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.

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.

Cryptococcal meningitis is the most common cause of opportunistic meningitis in HIV-infected patients in Brazil and causes unacceptable high mortality rates. In this study, HIV-infected patients with a first episode of culture-proven cryptococcal meningitis in cerebrospinal fluid (CSF) were prospectively included in order to evaluate sensitivity of cryptococcal antigen (CrAg) lateral flow assay (LFA) in serum, CSF, whole blood (fingerstick), and fresh urine. In addition, HIV-infected patients with other neurological confirmed diseases were included in order to evaluate the specificity of CrAg LFA in serum. Twenty patients with cryptococcal meningitis were included and in 19 of them, CrAg LFA in CSF, serum, and whole blood were positive (95% sensitivity). In 18 patients, India ink test was positive in CSF (90% sensitivity), and in 16 cases, CrAg LFA was positive in urine (80% sensitivity). Thirty-six HIV-infected patients with other neurological diseases had negative results of CrAg LFA in serum (100% specificity). In conclusion, CrAg LFA in serum, CSF, and whole blood showed high sensitivity and specificity. Whole blood CrAg LFA seems to be a good and reliable strategy to improve AIDS-related cryptococcal meningitis diagnosis in Brazil.

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BackgroundToxoplasmosis was recently included as a neglected disease by the Center for Disease Control. Ocular toxoplasmosis is one clinical presentation of congenital or acquired infection. The laboratory diagnosis is being used worldwide to support the clinical diagnosis and imaging. The aim of this study was to evaluate the use of serology and molecular methods to monitor acute OT in immunocompetent patients during treatment.MethodsFive immunocompetent patients were clinically diagnosed with acute OT. The clinical evaluation was performed by ophthalmologic examination using the Early Treatment Diabetic Retinopathy Study, best-corrected visual acuity, slit lamp biomicroscopy, fundoscopic examination with indirect binocular ophthalmoscopy color fundus photography, fluorescein angiography and spectral optical coherence tomography (OCT). Serology were performed by ELISA (IgA, IgM, IgG) and confirmed by ELFA (IgG, IgM). Molecular diagnoses were performed in peripheral blood by cPCR using the Toxoplasma gondiiB1 gene as the marker. Follow-up exams were performed on day +15 and day +45.ResultsOnly five non-immunocompromised male patients completed the follow up and their data were used for analysis. The mean age was 41.2 ± 11.3 years (median: 35; range 31–54 years). All of them were positive for IgG antibodies but with different profiles for IgM and IgA, as well as PCR. For all patients the OCT exam showed active lesions with the inner retinal layers being abnormally hyper-reflective with full-thickness disorganization of the retinal reflective layers, which assumed a blurred reflective appearance and the retina was thickened.ConclusionsThe presence of IgA and IgM confirmed the acute infection and thus was in agreement with the clinical evaluation. Our results show the adopted treatment modified the serological profile of IgM antibodies and the PCR results, but not the IgG and IgA antibodies and that imaging is a good tool to follow-up patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-015-1650-6) contains supplementary material, which is available to authorized users.