The marine dinoflagellate genusAlexandriumincludes a number of species which produce neurotoxins responsible for paralytic shellfish poisoning (PSP), which in humans may cause muscular paralysis, neurological symptoms, and, in extreme cases, death.A. minutumis the most widespread toxic PSP species in the western Mediterranean basin. The monitoring of coastal waters for the presence of harmful algae also normally involves microscopic examinations of phytoplankton populations. These procedures are time consuming and require a great deal of taxonomic experience, thus limiting the number of specimens that can be analyzed. Because of the genetic diversity of different genera and species, molecular tools may also help to detect the presence of target microorganisms in marine field samples. In this study, we developed a real-time PCR-based assay for rapid detection of all toxic species of theAlexandriumgenus in both fixative-preserved environmental samples and cultures. Moreover, we developed a real-time quantitative PCR assay for the quantification ofA. minutumcells in seawater samples.Alexandriumgenus-specific primers were designed on the 5.8S rDNA region. Primer specificity was confirmed by using BLAST and by amplification of a representative sample of the DNA of other dinoflagellates and diatoms. Using a standard curve constructed with a plasmid containing the ITS1-5.8S-ITS2A. minutumsequence and culturedA. minutumcells, we determined the absolute number of 5.8S rDNA copies per cell. Consequently, after quantification of 5.8S rDNA copies in samples containingA. minutumcells, we were also able to estimate the number of cells. Several fixedA. minutumbloom sea samples from Arenys Harbor (Catalan Coast, Spain) were analyzed using this method, and quantification results were compared with standard microscopy counting methods. The two methods gave comparable results, confirming that real-time PCR could be a valid, fast alternative procedure for the detection and quantification of target phytoplankton species during coastal water monitoring.

Leishmaniasis is a vector-borne disease caused by many Leishmania species, which can infect both humans and other mammals. Leishmaniasis is a complex disease, with heterogeneous clinical manifestations ranging from asymptomatic infections to lesions at cutaneous sites (cutaneous leishmaniasis), mucosal sites (mucocutaneous leishmaniasis) or in visceral organs (visceral leishmaniasis), depending on the species and host characteristics. Often, symptoms are inconclusive and leishmaniasis can be confused with other co-endemic diseases. Moreover, co-infections (mainly with HIV in humans) can produce atypical clinical presentations. A correct diagnosis is crucial to apply the appropriate treatment and the use of molecular techniques in diagnosis of leishmaniasis has become increasingly relevant due to their remarkable sensitivity, specificity and possible application to a variety of clinical samples. Among them, real-time PCR (qPCR)-based approaches have become increasingly popular in the last years not only for detection and quantification of Leishmania species but also for species identification. However, despite qPCR-based methods having proven to be very effective in the diagnosis of leishmaniasis, a standardized method does not exist. This review summarizes the qPCR-based methods in the diagnosis of leishmaniasis focusing on the recent developments and applications in this field.