High prevalence of Cryptococcal antigenemia using a finger-prick lateral flow assay in individuals with advanced HIV disease in Santarém Municipality, Brazilian Amazon Basin.

There is scarce information about HIV-related cryptococcosis in the Brazilian Amazon basin where laboratory infrastructure is limited. The serum cryptococcal antigen (CrAg) lateral flow assay (LFA) has simplified diagnosis of cryptococcosis and is recommended for screening in advanced HIV disease. We evaluated the prevalence of cryptococcal antigenemia using finger-prick CrAg LFA in the Brazilian Amazon basin. We enrolled a prospective cohort of outpatients and hospitalized individuals with advanced HIV disease at two centers in Santarém Municipality, Northern Brazil. All individuals were > 18 years old with advanced HIV disease, regardless of antiretroviral therapy (ART) status and with no prior or current history of confirmed cryptococcal meningitis. We tested CrAg LFA on finger-prick whole blood using an exact volume transfer pipette. From August 2018 to October 2019, 104 individuals were enrolled (outpatients 62 [60%] and hospitalized 42 [40%]). Median age was 38 years (interquartile range [IQR] 30-46), and 84 (81%) were male. Sixty-five (63%) individuals were ART-naïve. Prevalence of finger-prick CrAg LFA-positive was 10.6%; 95% CI, 5.4 to 18.1%. Prevalence of finger-prick CrAg LFA-positive among individuals without neurological symptoms was 6.0%; 95% CI, 1.7-14.6%. The number needed to test to detect one CrAg-positive individual was 9.4 persons (95% CI, 5.5-18.5). Prevalence of cryptococcal antigenemia using finger-prick whole blood CrAg LFA was high. Point-of-care approach was important for the diagnosis and screening of cryptococcosis in resource-limited settings. Screening and preemptive therapy strategy should be urgently implemented in individuals with advanced HIV disease in the Brazilian Amazon basin.

This prospective cohort study was carried-out in the Brazilian Amazon basin. We used a cryptococcal rapid test in patients with AIDS. We included 104 participants, and 11 (10.6%) of them had positive results showing a high prevalence of cryptococcal antigenemia.

Clinical features and Outcomes of Cryptococcemia patients with and without HIV infection.

BACKGROUND: The effects of cryptococcemia on patient outcomes in those with or without HIV remain unclear. METHODS: One hundred and seventy-nine cryptococcemia patients were enrolled in this retrospective study. Demographic characteristics, blood test results and outcome were compared between the two groups. RESULTS: The diagnosis time of Cryptococcus infection was 2.0(0-6.0) days for HIV-infected patients, 5.0 (1.5-8.0) days for HIV-uninfected patients (p = .008), 2.0 (1.0-6.0) days for cryptococcal meningitis (CM) patients and 6.0 (5.0-8.0) days for non-CM patients (p < .001). HIV infection [adjusted odds ratio (AOR) (95% confidence interval): 6.0(2.3-15.9)], CRP < 15 mg/L [AOR:3.7(1.7-8.1)) and haemoglobin > 110 g/L [AOR:2.5(1.2-5.4)] were risk factors for CM development. Forty-six (25.7%) patients died within 90 days. ICU stay [AOR:2.8(1.1-7.1)], hypoalbuminemia [AOR:2.7(1.4-5.3)], no anti-cryptococcal treatment [AOR:4.7(1.9-11.7)] and altered consciousness [AOR:2.4(1.0-5.5)] were independent risk factors for 90-day mortality in all patients. HIV infection did not increase the 90-day mortality of cryptococcemia patients when anti-Cryptococcus treatment was available. Non-Amphotericin B treatment [AOR:3.4(1.0-11.2)] was associated with 90-day mortality in HIV-infected patients, but age ≥ 50.0 years old [AOR:2.7(1.0-2.9)], predisposing disease [AOR:4.1(1.2-14.2)] and altered consciousness [AOR:3.7(1.1-12.9)] were associated with 90-day mortality in HIV-uninfected patients who accepted anti-Cryptococcus treatment. CONCLUSION: HIV infection increased the incidence of CM rather than mortality in cryptococcemia patients. The predictive model was completely divergent in HIV-infected and HIV-uninfected patients, suggesting that novel strategies for diagnosis and treatment algorithms are urgently needed.

Clinical Significance of Low Serum Cryptococcal Antigen Titers by Lateral Flow Assay in Immunocompromised Patients: a Retrospective Case-Control Study.

Cryptococcus species are associated with invasive fungal infections in immunosuppressed individuals. The clinical significance of low-titer cryptococcal antigen (CrAg) by lateral flow assay is frequently uncertain. We investigated the correlation of low CrAg titers with disease in an immunocompromised patient population. Patients with first-time positive CrAg results with low serum titers (≤1:10) at two medical centers (Los Angeles, CA) from April 2014 to July 2018 were included. Age-matched controls with high (≥1:20) and negative titers were selected. We extracted medical records for pertinent clinical, radiologic, and laboratory data for cryptococcal disease. From 2,196 serum samples submitted for CrAg testing, 96 cases were included (32 each in low-titer, high-titer, and negative-titer groups). One or more immunocompromising condition was identified in 95% of patients, including HIV infection (45%), solid organ transplant (26%), and cirrhosis (22%). Pulmonary cryptococcosis was diagnosed in 9 (28%) low-titer and 8 (25%) high-titer patients (P = 1.00). Disseminated cryptococcosis occurred in 7 (22%) low-titer and 15 (47%) high-titers cases (P = 0.064). Titers ≤1:10 more frequently represented isolated antigenemia in HIV-positive than non-HIV, immunocompromised patients (P < 0.001). Follow-up testing in patients with ≤1:5 titers (n = 21) showed persistently low titers in 6 of 12 instances and increased titers in 2 cases. Twenty-seven patients with low CrAg titers were treated with antifungal therapy and 22 (81%) responded well clinically. Low-serum CrAg titers (≤1:10) correlated with cryptococcal disease in a substantial proportion of non-HIV immunocompromised patients and should prompt careful clinical workup for cryptococcal infection.

Prevalence of cryptococcal antigenemia in hospitalized patients with liver cirrhosis.

The benefits of screening for cryptococcal antigenemia and of preemptive antifungal treatment in HIV-infected patients have been proven. Liver cirrhosis is an important risk factor for cryptococcal infections. Cryptococcal infections are rapidly fatal in patients with liver cirrhosis, especially when diagnosis is delayed. However, screening for cryptococcal antigenemia has not been investigated in these patients. The aim of this study was to investigate the prevalence of cryptococcal antigenemia in hospitalized patients with liver cirrhosis. This prospective study was conducted at Seoul National University Hospital from July 2017 to January 2018. We included patients with liver cirrhosis who were admitted regardless of symptoms or signs suggesting cryptococcal infections. The severity of cirrhosis was evaluated from Child-Pugh and model for end-stage liver disease (MELD) scores. Serum cryptococcal antigenemia was determined using a latex agglutination test. A total of 294 patients were included in the analysis, comprising 104 (35.4%), 100 (34.0%), and 90 (30.6%) patients in Child-Pugh classes A, B, and C, respectively. There were 21 cases of spontaneous bacterial peritonitis, and 14 of hepatic encephalopathy, but none of cryptococcal peritonitis or meningitis. In addition, none of the patient specimens tested positive in the serum cryptococcal latex agglutination test (one-sided 97.5% confidence interval, 0% ∼ 1.2%). Liver cirrhosis is a major risk factor for cryptococcal infections, but the prevalence of serum cryptococcal antigen positivity in patients with liver cirrhosis is very low. Therefore, screening for cryptococcal antigenemia and preemptive antifungal treatment in cirrhotic patients might not be beneficial.

Comparing immunochromatography with latex antigen agglutination testing for the diagnosis of cryptococcosis in cats, dogs and koalas.

Although the point-of-care cryptococcal antigen lateral flow assay (LFA) has revolutionized the diagnosis of cryptococcosis in human patients, to date there has been no large-scale examination of this test in animals. We therefore assessed the LFA in cats, dogs and koalas suspected of having cryptococcosis. In sum, 528 serum specimens (129 from cats, 108 from dogs, 291 from koalas) were tested using the LFA and one of two commercially available latex cryptococcal antigen agglutination test (LCAT) kits. The LCAT is a proven and well-accepted method in veterinary patients and therefore taken as the "gold standard" against which the LFA was compared. The LFA achieved a sensitivity of 92%, 100%, and 98% in cats, dogs, and koalas, respectively, with corresponding negative predictive values of 94%, 100%, and 98%. The specificity of the LFA was 81%, 84%, and 62% for cats, dogs, and koalas, respectively, with corresponding positive predictive values of 76%, 48%, and 69%. These findings suggest the most appropriate role for the LFA is as a screening test to rule out a diagnosis of cryptococcosis in cats, dogs, and koalas. Point-of-care accessibility makes it equally suited for use in the field and as a cage-side test in veterinary hospitals. The suboptimal specificity of the LFA makes it less suited to definitive confirmation of cryptococcosis in animals; therefore, all LFA-positive test results should be confirmed by LCAT testing. The discrepancy between these observations and the high specificity of the LFA in humans may reflect differences in the host-pathogen interactions amongst the species.

Multi-locus Sequence Typing and Whole Genome Sequence Analysis of Cryptococcus neoformans Isolated from Clinical Specimens in Vajira Hospital, Bangkok, Thailand.

The basidiomycete yeast Cryptococcus neoformans causes disease in immunocompromized patients. Whole genome sequencing (WGS) technology provides insights into the molecular epidemiology of C. neoformans. However, the number of such studies is limited. Here we used WGS and multilocus sequence typing (MLST) to determine the genetic diversity of C. neoformans isolates and genetic structures of their populations among patients admitted to a single hospital in Bangkok, Thailand. Seven isolates from six patients collected during 1 year were identified as C. neoformans sensu stricto according to colony morphology, microscopy, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nucleotide sequence analysis of internal transcribed sequences. These isolates were sensitive to the antifungal drugs amphotericin B, fluconazole, 5-flucytosine, voriconazole, itraconazole and posaconazole and were mating type α and molecular type VNI. MLST analysis identified ST4, ST5 and ST6. We further employed WGS to determine the genetic diversity and relationships of C. neoformans isolated here combined with C. neoformans sequences data acquired from a public database (n = 42). We used the data to construct a phylogenetic tree. WGS provided additional genomics data and achieved high discriminatory power for identifying C. neoformans isolates isolated in Thailand. This report further demonstrates the applicability of WGS analysis for conducting molecular epidemiology and provides insight into the genetic diversity of C. neoformans isolates from one hospital in Thailand.

Serum Cryptococcal Antigen Confirmed Pulmonary Cryptococcosis in an Immune-competent Adult Firstly Misdiagnosed of Tuberculosis from a Lung Biopsy: a Case Report and Literature Review.

Background: Cryptococcal pneumonia is an uncommon lesion in immune-competent adults. Histological evidence of Cryptococcus neoformans is a gold criterion for diagnosis. Here we report a case firstly misdiagnosed as tuberculosis from a lung biopsy. Methods: Chest computed tomography (CT) scan and CT-guided puncture were performed for diagnosis and blood tests explored for the latent etiology. Results: Chest CT scan images showed multiple nodules in the left peripheral lower lobe. Histopathology demonstrated multiple granulomatous inflammatory response lacking evidence of Cryptococcus neoformans, acid-fast staining was negative, serum cryptococcal antigen was positive. Conclusions: Serum cryptococcal antigen has high specificity in cryptococcal pneumonia.

Efficacy of Prosopilosidine from Prosopis glandulosa var. glandulosa against Cryptococcus neoformans Infection in a Murine Model.

In this study, 2,3-dihydro-1H-indolizinium alkaloid-prosopilosidine (PPD), that was isolated from Prosopis glandulosa, was evaluated against C. neoformans in a murine model of cryptococcosis. In vitro and in vivo toxicity of indolizidines were also evaluated. Mice were infected via the tail vein with live C. neoformans. Twenty-four hours post-infection, the mice were treated with PPD once a day (i.p.) or twice a day (bid) orally, or with amphotericin B (Amp B) intraperitoneally (IP), or with fluconazole (Flu) orally for 5 days. The brains of all of the animals were aseptically removed and the numbers of live C. neoformans were recovered. In vitro toxicity of indolizidine alkaloids was determined in HepG2 cells. PPD showed to be potent in vivo activity against C. neoformans at a dose of 0.0625 mg/kg by eliminating ~76% of the organisms compared to ~83% with Amp B (1.5 mg/kg). In addition, PPD was found to be equally efficacious, but less toxic, at either 0.125 or 0.0625 mg/kg compared to Amp B (1.5 mg/kg) when it was administered bid (twice a day) by an i.p. route. When tested by an oral route, PPD (10 mg/kg) showed potent activity in this murine model of cryptococcosis with ~82% of organisms eliminated from the brain tissue, whereas Flu (15 mg/kg) reduced ~90% of the infection. In vitro results suggest that quaternary indolizidines were less toxic as compared to those of tertiary bases. PPD (20 mg/kg) did not cause any alteration in the plasma chemistry profiles. These results indicated that PPD was active in eliminating cryptococcal infection by oral and i.p. routes at lower doses compared to Amp B. or Flu.

Plasma But Not Cerebrospinal Fluid Interleukin 7 and Interleukin 5 Levels Pre-Antiretroviral Therapy Commencement Predict Cryptococcosis-Associated Immune Reconstitution Inflammatory Syndrome.

BACKGROUND: Patients with human immunodeficiency virus/AIDS-associated cryptococcal meningitis (CM) frequently experience clinical deterioration, known as cryptococcosis-associated immune reconstitution inflammatory syndrome (C-IRIS), upon initiation of antiretroviral therapy (ART). The immunological mechanisms underlying C-IRIS are incompletely defined and no reliable predictive biomarkers exist. We investigated whether plasma or cerebrospinal fluid (CSF) levels of cytokines and chemokines predicted C-IRIS and are potential predictive biomarkers. METHODS: Patients with CM who experienced C-IRIS (N = 27) upon ART initiation were compared to CD4+ T-cell count-matched patients without C-IRIS (N = 27). Plasma and CSF collected pre-ART were assayed for cytokines and chemokines using a 17-plex Luminex kit or enzyme-linked immunosorbent assay. Cox proportional hazards regression and principal component analyses were also performed. RESULTS: Plasma interleukin (IL) 2, IL-4, IL-5, IL-7, IL-17, interferon-γ, and tumor necrosis factor-α levels were higher in C-IRIS patients compared to controls (all P < .05), with IL-5 and IL-7 significant after Bonferroni-Holm correction. In multivariate Cox proportional hazards regression, high IL-5 (hazard ratio [HR], 5.76 [95% confidence interval {CI}, .77-43.0]; P = .088) and IL-7 (HR, 9.30 [95% CI, 1.96-44.0]; P = .005) were predictive of C-IRIS. Plasma IL-5 (P = .0008) and IL-10 (P = .0089) were lower in those who achieved CSF cryptococcal culture negativity compared to those with positive cultures pre-ART. There were no significant differences in CSF cytokine or chemokine levels between cases and controls. CONCLUSIONS: High plasma IL-5 and IL-7 levels pre-ART were associated with increased risk of developing C-IRIS. High IL-5 levels may reflect a Th2 environment associated with impaired clearance of cryptococci while high IL-7 levels may reflect IL-7/IL-7R pathway dysfunction in T cells, both of which could be associated with C-IRIS immunopathogenesis.

Clinical features of pulmonary cryptococcosis among patients with different levels of peripheral blood CD4+ T lymphocyte counts.

BACKGROUND: The clinical manifestation of pulmonary cryptococcosis varies notably between immunocompromised and immunocompetent patients. To better understand pulmonary cryptococcosis, we compared the clinical features of pulmonary cryptococcosis patients with or without decreased peripheral blood CD4+ T cell counts. METHODS: We retrospectively reviewed the medical records of 80 patients with cryptococcosis who had been treated in Jingling Hospital from January 2011 to January 2016. According to the normal range of peripheral blood CD4 + T-lymphocyte counts in our population, we chose CD4 = 378/µL as a cut-off value. RESULTS: The proportion of fever in the patients with decreased CD4+ T cells was higher than that of the patients with a normal amount of CD4+ T cells (86.7% vs 28.6%, P < 0.001). The incidence of clinical symptoms, such as cough (60.6% vs 64.7%, P = 0.729), chest pain (9.1% vs 26.5%, P = 0.064), and dyspnea (27.3% vs 23.5%, P = 0.725) showed no difference between patients with low CD4+ T cell counts and those with normal CD4+ T cell counts. The number of asymptomatic patients in the CD4+ T cell normal group was higher than that in the decreased CD4+ T cell group (17.1% vs 0%, P = 0.005). Nodules, masses, and halo signs were more common in the CD4+ T cell normal patients than in the low-CD4+ T cell patients (79.4% vs 54.5%, P = 0.03). The opposite trend was observed for cavitations (14.7% vs 51.5%, P = 0.001). The other CT findings, including pulmonary consolidation (P = 0.205), and pleural effusion (P = 0.641), did not differ significantly between the two groups. CONCLUSIONS: CD4+ T lymphocytes have a significant impact on the clinical and radiological characteristics of pulmonary cryptococcosis. The patients with normal CD4+ T cell counts were found to have less fever and more nodule-like radiographic findings. TRIAL REGISTRATION: 2011NJKY-023-01. Registered on January 10, 2011.

Cryptococcal infections in solid organ transplant recipients over a 15-year period at a state transplant center.

BACKGROUND: The aim of this research paper was to determine the incidence, risk factors, and clinical outcome of solid organ transplant (SOT) recipients diagnosed and treated for cryptococcosis at our institution. METHODS: Retrospective analysis of all patients with SOT diagnosed and treated for cryptococcal infection occurring between January 2001 and December 2015. RESULTS: Of 102 patients diagnosed with cryptococcal infection, 23 were SOT recipients. Renal transplant accounted for 22/23 cases, of which 13 had meningitis. The annual incidence of infection has risen significantly, and is now greater than 2/1000 prevalent renal transplant recipients. As expected, biochemical factors associated with meningitis include lower glucose on cerebrospinal fluid (CSF) analysis, median 2.4 vs 4.5 mmol/L (P=.02); CSF white blood cell median 50 vs 1/µL (P<.001); CSF protein, median 950 vs 335 mg/L (P=.04). Serum cryptococcal antigen titers were higher in the meningitis cohort, median 512 vs 32 (P=.03). Clinically, headache on admission (odds ratio: 9 [1.29-63.03], P=.03) and a prolonged length of stay (median of 36 vs 13 days) in the meningitis cohort (P=.02) were significant. CONCLUSION: Cryptococcal infection in SOT recipients remains rare; however, there has been a marked increase in cases since 2014. This study reveals a need for increased vigilance for a potential emerging infectious disease. It furthermore highlights the need for ongoing research to further aid early diagnosis, prognostication, management, and screening cost-effectiveness.

Cryptococcus laurentii diarrhea post hematopoietic stem cell transplant.

We report the recent isolation of Cryptococcus laurentii from the feces of a patient with Hodgkin's lymphoma who underwent autologous hematopoietic stem cell transplant (HSCT). The organism was identified using microscopic morphology, cultural characteristics, and biochemical tests including sugar assimilation. Minimum inhibitory concentration of various antifungals was determined by microbroth dilution method. The recovery of pure culture of C. laurentii from stool culture, and the patient's response to treatment with voriconazole support its potential etiological role. To the best of our knowledge, we report the first case of diarrhea caused by C. laurentii in an HSCT recipient.

Clinical characteristics and prognosis of pediatric cryptococcosis in Beijing Children's Hospital, 2002-2014.

Cryptococcosis is a rare pediatric disease. The aim of the study is to describe clinical characteristics and prognosis of pediatric cryptococcosis from 2002 to 2014 in Beijing Children's Hospital. A total of 53 cases of cryptococcosis were identified, 69.8% of which were males. The mean age was 7 years. Forty-one (77.4%) patients had no underlying conditions. Fever, headache, and vomiting were the most common symptoms. The most common sites were the central nervous system (CNS), followed by the lungs. Most patients received a combination of amphotericin B and fluconazole with or without flucytosine as their initial regimen. Twenty-seven patients received a follow-up and six patients (22.2%) had died. The factors associated with neurological complications or death were headache (P = 0.008), seizures (P = 0.006), visual impairment (P = 0.011), neck stiffness (P = 0.008), low erythrocyte sedimentation rate (ESR) (P = 0.024), and a cerebral spinal fluid (CSF) cryptococcal antigen titer ≥ 1:1024 (P = 0.038). CONCLUSIONS: The majority of cryptococcosis cases in China occurred in children without underlying conditions, causing multiple organ damage. The CNS was the most common site. Patients who had headaches, seizures, or high CSF antigen titers experienced neurological complications or died. What is known: • Cryptococcosis is a rare cause of infection in children. What is new: • This review gives a brief overview over pediatric cryptococcosis in China.

Combined antifungal therapy against systemic murine infections by rare Cryptococcus species.

Cryptococcus albidus and Cryptococcus laurentii are uncommon species of this genus that in recent decades have increasingly caused opportunistic infections in humans, mainly in immunocompromised patients; the best therapy for such infection being unknown. Using a murine model of systemic infection by these fungi, we have evaluated the efficacy of amphotericin B (AMB) at 0.8 mg/kg, administered intravenously, fluconazole (FLC) or voriconazole (VRC), both administered orally, at 25 mg/kg and the combination of AMB plus VRC against three C. albidus and two C. laurentii strains. All the treatments significantly reduced the fungal burden in all the organs studied. The combination showed a synergistic effect in the reduction in fungal load, working better than both monotherapies. The histopathological study confirmed the efficacy of the treatments.

Clinical features of pulmonary cryptococcosis in non-HIV patients in Japan.

OBJECTIVE: To clarify the clinical features of pulmonary cryptococcosis in Japanese non-HIV population. METHODS: Retrospective investigation of 151 pulmonary cryptococcosis cases between 1977 and 2012 was executed. The underlying disease (UDs), aggravating factors, radiological characteristics, and treatment were examined. RESULTS: Sixty-seven patients (44.4%) had no UDs. The common UDs were diabetes (32.1%) followed by hematologic disease (22.6%), and collagen disease (22.6%). Peripherally distributed pulmonary nodules/masses were most commonly seen. Lesions in the right middle lobe (p = 0.01) and air bronchogram (P = 0.05) were significantly more frequent, respectively, in patients with UDs than patients without them. Azoles were mainly selected for the patients without meningoencephalitis. Mean treatment duration for patients with and without UDs was 6.64 and 2.87 months, respectively. Patients whose pulmonary nodules improved after treatment continued to experience gradual reduction of cryptococcosis antigen titers, even if antigen titers were positive at the time of treatment cessation. The average time for antigen titers to become negative after treatment cessation was 13.1 and 10.7 months for patients with and without UDs, respectively. When groups were compared according to the presence of meningoencephalitis complications, deaths, and survivals, factors contributing to cryptococcosis prognosis included higher age, hypoproteinemia, hypoalbuminemia, steroid use, high C-reactive protein levels, and meningoencephalitis complications. CONCLUSIONS: It is crucial to consider the presence of UDs and meningoencephalitis for the choice of antifungals and treatment duration for cryptococcosis in non-HIV patients. Three- and six months-administration of azoles for pulmonary cryptococcosis with or without UDs, respectively is reasonable.

A 61-year-old man with disseminated intravascular coagulation: a case report.

BACKGROUND: Disseminated intravascular coagulation (DIC) can frequently be observed in patients with severe inflammatory response. It is still correlated with a poor prognosis. Activation of coagulation activity leads to occlusions of small vessels resulting in various organ failure symptoms. In addition, secondary fibrinolysis leads to an increased risk of bleedings and means a therapeutic dilemma. Here, we present a case of a 61-year-old Caucasian man with a severe case of DIC and its clinical complications. METHODS: We report the case of a man with a severe case of DIC. Data collection was performed retrospectively. RESULTS: We report the case of a 61-year-old Caucasian man with contact to pigeon droppings in his medical history. This was followed by a rhinopharyngitis, an exanthema, and a recurring priapism. Thrombotic occlusions were predominant on admission, and necrosis of the lower legs, the hands, and the genital resulted in amputation. Hypoperfusion of the rectum and the bladder lead to the creation of a descendostoma and an uretrostoma. Anticoagulation was managed by continuous infusion of unfractionated heparin and activated protein C supplementation. Long-term anticoagulation is managed with rivaroxaban. CONCLUSIONS: Cryptococcus soil inhalation may cause severe DIC resulting in extremity amputations; however, effective anticoagulation and activated protein C supplementation might extenuate the progress. As multiple complications might occur, an interdisciplinary cooperation is essential.

A sandwich chemiluminescent magnetic microparticle immunoassay for cryptococcal antigen detection.

BACKGROUND: Cryptococcosis is a global invasive mycosis associated with significant morbidity and mortality. Cryptococcal antigen (CrAg) testing from serum and cerebrospinal fluid (CSF) has been regarded as a gold standard for early diagnosis. This study aimed to develop and validate a rapid and sensitive sandwich chemiluminescent magnetic microparticle immunoassay (CMIA) for quantitative detection of CrAg in sera. RESEARCH DESIGN AND METHODS: CMIA is based on magnetic beads modified with capture antibodies and biotinylated antibodies and Streptavidin-polyHRP, where biotinylated antibodies functioned as the recognition element and Streptavidin-polyHRP as the signal component. Assay parameters were first optimized, and then assay performances were evaluated. RESULTS: Under optimized conditions, the total runtime of the CMIA was 22 min. The assay had a wide linear range (2 -10,000 ng/mL) and high analytical sensitivity (0.24 ng/mL), together with acceptable reproducibility, accuracy, and stability. Besides, it exhibited no cross-reactivity with other pathogens. Importantly, the assay showed 92.91% (95% CI, 80.97-93.02%) overall qualitative agreement with a commercial ELISA kit in a retrospective cohort of 55 cases with confirmed cryptococcal infection, and 72 controls without evidence of invasive fungal disease (IFD). CONCLUSION: These results demonstrated that the present study paved a novel strategy for reliable quantitative detection of CrAg in sera.

Changes of Th1/Th2 cytokines in immunocompetent patients with pulmonary cryptococcosis.

The aim of this study was to investigate the changes of Th1/Th2 cytokines in immunocompetent patients with pulmonary cryptococcosis (PC). Twenty immunocompetent patients with PC were identified by histopathological examination and were enrolled in the study along with the age- and gender-matched healthy controls. The serum concentrations of interferon-γ (IFN-γ), interleukin-4 (IL-4), and interleukin-12 (IL-12) were measured by enzyme-linked immunosorbent assay (ELISA). Peripheral blood mononuclear cells (PBMCs) in both groups were isolated and incubated with or without recombinant human IL-12 (rhIL-12) for 48 h, and the concentrations of IFN-g and IL-4 in the supernatant were measured by ELISA. Serum IFN-γ levels were greatly decreased in the patients compared with control groups (P < 0.01), whereas no significant difference was observed in serum IL-4 and IL-12 levels. The concentrations of IFN-γ and IL-4 in the supernatant of PBMCs without the stimulation of rhIL-12 showed no differences between the two groups. Treatment with rhIL-12 stimulated the release of IFN-γ, but not IL-4, into the supernatant of PBMCs in both groups, with a lower increase observed in the patients (4.3-fold) compared to that of controls (7.9-fold) (P < 0.01). Serum IFN-γ levels may be dampened in immunocompetent patients with PC with no significant changes in serum IL-4 and IL-12 levels. The deficiency in the response to IL-12 stimulation of Th1 cells may be one of the underlying mechanisms for the decline in serum IFN-γ levels.

[Changes of Th1/Th2 cytokines in immunocompetent patients with pulmonary cryptococcosis].

OBJECTIVE: To study the changes of Th1/Th2 cytokines in immunocompetent patients with pulmonary cryptococcosis (PC). METHODS: Twenty immunocompetent patients with PC were identified by histopathological examination and enrolled along with the age- and gender-matched healthy controls. The serum concentrations of interferon-γ (IFN-γ), interleukin-4 (IL-4) and interleukin-12 (IL-12) were measured by enzyme linked immunosorbent assay (ELISA). Peripheral blood mononuclear cells (PBMC) in both groups were isolated and incubated with or without recombinant human IL-12 (rhIL-12) for 48 hours, and the concentrations of IFN-γ and IL-4 in the supernatant were measured by ELISA. RESULTS: (1) Serum IFN-γ levels were significantly decreased in the patients compared with the control group [(14.5 ± 2.7) vs (81.8 ± 9.8) ng/L (t = 6.590, P < 0.01)], while no significant difference was observed in serum IL-12 and IL-4 levels [(2.5 ± 0.5) vs (2.52 ± 0.6) ng/L and (6.9 ± 1.3) vs (7.3 ± 1.5) ng/L, (t = 0.0035 and 0.2136, P > 0.05) ]. (2) The concentrations of IFN-γ and IL-4 in the supernatant of PBMC without rhIL-12 stimulation showed no differences between the 2 groups [(55.7 ± 13.6) vs (51.1 ± 17.5) ng/L and (5.1 ± 0.7) vs (5.0 ± 0.6) ng/L (t = 0.2979 and 0.0325, P > 0.05) ]. (3) Treatment with rhIL-12 stimulated the release of IFN-γ, but the increase in the patients [(4.3 ± 0.5) folds] was less compared with that in the controls [(7.9 ± 1.1) folds] (t = 3.01, P < 0.01) , while IL-4 concentration in the supernatant of PBMC was not increased in both groups[ (0.9 ± 0.4) vs (1.3 ± 0.4) folds (t = 0.7240, P > 0.05) ]. CONCLUSIONS: Serum Th1 cytokine (IFN-γ) levels may be dampened in immunocompetent patients with PC, without significant change in serum levels of Th2 cytokines (IL-4). Deficiency in the response to IL-12 stimulation of Th1 cells may be one of the underlying mechanisms for the decline in serum IFN-γ levels.