A comparative study on the detection of Mycobacterium leprae DNA in urine samples of leprosy patients using Rlep-PCR with other conventional samples.

BACKGROUND: Mycobacterium leprae causes leprosy that is highly stigmatized and chronic infectious skin disease. Only some diagnostic tools are being used for the identification M. leprae in clinical samples, such as bacillary detection, and histopathological tests. These methods are invasive and often have low sensitivity. Currently, the PCR technique has been used as an effective tool fordetecting M. leprae DNA across different clinical samples. The current study aims to detect M. leprae DNA in urine samples of untreated and treated leprosy patients using the Rlep gene (129 bp) and compared the detection among Ridley-Jopling Classification. METHODS: Clinical samples (Blood, Urine, and Slit Skin Smears (SSS)) were collected from leprosy and Non-leprosy patients. DNA extraction was performed using standard laboratory protocol and Conventional PCR was carried out for all samples using Rlep gene target and the amplicons of urine samples were sequenced by Sanger sequencing to confirm the Rlep gene target. RESULTS: The M. leprae DNA was successfully detected in all clinical samples across all types of leprosy among all the study groups using RLEP-PCR. Rlep gene target was able to detect the presence of M. leprae DNA in 79.17% of urine, 58.33% of blood, and 50% of SSS samples of untreated Smear-Negative leprosy patients. The statistical significant difference (p = 0.004) was observed between BI Negative (Slit Skin Smear test) and RLEP PCR positivity in urine samples of untreated leprosy group. CONCLUSION: The PCR positivity using Rlep gene target (129 bp) was highest in all clinical samples among the study groups, across all types of leprosy. Untreated tuberculoid and PNL leprosy patients showed the highest PCR positivity in urine samples, indicating its potential as a non-invasive diagnostic tool for leprosy and even for contact screening.

Tools for non-invasive sampling of metal accumulation and its effects in Mediterranean pond turtle populations inhabiting mining areas.

Among reptiles, freshwater turtle species have high potential for metal accumulation because of their long lifespan or their aquatic and terrestrial habits. In order to monitor metal bioaccumulation, determine potential toxic effects, and investigate tools for non-invasive metal sampling in reptiles, we studied lead (Pb) and mercury (Hg) accumulation in Mediterranean pond turtles (Mauremys leprosa) inhabiting two former mining areas, one of them with high environmental concentrations of Pb (Sierra Madrona-Alcudia Valley district) and the other one with high environmental concentrations of Hg (Almadén district). Individuals from the Pb mining area showed mean blood concentrations (i.e. 5.59 µg Pb/g dry weight, d.w.) that were higher than those measured in other populations. Blood Hg concentrations were highest (8.83 µg Hg/g d.w.) in the site close to the former Hg mines, whereas blood Hg concentrations in terrapins from another site of Almadén district, located ∼28 km downstream, were not different from locations at the non-mining area. Animals from the Pb-contaminated site showed evidence of oxidative stress, whereas those from the Hg-contaminated site showed increased activity of the antioxidant enzyme glutathione peroxidase, as well as reduced circulating levels of the main endogenous antioxidant peptide, glutathione. Concentrations measured in feces and carapace scutes were useful indicators to monitor blood concentrations of Pb, but not of Hg. Our results provide evidence of the usefulness of freshwater turtles as sentinels of chronic metal pollution, and validate non-invasive tools to advance Pb monitoring in reptiles.