Evaluation of preservation methods and simulated multiple infection on the fidelity of real-time PCR detection of Leishmania DNA

To detail the evaluation of a real-time polymerase chain reaction [PCR]-based approach to Leishmania detection. Also to test the fidelity of PCR diagnostics in a series of experiments mimicking infection by two species of Leishmania. Leishmania major [a causal agent of cutaneous leishmaniasis] infected Phlebotomus papatasi sandflies were generated to test the effect of four preservation methods on the fidelity of real-time PCR detection of Leishmania DNA. There was no effect of preservation methods on the sensitivity or specificity of two different assays. The ability of these assays to correctly diagnose cases of multiple infection was tested with artificial double infections created by combining DNA from pairs of Leishmania species [L, major, L. tropica and L, panamensis]. One assay failed to properly diagnose certain double infections but overall the PCR methodologies were robust. These findings provide important reassurances for subsequent real world investigations of Leishmania in vector, reservoir and human populations

Rapid enzyme-linked immunosorbent assay for the diagnosis of human brucellosis in surveillance and clinical settings in Egypt

To optimize and standardize an enzyme-linked immunosorbent assay [ELISA] for rapid diagnosis of human brucellosis in clinical cases identified during a surveillance study for acute febrile illness [AFI]. Serum samples from patients presenting with AFI at 13 fever hospitals across Egypt between 1999 and 2003 were kept frozen at NAMRU-3 and used in this study. The assay was evaluated in 5 subject groups: brucellosis cases confirmed by blood culture [group I, n=202] 87% positive by standard tube agglutination test [TA], brucellosis cases exclusively confirmed by TA [group II, n=218], blood cultures from AFI cases positive for bacterial species other than Brucella [group III, n=103], AFI cases with unexplained etiologies [group IV, n=654], and healthy volunteers [group V, n=50]. All members of groups III-V were negative for brucellosis by TA. Sensitivity and specificity of ELISA for total specific antibodies were >=96% versus 87% for TA as compared to microbial culture, the current gold standard method for Brucella identification. Assessment of Brucella antibody classes by ELISA in random subsets of the 5 groups showed significantly high [p>0.001] levels of anti Brucella IgG [>=81%] and IgM [>=90%] in groups I and II only. The obtained sensitivity and specificity results indicate that our ELISA is more suitable for AFI surveillance and clinical settings than blood culture and TA. The developed assay is also cost-effective, easier to use, faster, and the coated plates can be stocked for at least 8 months, providing a potential for field use and automation