-mediated immune responses in patients who had recovered from cryptococcosis were compared to those of healthy subjects. Cryptococcal patients were mildly lymphopenic but showed no defect in percentage of thymus-derived lymphocytes. One-third had positive delayed skin test reactions to cryptococcal antigen. Their skin test reactivity to two commonly used noncryptococcal antigens was less intense than healthy control subjects. Strongly positive and specific lymphocyte transformation occurred in the presence of an extract of Cryptococcus neoformans (cryptococcin) in half of the patients. In contrast, few healthy subjects had positive transformation responses to cryptococcin. One patient who was followed sequentially through treatment of cryptococcal meningitis acquired strong cryptococcin reactivity during the course of treatment. Cellular immunologic response to cryptococcin identifies many subjects who have had C. neoformans exposure, and may be of value for assessing immunologic status of patients undergoing therapy. These studies also indicate that most patients with cryptococcosis have a degree of deficiency in cell-mediated response to fungal antigens even when a specific underlying disease process cannot be identified.     

Enzyme-linked immunosorbent assay for detection and quantification of Cryptococcus neoformans antigen

An enzyme-linked immunosorbent assay was standardized for the detection of cryptococcal antigen in serum and cerebrospinal fluid. The system was evaluated in clinical samples from patients infected by human immunodeficiency virus with and without previous cryptococcosis diagnosis. The evaluated system is highly sensitive and specific, and when it was compared with latex agglutination there were not significant differences. A standard curve with purified Cryptococcus neoformans antigen was settled down for the antigen quantification in positive samples.