Development and Clinical Evaluation of a Rapid Diagnostic Test for Yellow Fever Non-Structural Protein 1.

A rapid diagnostic test (RDT) kit was developed to detect non-structural protein 1 (NS1) of yellow fever virus (YFV) using monoclonal antibody. NS1 protein was purified from the cultured YFV and used to immunize mice. Monoclonal antibody to NS1 was selected and conjugated with colloidal gold to produce the YFV NS1 RDT kit. The YFV RDTs were evaluated for sensitivity and specificity using positive and negative samples of monkeys from Brazil and negative human blood samples from Korea. Among monoclonal antibodies, clones 3A11 and 3B7 proved most sensitive, and used for YFV RDT kit. Diagnostic accuracy of YFV RDT was fairly high; Sensitivity was 0.0% and specificity was 100% against Dengue viruses type 2 and 3, Zika, Chikungunya and Mayaro viruses. This YFV RDT kit could be employed as a test of choice for point-of-care diagnosis and large scale surveys of YFV infection under clinical or field conditions in endemic areas and on the globe.

In vitro susceptibility testing of amphotericin B for Cryptococcus neoformans variety grubii AFLP1/VNI and Cryptococcus gattii AFLP6/VGII by CLSI and flow cytometry.

Cryptococcus neoformans var. grubii AFLP1/VNI is the main causative agent of cryptococcosis associated with AIDS in the world. Cryptococcus gattii AFLP6/VGII causes mainly endemic primary infection in immunocompetent hosts. To determine the minimum inhibitory concentrations (MICs) of C. neoformans var. grubii AFLP1/VNI and C. gattii AFLP6/VGII against amphotericin B (AMB) in a short period of time, flow cytometry (FCM) with FUN-1 fluorochrome was used to compare with broth microdilution method (CLSI M27-A3). The minimum incubation period was evaluated by minimum fungicidal concentration procedure. Seventeen clinical isolates of C. neoformans var. grubii AFLP1/VNI and 18 of C. gattii AFLP6/VGII were analysed. The time for the determination of MICs by FCM was 2 h against 72 h by CLSI M27-A3 and the comparison of MIC showed a positive significant correlation (P = 0.048). It is important to highlight the role of the FCM as an alternative method to determine the MICs for AMB in within a day, with positive cost-benefit.

Determination of the minimum inhibitory concentration of Cryptococcus neoformans and Cryptococcus gattii against fluconazole by flow cytometry.

Recent studies have used flow cytometry (FCM) as an important alternative method to determine the antifungal susceptibility of yeasts compared to the broth microdilution Clinical and Laboratory Standards Institute (CLSI) reference procedure. We present a comparative study of the broth microdilution method and flow cytometry to assess the in vitro antifungal susceptibility of Cryptococcus neoformans (n = 16) and C. gattii (n = 24) to fluconazole. The minimum inhibitory concentration (MIC) assays by flow cytometry were defined as the lowest drug concentration that showed ∼50% of the count of acridine orange negative cells compared to that of the growth control. Categorical classification showed all C. neoformans isolates were susceptible to fluconazole. Three isolates of C. gattii were susceptible dose-dependent and the remaining 21 isolates were classified as susceptible. MICs comparison of both methodologies demonstrated 100% categorical agreement of the results obtained for C. neoformans and C. gattii. The MICs obtained with the CLSI-approved method and flow cytometry were compared by the Spearman correlation test and a significant Pv = 0.001. The flow cytometric method has the advantage of analyzing a large and constant number of cells in less time, i.e., 9 h incubation for fluconazole using acridine orange versus 72 h for broth microdilution method. In conclusion, the two methods were comparable and flow cytometry method can expedite and improve the results of in vitro susceptibility tests of C. neoformans and C. gattii against fluconazole and also allows comparative studies in vitro/in vivo more rapidly, which along with clinical data, could assist in selecting the most appropriate treatment choice.

Malaria associated apoptosis is not significantly correlated with either parasitemia or the number of previous malaria attacks.

The occurrence and intensity of lymphocyte apoptosis in blood samples from 79 outclinic patients with uncomplicated Plasmodium falciparum or Plasmodium vivax malaria and 30 healthy individuals were investigated. No difference in apoptosis percentages was detected between healthy individuals and malaria patients when ex vivo lymphocytes were analyzed. However, significantly increased apoptosis levels were observed in lymphocytes from both P. falciparum- and P. vivax-infected patients when the cells were cultured for 24 h. CD4(+)and CD8(+) T cells were affected to a comparable extent in P.falciparum- and P.vivax-infected patients. However, when we compared apoptosis values in infected and non-infected individuals it appeared that CD4(+) T cells were more susceptible than CD8(+) T cells. A significant increase in the sIL-2R plasma levels was observed in malaria patients when compared with healthy individuals and a positive correlation was observed between sIL-2R levels and apoptosis rates in infected patients presenting increased rates of apoptosis. An increased expression of Fas antigen was recorded after stimulation with P. falciparum antigen or anti-CD3 monoclonal antibody. These data show that a consistent proportion of the lymphocyte population dies by apoptosis during a malaria infection and that a period of time is necessary before in vivo activated cells can express the apoptotic process in vitro.