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Sensitivity is the main concern at the development of rapid test by lateral flow immunoassays. On the other hand, low limits of detection are often required at medical diagnostics and other field of analysis. To overcome this drawback, several enhancement protocols have been described. In this paper, we have selected different silver enhancement methods and one dual gold conjugation, and we critically compared the amplification produced when applied to a gold-nanoparticle based lateral flow immunoassay for the detection of prostate specific antigen (PSA). The highest amplification was obtained by using an immersion method based on a solution of silver nitrate and hydroquinone/citrate buffer in proportion 1:1. Under these conditions, the system is capable of detecting PSA within 20 min at levels as low as 0.1 ng/mL, with a 3-fold sensitivity improvement.

The occurrence of free-living amoebae (FLA) was investigated in 83 water samples from reservoirs and water treatment plants, with culture positive in 64 of them (77.1%). Polymerase chain reaction (PCR) of partial 18S rRNA gene and ITS region was performed in order to identify amoeba isolates, and the presence of Legionella pneumophila , Mycobacterium spp., Pseudomonas spp., and Microcystis aeruginosa was investigated in 43 isolates of amoebae by multiplex PCR. Of the isolated amoebae, 31 were Acanthamoeba spp., 21 were Hartmannella vermiformis, 13 were Naegleria spp., and one was Vanella spp. T2, T4, and T5 genotypes of Acanthamoeba have been identified, and T4 isolates were grouped into five subgenotypes and graphically represented with a Weblog application. Inside amoebae, L. pneumophila was detected in 13.9% (6/43) of the isolates, and Pseudomonas spp. and Mycobacterium spp. were detected in 32.6% (14/43) and 41.9% (18/43), respectively. No statistical correlation was demonstrated between FLA isolation and seasonality, but the presence of intracellular bacteria was associated with warm water temperatures, and also the intracellular presence of Mycobacterium spp. and Pseudomonas spp. were associated. These results highlight the importance of amoebae in natural waters as reservoirs of potential pathogens and its possible role in the spread of bacterial genera with interest in public and environmental health.

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