Cryptococcal Antigen Research Articles

Global burden of disease of HIV-associated cryptococcal meningitis: an updated analysis

Cryptococcus is the most common cause of meningitis in adults living with HIV in sub-Saharan Africa. Global burden estimates are crucial to guide prevention strategies and to determine treatment needs, and we aimed to provide an updated estimate of global incidence of HIV-associated cryptococcal disease. We used 2014 Joint UN Programme on HIV and AIDS estimates of adults (aged >15 years) with HIV and antiretroviral therapy (ART) coverage. Estimates of CD4 less than 100 cells per μL, virological failure incidence, and loss to follow-up were from published multinational cohorts in low-income and middle-income countries. We calculated those at risk for cryptococcal infection, specifically those with CD4 less than 100 cells/μL not on ART, and those with CD4 less than 100 cells per μL on ART but lost to follow-up or with virological failure. Cryptococcal antigenaemia prevalence by country was derived from 46 studies globally. Based on cryptococcal antigenaemia prevalence in each country and region, we estimated the annual numbers of people who are developing and dying from cryptococcal meningitis. We estimated an average global cryptococcal antigenaemia prevalence of 6·0% (95% CI 5·8-6·2) among people with a CD4 cell count of less than 100 cells per μL, with 278 000 (95% CI 195 500-340 600) people positive for cryptococcal antigen globally and 223 100 (95% CI 150 600-282 400) incident cases of cryptococcal meningitis globally in 2014. Sub-Saharan Africa accounted for 73% of the estimated cryptococcal meningitis cases in 2014 (162 500 cases [95% CI 113 600-193 900]). Annual global deaths from cryptococcal meningitis were estimated at 181 100 (95% CI 119 400-234 300), with 135 900 (75%; [95% CI 93 900-163 900]) deaths in sub-Saharan Africa. Globally, cryptococcal meningitis was responsible for 15% of AIDS-related deaths (95% CI 10-19). Our analysis highlights the substantial ongoing burden of HIV-associated cryptococcal disease, primarily in sub-Saharan Africa. Cryptococcal meningitis is a metric of HIV treatment programme failure; timely HIV testing and rapid linkage to care remain an urgent priority. None.

Examination of cryptococcal glucuronoxylomannan antigen in bronchoalveolar lavage fluid for diagnosing pulmonary cryptococcosis in HIV-negative patients.

We clarified the performance of a cryptococcal glucuronoxylomannan (GXM) antigen test using bronchoalveolar lavage fluid (BALF) samples, in an HIV-negative Japanese population. Between March 2008 and December 2014, we examined cryptococcal GXM antigens in both serum and BALF specimens from 429 cases at Nagasaki University hospital. The diagnoses, underlying diseases, chest computed tomography findings, and cryptococcal GXM antigen test results were retrospectively investigated. Twenty-three patients were confirmed to have pulmonary cryptococcosis, another six were clinically diagnosed with cryptococcosis because they were seropositive for the GXM antigen, and five possible cryptococcosis cases had BALF samples that were positive for the GXM antigen and serum samples that were negative. The test's sensitivities for detecting cryptococcal GXM antigens in serum and BALF samples, for confirmed cases, were 73.9% and 82.6%, respectively, and their respective specificities were 98.5% and 97.8%. Three of the five putative patients with cryptococcosis were treated with antifungal agents; the pulmonary lesions decreased in size in all treated patients. Both the BALF and serum GXM antigen titers showed positive correlations with the lesion sizes; however, the serum antigen titers showed a higher correlation (r = 0.490, P = .0033) than did the BALF titres (r = 0.312, P = .0724). The rate of GXM-positive BALF samples was higher than the rate for serum samples, especially for patients with pulmonary lesion diameters ≤25mm. Testing for the presence of the cryptococcal GXM antigen in BALF specimens might contribute to the early diagnosis of pulmonary cryptococcosis.

Cost-effectiveness of CRAG-LFA screening for cryptococcal meningitis among people living with HIV in Uganda

BackgroundCryptococcal meningitis (CM) constitutes a significant source of mortality in resource-limited regions. Cryptococcal antigen (CRAG) can be detected in the blood before onset of meningitis. We sought to determine the cost-effectiveness of implementing CRAG screening using the recently developed CRAG lateral flow assay in Uganda compared to current practice without screening.MethodsA decision-analytic model was constructed to compare two strategies for cryptococcal prevention among people living with HIV with CD4 < 100 in Uganda: No cryptococcal screening vs. CRAG screening with WHO-recommended preemptive treatment for CRAG-positive patients. The model was constructed to reflect primary HIV clinics in Uganda, with a cohort of HIV-infected patients with CD4 < 100 cells/uL. Primary outcomes were expected costs, DALYs, and incremental cost-effectiveness ratios (ICERs). We evaluated varying levels of programmatic implementation in secondary analysis.ResultsCRAG screening was considered highly cost-effective and was associated with an ICER of $6.14 per DALY averted compared to no screening (95% uncertainty range: $-20.32 to $36.47). Overall, implementation of CRAG screening was projected to cost $1.52 more per person, and was projected to result in a 40% relative reduction in cryptococcal-associated mortality. In probabilistic sensitivity analysis, CRAG screening was cost-effective in 100% of scenarios and cost saving (ie cheaper and more effective than no screening) in 30% of scenarios. Secondary analysis projected a total cost of $651,454 for 100% implementation of screening nationally, while averting 1228 deaths compared to no screening.ConclusionCRAG screening for PLWH with low CD4 represents excellent value for money with the potential to prevent cryptococcal morbidity and mortality in Uganda.

Clinical characteristics and prognosis of disseminated cryptococcosis in children

Objective To analyze the clinical characteristics and prognosis of disseminated cryptococcosis in children. Methods The data of disseminated cryptococcosis inpatients were reviewed retrospectively at Beijing Children′s Hospital, Capital Medical University, from January 2002 to September 2014.The demographic data, clinical manifestations, laboratory findings, imaging, antifungal treatments and outcomes of all the patients were analyzed. Results Overall 25 children with disseminated cryptococcosis were enrolled including 17 boys(68.0%). The average age was 7 years old.Four cases (16.0%) had underlying conditions, among them 1 case had human immunodeficiency virus(HIV)-positive.The median time to diagnosis was 32 (23-47) days, 44.0% of the patients (11 cases) were misdiagnosed, and 8 cases misdiagnosed as tuberculosis.All patients had fever.Other common clinical manifestations included cough(16 cases), headache(10 cases), vomiting(10 cases), altered mental status (6 cases) and stomachache (6 cases). Respiratory system involvement was seen in all cases, central nervous system was involved in 18 cases, other organ involvement included lymph nodes, spleen, liver, kidney, skin, skeleton and costicartilage.Amphotericin B (AmB) + Fluconazole (Flu) ±5-flucytosine (5-FC) was the most common therapy (15 cases), Flu±5-FC for 6 cases, AmB±5-FC for 2 cases, Voricanazole (VOR) for 1 case.Sixteen cases (66.7%) got recovery/improved on discharge, 8 cases (33.3%) rejected to the advise and discharged with treatment failure, and the HIV-infected patient transferred to a special hospital.Fifteen patients (60.0%) were followed up, and 13 cases (87.0%) showed recovery, but 2 died in the long-term prognosis. Conclusions Disseminated cryptococcosis in children is more common in school-age boys.Most patients are without underlying conditions.Disseminated cryptococcosis can cause multiple organ damage.Patients with prolonged fever, cough, headache, with or without underlying diseases, can be suspected as disseminated cryptococcosis.Blood and cerebrospinal fluid culture for fungus, cryptococcal antigen should be performed for early diagnosis and treatment. Key words: Cryptococcus; Disseminated cryptococcosis; Clinical feature; Prognosis

Looking for fungi in all the right places: screening for cryptococcal disease and other AIDS-related mycoses among patients with advanced HIV disease.

As HIV treatment programmes scale up to meet the UNAIDS 90-90-90 goals, care must be taken to start antiretroviral treatment safely in patients with advanced disease (CD4 counts <200 cells/μl) who are simultaneously at risk for opportunistic infections and immune reconstitution inflammatory syndrome. Invasive fungal diseases pose a great threat at this critical time point, though the development of inexpensive and highly accurate rapid diagnostic tests has changed the approach HIV programmes are taking to reduce the high mortality associated with these opportunistic infections. This article summarizes recent advances and findings in fungal opportunistic infection diagnostics with a focus on screening to prevent cryptococcal meningitis. Cryptococcal antigen (CrAg) screening using a lateral flow assay platform is cost-effective and feasible to implement as either a laboratory reflex or point-of-care test. Recent CrAg screening pilots have elucidated the varying prevalence of cryptococcal antigenemia across geographic regions, which may aid programme planning. Evidence from recently completed clinical trials provides a strong motivation for the use of CrAg titer to refine treatment options for patients with subclinical cryptococcal disease. Although several operational barriers to programme effectiveness still need to be addressed, the utility of CrAg screening using inexpensive and accurate antigen assays has been demonstrated in real-world HIV programmes, paving the way for development and testing of other fungal opportunistic infection screening strategies and for an integrated advanced HIV disease testing package to reduce AIDS mortality and ensure safe antiretroviral treatment initiation.

Establishing a cost-per-result of laboratory-based, reflex Cryptococcal antigenaemia screening (CrAg) in HIV+ patients with CD4 counts less than 100 cells/μl using a Lateral Flow Assay (LFA) at a typical busy CD4 laboratory in South Africa.

IntroductionCryptococcal meningitis is a major cause of mortality and morbidity in countries with high HIV prevalence, primarily affecting patients whose CD4 are < = 100 cells/μl. Routine Cryptococcal Antigen (CrAg) screening is thus recommended in the South African HIV treatment guidelines for all patients with CD4 counts < = 100 cells/μl, followed by pre-emptive anti-fungal therapy where CrAg results are positive. A laboratory-based reflexed CrAg screening approach, using a Lateral Flow Assay (LFA) on remnant EDTA CD4 blood samples, was piloted at three CD4 laboratories.ObjectivesThis study aimed to assess the cost-per-result of laboratory-based reflexed CrAg screening at one pilot CD4 referral laboratory.MethodsCD4 test volumes from 2014 were extracted to estimate percentage of CD4 < = 100 cells/μl. Daily average volumes were derived, assuming 12 months per/year and 21.73 working days per/month. Costing analyses were undertaken using Microsoft Excel and Stata with a provider prospective. The cost-per-result was estimated using a bottom-up method, inclusive of test kits and consumables (reagents), laboratory equipment and technical effort costs. The ZAR/$ exchange of 14.696/$1 was used, where applicable. One-way sensitivity analyses on the cost-per-result were conducted for possible error rates (3%– 8%, reductions or increases in reagent costs as well as test volumes (ranging from -60% to +60%).ResultsThe pilot CD4 laboratory performed 267000 CD4 tests in 2014; ~ 9.3% (27500) reported CD4< = 100 cells/μl, equivalent to 106 CrAg tests performed daily. A batch of 30-tests could be performed in 1.6 hours, including preparation and analysis time. A cost-per-result of $4.28 was reported, with reagents contributing $3.11 (72.8%), while technical effort and laboratory equipment overheads contributed $1.17 (27.2%) and $0.03 (<1%) respectively. One-way sensitivity analyses including increasing or decreasing test volumes by 60% revealed a cost-per-result range of $3.84 to $6.03.ConclusionA cost-per-result of $4.28 was established in a typical CD4 service laboratory to enable local budgetary cost projections and programmatic cost-effectiveness modelling. Varying reagent costs linked to currency exchange and varying test volumes in different levels of service can lead to varying cost-per-test and technical effort to manage workload, with an inverse relationship of higher costs expected at lower volumes of tests.

Differences in Immunologic Factors Among Patients Presenting with Altered Mental Status During Cryptococcal Meningitis.

Altered mental status in cryptococcal meningitis results in poorer survival, but underlying causes of altered mentation are poorly understood. Within two clinical trials, we assessed risk factors for altered mental status (GCS score<15) considering baseline clinical characteristics, CSF cytokines/chemokines, and antiretroviral therapy. Among 326 enrolled participants, 97 (30%) had GCS<15 and these patients had lower median CSF cryptococcal antigen titers (P = .042) and CCL2 (P = .005) but higher opening pressures (320 vs. 269 mm H2O; P = .016), IL-10 (P = .044), and CCL3 (P = .008) compared with persons with GCS=15. Altered mental status may be associated with host immune response rather than Cryptococcus burden.

Primary Prophylaxis for Cryptococcosis With Fluconazole in Human Immunodeficiency Virus-Infected Patients With CD4 T-Cell Counts

A prospective observational cohort study was conducted in 302 human immunodeficiency virus-infected patients who had a CD4 T-cell count <100 cells/µL and negative serum cryptococcal antigen initiating antiretroviral therapy in a resource-limited setting. During 2-year follow-up, there were no differences of survival rates and occurrences of newly diagnosed cryptococcosis between patients with and without fluconazole for primary prophylaxis of cryptococcosis.

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

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. Retrospective analysis of all patients with SOT diagnosed and treated for cryptococcal infection occurring between January 2001 and December 2015. 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 4.5mmol/L (P=.02); CSF white blood cell median 50 vs 1/μL (P<.001); CSF protein, median 950 vs 335mg/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 13days) in the meningitis cohort (P=.02) were significant. 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.

Cryptococcal neorformans Antigenemia among HIV-Infected Patients in North Eastern Nigeria

Background: Cryptococcus neoformans infection is a common fungal infection that is cosmopolitan in distribution and causes a life-threatening disease among HIV-infected patients especially in advanced disease. Screening for cryptococcal antigen (CrAg) in patients enrolling in ART programs may identify those at risk of cryptococcal meningitis and permit targeted use of pre-emptive therapy. Methods: This was a cross survey study of 215 consecutive HIV-infected patients at an ART treatment centre were screened for cryptococcal antigenaemia using the cryptococcalantigen lateral flow assay (CrAg LFA). Study subjects were assessed for clinical features of Cryptococcus neoformans infection. CD4+ cell count, haemoglobin concentration as well as HIV-1 RNA viral load was also compared between cryptococcalantigen positive and negatives. Results: 37 (16.7%) patients were positive for cryptococcal antigenaemia. Cryptococcal antigen positive subjects had a lower median CD4+ cell count (58 cells/uL vs. 273 cells/uL, p<0.001) and higher median viral load (log 3.6 copies/mL vs. log2.3 copies/mL, p=0.05) compared to cryptococcal antigen negative subjects. The commonest predictors of positive CrAg status in the study were; low BMI, low Hb, fever and cough. Conclusion: The study has shown the prevalence of cryptococcal antigenaemia to be high among the study subjects attending this service. Therefore, screening of patients receiving care at this centre may help in identifying individuals who are at risk of cryptococcalmeningitis for prophylactic treatment.

Survival in HIV-infected Asymptomatic Cryptococcal Antigenemia without CSF Positivity Treated with Fluconazole Did Not Differ from Cryptococcal Antigen (CrAg) Negative with CD4 &lt;150.

Survival in HIV-infected Asymptomatic Cryptococcal Antigenemia without CSF Positivity Treated with Fluconazole Did Not Differ from Cryptococcal Antigen (CrAg) Negative with CD4 &lt;150.

High Prevalence of Cryptococcal Antigenaemia and Disseminated Cryptococcal Disease Amongst Patients with Advanced HIV Disease in Pune, India

Background The World Health Organization (WHO) recommends routine cryptococcal antigen (CrAg) screening in patients with advanced HIV disease initiating antiretroviral treatment (ART). India has yet to adopt this strategy as the burden of cryptococcal disease is unknown.Methods This was a prospective cohort study conducted between March 1, 2010 and March 1, 2017 at three private hospitals in Pune, India. All HIV-positive patients (symptomatic and asymptomatic) with CD4 counts ≤ 200 cells/µL were screened for serum cryptococcal antigen. Serum CrAg was measured using latex agglutination (LA) test. Both, ART naïve and ART experienced patients were included in the study. All HIV infected patients who were CrAg-positive were offered lumbar puncture (LP) and worked up for disseminated cryptococcal disease.ResultsA total of 785 HIV-positive patients (24.2% females) were included. Median age of cohort was 42 years (IQR, 35–49) and median CD4 count was 79 cells/mm3 (IQR, 37–82). 182/785(23.2%) patients were ART experienced. A total of 6.75% (53/785) of patients were CrAg positive in serum. Thirty-nine of 53 (73.6%) patients with positive serum cryptococcal antigen test had CD4 count ≤100 cells/mm3 while 14/53 (26.4%) had CD4 between 100 and 200 cells/mm3. Cerebrospinal fluid (CSF) CrAg was positive in 44/53(83%) patients. Two of 53(3.78%) had non-CNS, diffuse pulmonary cryptococcal disease and 7/53(13.2%) patients had isolated cryptococcal antigenemia. Patients with cryptococcal meningitis and crptococcal pulmonary disease were treated with Amphotericin-B plus oral Fluconazole. Patients with isolated cryptococcal antigenemia were treated with oral Fluconazole. Mortality at 6 months for patients with positive CrAg test was 22.6% (12/53).Conclusion We found 6.75% prevalence of cryptococcaemia amongst HIV patients with CD4 <200 cells/mm3. Given the high fatality rates observed, routine CrAg screening should be considered for all Indians with advanced HIV disease.Disclosures All authors: No reported disclosures.

Fungal Opportunistic Pneumonias in HIV/AIDS Patients: An Indian Tertiary Care Experience.

Opportunistic pneumonias are a major cause of mortality and morbidity in Human Immunodeficiency Virus (HIV) reactive patients. Despite the significant role that fungi play in causation of this opportunistic mycoses, very few Indian studies have attempted to investigate the burden and aetiological spectrum of HIV/AIDS-associated fungal pneumonias. To document the prevalence of fungal aetiology in HIV/AIDS-related opportunistic pneumonias in an Indian setting; and to elucidate the various fungal opportunists responsible for the same. The present study was a prospective, cross-sectional analysis conducted at Maulana Azad Medical College and associated Lok Nayak Hospital, New Delhi from October 2008 to September 2011. Expectorated sputa were collected from 71 HIV reactive patients with a clinical diagnosis of pneumonia and subjected to direct microscopic examination employing Gram stain, 10% KOH wet mount and India ink preparation. In addition, direct immunofluorescence of sputum samples was performed for detection of cysts and trophozoites of Pneumocystis carinii. Also, each sputum sample was inoculated in duplicate onto Sabouraud Dextrose Agar (SDA) for culture. A blood sample was drawn from each patient and a battery of serological tests was performed, including Cryptococcal Antigen Latex Agglutination System (CALASTM) for detection of cryptococcal capsular polysaccharide antigen; Platelia™ Aspergillus EIA for detection of Aspergillus galactomannan antigen; SERION ELISA antigenCandida for detection of Candida antigen and Histoplasma DxSelect™ for detecting antibodies to Histoplasma species. Descriptive statistics were employed to depict results as proportions and figures. Further, arithmetic mean and standard deviation were calculated for central tendencies and median for non-normal/skewed distributions. A definite fungal aetiology was established in 25 (35.2%) of 71 HIV reactive patients with pneumonic involvement. Of these, sputa of 21 patients yielded single fungal isolates, while mixed fungal isolates were reported in four patients. Pneumocystis carinii was the predominant fungal pathogen isolated in our study and was reported in 14 (19.7%) patients. Pulmonary aspergillosis was reported in 7 (9.9%) patients, with Aspergillus flavus (4), Aspergillus fumigatus (2) and Aspergillus niger (1) being the commonly recovered Aspergillus species. Candida pneumonia was documented in 6 (8.5%) patients and the Candida species isolated included Candida albicans in four, Candida glabrata in one and Candida tropicalis in one of these six patients respectively. Pulmonary cryptococcosis was diagnosed in 2 (2.8%) patients; a coexisting cryptococcal meningitis was documented in one of them. Furthermore, antibodies against Histoplasma species were detected in 21 (29.6%) cases suggesting its possible aetiological role. Fungal opportunistic pneumonias are common in HIV reactive patients in Indian setting and warrant a prompt and accurate diagnostic evaluation in the form of a combination of microbiological, serological and histopathological techniques, for an effective prophylactic and therapeutic management.

Cryptococcal meningitis in HIV (AIDS) patients: A Mini Review

Meningitis is a major health problem causing inflammation of the protective membranes covering the brain and spinal cord, collectively known as the meninges . Meningitis is generally caused by Meningococcus , Streptococcus pyogenes bacterial species causing bacterial meningitis: BM), Mycobacterium tuberculosis (Tubercular meningitis: TBM), Cryptococcus neoformans (fungal meningitis: CM). In recent times the CM and TBM are two most common types of chronic infectious meningitis. In adults, HIV-associated Cryptococcal meningitis has been reported in many areas of the world having high HIV seroprevalence i.e. in Sub-Saharan Africa. Cryptococcal meningitis is the fourth most commonly recognized cause of life-threatening infection among AIDS patients.Despite availability of antiretroviral therapy, the rise in rate of infections due to HIV/AIDS, suppress the cell mediated immune system and subjects are predisposed for opportunistic infections. Additionally, In AIDS patients, it is characterized by lack of meningeal signs and diminished inflammatory response. Diagnosis of CM is generally based on the collection of CSF sample and/or blood samples. Initially the CSF samples are processed for ‘gold standard’ culture methods, microscopy and India ink preparation. Serologically, Latex agglutination test (LAT) is the most commonly used for detection of Cryptococcal capsular antigen. Enzyme immunoassay (EIA) is another serological tool for detection of capsular polysaccharide antigens of C. neoformans in CSF. DOI: 10.21276/AABS.2017.1344

Screening of Invasive Fungal Infections by a Real-Time Panfungal (pan-ACF) Polymerase Chain Reaction Assay in Patients with Haematological Malignancy

Background: Invasive fungal infection (IFI) is a fatal infection in haematology patients. There is an urgent need for reliable screening methods facilitating timely diagnosis and treatment. A real-time panfungal polymerase chain reaction (PCR) assay based on TaqMan technology targeting 18S ribosomal RNA gene was used to screen whole blood specimen obtained from series of Haematology malignancy patients for IFIs. Materialsand Methods: The panfungal (Pan-ACF) assay was employed to investigate specimen from 133 patients in duplicate with suspected IFI. In addition twenty healthy subjects and twenty patients with bacterial infections were taken as control. The patients with suspected IFI were also diagnosed by conventional methods including direct microscopy, culture techniques and antigen detection (galactomannan antigen ELISA and latex agglutination for cryptococcal antigen). The results of molecular testing were evaluated in relation to the criteria proposed by the European Organization for Research and Treatment of Cancer and patients were classified as having proven and probable IFD. Results: Of 133 patients, 89 had proven, 18 had probable and 26 had possible IFI. One hundred four samples were reverse transcription-PCR positive. Of 89 proven cases, 84 were panfungal PCR positive. These 84 cases included 82 cases which revealed growth on fungal blood culture and two cases were negative on fungal blood culture. Of the 82 cases which revealed growth on culture: 74 grew Candida in culture, 3 grew Fusarium solani, 5 grew Aspergillus species on blood culture. The later five were also galactomannan antigen positive. The five specimen which were negative on panfungal PCR, two grew Trichosporon asahii, one grew Candida rugosa and two grew as Cryptococcus neoformans var. neoformans. Of the 18 probable cases, 18 were panfungal PCR positive. These were also galactomannan antigen positive. The sensitivity and specificity of panfungal PCR in proven cases were 94.3% and 95.2%, respectively. The positive and negative predictive values proven cases were 97.6% and 88.9%, respectively. Conclusions: The panfungal (Pan-ACF) real-time PCR assay can detect common fungal genera and it may be used as an adjunct to conventional methods for screening of IFI.

In Vitro Characterization of the Neurapheresis™ System for the Treatment of Cryptococcal Meningitis

BackgroundCryptococcal Meningitis (CM) is the most common cause of fungal meningitis in adults. Treatment for CM is an induction, consolidation, and maintenance approach with antifungal agents. but is associated with continued high morbidity and mortality. Here we describe the in vitro characterization of a catheter-based extracorporeal filtration system (Neurapheresis™) as an alternative mechanical intervention for the filtration of C. neoformans cells, polysaccharide antigen, and inflammatory mediators from infected cerebrospinal fluid (CSF).MethodsH99, a clinical strain of C. neoformans, was grown overnight in YPD before being transferred to diluted Saboraud/MOPS media for 24 hours to induce cell proliferation and capsule growth, respectively. Cells were diluted to clinically relevant concentrations (1 × 107 and 1 × 105 cells/mL) in 150 mL of Saboraud/MOPS and passed through the closed-loop system with either 100 or 5 kDa tangential flow filters. Samples were taken every full CSF volume filtration cycle (150 mL) for quantification of yeast load, antigen, and cytokines. Infected human CSF was used to obtain cytokine data.ResultsBoth tangential flow filter sizes thoroughly cleared yeasts. Over 24 cycles, we consistently observed a 5-log drop (≥99%) in colony forming units (CFUs), which resulted in complete elimination at a starting concentration of 1 × 105 cells/mL. Both 100 and 5kDa achieved a substantial antigen reduction (using CrAg LFA [initial titer]-[final titer]; [1:105]–[1:104] and [1:105]–[1:102], respectively). A similar reduction in cytokine levels (IL-1ra, IL-6, TNF, CRP, and CXCL10) in infected human CSF was also achieved (100 kDa reduced all cytokines except IL-1ra by >95% baseline, and 5kDa removed >95% of all quantified cytokines).ConclusionContinuous filtration via Neurapheresis is capable of eliminating CSF CFU burden in an in vitro CM model. Future iterations may include adjunctive infusions with drug therapies to further accelerate eradication of yeasts. Significant reduction of cryptococcal antigen and inflammatory cytokines also has potential for controlling the neuro-inflammatory storm that accompanies CM.Disclosures B. Hedstrom, Minnetronix, Inc.: Employee, Salary; L. Zitella Verbick, Minnetronix, Inc.: Employee, Salary; A. Mccabe, Minnetronix, Inc.: Employee, Salary; S. P. Lad, Minnetronix, Inc.: Collaborator and Scientific Advisor, Licensing agreement or royalty, Research grant and Research support

High Prevalence of Cryptococcal Antigenaemia amongst Asymptomatic Advanced HIV Patients in Pune, India

Background: The World Health Organization recommends routine cryptococcal antigen (CrAg) screening in advanced AIDS patients initiating antiretroviral treatment (ART). India has yet to adopt this strategy as the burden of cryptococcal antigenaemia is unknown. Methods: A prospective study was conducted in a large public sector ART centre and the inpatient wards of Sassoon Hospital, Pune, India. All consenting patients >18 years of age with CD4 count <100 cells/mm3 were screened for CrAg by latex agglutination assay. Those with positive CrAg underwent cerebrospinal fluid analysis, chest radiograph and abdominal ultrasound to rule out cryptococcal disease. The impact of CrAg positivity on all-cause mortality was assessed by logistic regression analysis. Results: Amongst 208 AIDS patients with CD4 cells <100 cells/mm3 who underwent CrAg testing, median age was 40 (interquartile range [IQR], 35–49) years, 134 (64%) were male and median CD4 count was 64.5 cells/mm3 (IQR, 37–82). Overall, 16 (8%, 95% confidence interval [CI], 4–12) patients were positive for CrAg, of which 8 (50%) had CD4 cells <50 cells/mm3 and 3 (19%) CrAg-positive patients had incidental cryptococcal meningitis. At 6-month follow-up, the case fatality rate was higher amongst CrAg-positive patients (38%) compared with CrAg-negative patients (18%) (P = 0.06). After adjusting for age, sex, CD4 count and ART, there was a trend towards increased all-cause mortality (adjusted OR, 3.18, 95% CI, 0.60–16.88, P = 0.17). Conclusions: We found an 8% prevalence of cryptococcaemia amongst adult AIDS patients with CD4 cells <100 cells/mm3. Given the high fatality rates observed, routine screening for CrAg should be considered for all Indian persons with advanced HIV disease.

Methodology for Anti-Cryptococcal Vaccine Development.

Cryptococcus neoformans and Cryptococcus gattii, the predominant etiological agents of cryptococcosis, are fungal pathogens that cause disease ranging from a mild pneumonia to life-threatening infections of the central nervous system (CNS). C. neoformans is widely considered an opportunistic fungal pathogen which targets individuals with impaired immune systems, while C. gattii is predominantly associated with fungal infections in immunocompetent individuals. However, C. neoformans and C. gattii have certainly been identified as the causative agent of cryptococcosis in both immune compromised and immune competent individuals. Cell-mediated immunity (CMI) by T-helper (Th) 1-type CD4+ T cells is the predominant host defense mechanism against cryptococcosis. Consequently, there has been great interest in identifying cryptococcal antigens that elicit protective CMI against Cryptococcus infection. Although many different cryptococcal proteins have been shown to stimulate potent cellular responses, there remains no standardized vaccine available for the prevention of cryptococcal infections in humans. Several studies have identified immunodominant antigens that may serve as attractive candidates for the development of novel subunit vaccines for the treatment and/or the prevention of cryptococcosis. The purpose of this chapter is to describe one methodology to screen and isolate cryptocococcal proteins that induce protective immune responses against cryptococossis.

Integrated therapy for HIV and cryptococcosis.

Cryptococcosis has been one of the most common opportunistic infections and causes of mortality among HIV-infected patients, especially in resource-limited countries. Cryptococcal meningitis is the most common form of cryptococcosis. Laboratory diagnosis of cryptococcosis includes direct microscopic examination, isolation of Cryptococcus from a clinical specimen, and detection of cryptococcal antigen. Without appropriate treatment, cryptococcosis is fatal. Early diagnosis and treatment is the key to treatment success. Treatment of cryptococcosis consists of three main aspects: antifungal therapy, intracranial pressure management for cryptococcal meningitis, and restoration of immune function with antiretroviral therapy (ART). Optimal integration of these three aspects is crucial to achieving successful treatment and reducing the mortality. Antifungal therapy consists of three phases: induction, consolidation, and maintenance. A combination of two drugs, i.e. amphotericin B plus flucytosine or fluconazole, is preferred in the induction phase. Fluconazole monotherapy is recommended during consolidation and maintenance phases. In cryptococcal meningitis, intracranial pressure rises along with CSF fungal burden and is associated with morbidity and mortality. Aggressive control of intracranial pressure should be done. Management options include therapeutic lumbar puncture, lumbar drain insertion, ventriculostomy, or ventriculoperitoneal shunt. Medical treatment such as corticosteroids, mannitol, and acetazolamide are ineffective and should not be used. ART has proven to have a great impact on survival rates among HIV-infected patients with cryptococcosis. The time to start ART in HIV-infected patients with cryptococcosis has to be deferred until 5 weeks after the start of antifungal therapy. In general, any effective ART regimen is acceptable. Potential drug interactions between antiretroviral agents and amphotericin B, flucytosine, and fluconazole are minimal. Of most potential clinical relevance is the concomitant use of fluconazole and nevirapine. Concomitant use of these two drugs should be cautious, and patients should be monitored closely for nevirapine-associated adverse events, including hepatotoxicity. Overlapping toxicities of antifungal and antiretroviral drugs and immune reconstitution inflammatory syndrome are not uncommon. Early recognition and appropriate management of these consequences can reinforce the successful integrated therapy in HIV-infected patients with cryptococcosis.

Cryptococcal meningitis: epidemiology, immunology, diagnosis and therapy.

HIV-associated cryptococcal meningitis is by far the most common cause of adult meningitis in many areas of the world that have high HIV seroprevalence. In most areas in Sub-Saharan Africa, the incidence of cryptococcal meningitis is not decreasing despite availability of antiretroviral therapy, because of issues of adherence and retention in HIV care. In addition, cryptococcal meningitis in HIV-seronegative individuals is a substantial problem: the risk of cryptococcal infection is increased in transplant recipients and other individuals with defects in cell-mediated immunity, and cryptococcosis is also reported in the apparently immunocompetent. Despite therapy, mortality rates in these groups are high. Over the past 5 years, advances have been made in rapid point-of-care diagnosis and early detection of cryptococcal antigen in the blood. These advances have enabled development of screening and pre-emptive treatment strategies aimed at preventing the development of clinical infection in patients with late-stage HIV infection. Progress in optimizing antifungal combinations has been aided by evaluation of the clearance rate of infection by using serial quantitative cultures of cerebrospinal fluid (CSF). Measurement and management of raised CSF pressure, a common complication, is a vital component of care. In addition, we now better understand protective immune responses in HIV-associated cases, immunogenetic predisposition to infection, and the role of immune-mediated pathology in patients with non-HIV associated infection and in the context of HIV-associated immune reconstitution reactions.