Asymptomatic cryptococcal antigen prevalence detected by lateral flow assay in hospitalised HIV-infected patients in São Paulo, Brazil.

OBJECTIVE: To determine the prevalence of asymptomatic cryptococcal antigen (CRAG) using lateral flow assay (LFA) in hospitalised HIV-infected patients with CD4 counts <200 cells/µl. METHODS: Hospitalised HIV-infected patients were prospectively recruited at Instituto de Infectologia Emilio Ribas, a tertiary referral hospital to HIV-infected patients serving the São Paulo State, Brazil. All patients were >18 years old without prior cryptococcal meningitis, without clinical suspicion of cryptococcal meningitis, regardless of antiretroviral (ART) status, and with CD4 counts <200 cells/µl. Serum CRAG was tested by LFA in all patients, and whole blood CRAG was tested by LFA in positive cases. RESULTS: We enrolled 163 participants of whom 61% were men. The duration of HIV diagnosis was a median of 8 (range, 1-29) years. 26% were antiretroviral (ART)-naïve, and 74% were ART-experienced. The median CD4 cell count was 25 (range, 1-192) cells/µl. Five patients (3.1%; 95%CI, 1.0-7.0%) were asymptomatic CRAG-positive. Positive results cases were cross-verified by performing LFA in whole blood. CONCLUSIONS: 3.1% of HIV-infected inpatients with CD4 <200 cells/µl without symptomatic meningitis had cryptococcal antigenemia in São Paulo, suggesting that routine CRAG screening may be beneficial in similar settings in South America. Our study reveals another targeted population for CRAG screening: hospitalised HIV-infected patients with CD4 <200 cells/µl, regardless of ART status. Whole blood CRAG LFA screening seems to be a simple strategy to prevention of symptomatic meningitis.

Toxoplasma gondii antigens: recovery analysis of tachyzoites cultivated in Vero cell maintained in serum free medium.

Vero cells have been used successfully in Toxoplasma gondii maintenance. Medium supplementation for culture cells with fetal bovine serum is necessary for cellular growth. However, serum in these cultures presents disadvantages, such as the potential to induce hypersensitivity, variability of serum batches, possible presence of contaminants, and the high cost of good quality serum. Culture media formulated without any animal derived components, designed for serum-free growth of cell lines have been used successfully for different virus replication. The advantages of protozoan parasite growth in cell line cultures using serum-free medium remain poorly studied. Thus, this study was designed to determine whether T. gondii tachyzoites grown in Vero cell cultures in serum-free medium, after many passages, are able to maintain the same antigenic proprieties as those maintained in experimental mice. The standardization of Vero cell culture in serum-free medium for in vitro T. gondii tachyzoite production was performed establishing the optimal initial cell concentration for the confluent monolayer formation, which was 1×10(6) Vero cell culture as initial inoculum. The total confluent monolayer formatted after 96 h and the best amount of harvested tachyzoites was 2.1×10(7) using parasite inoculum of 1.5×10(6) after 7 days post-infection. The infectivity of tachyzoites released from Vero cells maintained in serum-free medium was evaluated using groups of Swiss mice infected with cell-culture tachyzoites. The parasite concentrations were similar to those for mice infected with tachyzoites collected from other infected mice. The data from both in vivo and in vitro experiments showed that in at least 30 culture cell passages, the parasites maintained the same infectivity as maintained in vivo. Another question was to know whether in the several continued passages, immunogenic progressive loss could occur. The nucleotide sequences studied were the same between the different passages, which could mean no change in their viability in the lysate antigen. Thus, the antigen production by cell culture has clear ethical and cost-saving advantages. Moreover, the use of culture media formulated without any human or animal derived components, designed for serum-free growth of cell lines, successfully produced tachyzoites especially for antigen production.