Diagnostic Efficacy of Molecular Techniques for Detection and Identification of Leishmania Species in Human Whole Blood and Skin Samples from Ecuador.

Microscopic examination is the standard method for diagnosis of cutaneous and mucocutaneous leishmaniasis despite its low sensitivity. This study compared the diagnosis efficacy of microscopic examination versus polymerase chain reaction (PCR)-based methods and DNA sequencing using whole blood and skin lesion samples from patients with suspected leishmaniasis. The presence of Leishmania was determined by microscopy and amplification of 18S ribosomal RNA gene from blood and skin samples of 22 patients. Twenty individuals were positive for leishmaniasis. Microscopic analysis identified 85%, whereas PCR identified 100% of positive cases from skin and 90% from blood. Cytochrome b gene (cyt-b) amplification and sequencing identified Leishmania guyanensis, Leishmania shawi, and Leishmania naiffi from skin and blood samples. This study demonstrated the usefulness of whole blood and molecular techniques for the diagnosis and species identification of leishmaniasis.

Blood-meal identification in phlebotomine sand flies (Diptera: Psychodidae) from Valle Hermoso, a high prevalence zone for cutaneous leishmaniasis in Ecuador.

Cutaneous leishmaniasis is a neglected tropical disease transmitted by phlebotomine sand flies of the genus Lutzomyia. In South America, cutaneous leishmaniasis is endemic in the majority of countries. There are no previous reports of phlebotomine sand fly host feeding sources in Ecuador. We identified blood meal sources for phlebotomine sand fly species in Valle Hermoso, a hyper endemic area for leishmaniasis in Ecuador. Phlebotomine sand fly collections were carried out during the dry and rainy seasons. PCR and multiplex PCR were performed from DNA extracted from the abdomens of blood-fed females to specifically identify the avian and mammalian blood meal sources. Avian-blood (77%), mammalian-blood (16%) and mixed avian-mammalian blood (7%) were found in the samples. At the species level, blood from chickens (35.5%), humans (2.8%), cows (2.8%) and dogs (1.9%) was specifically detected. Nyssomyia trapidoi was the most common species of Lutzomyia found that fed on birds. The present results may aid the development of effective strategies to control leishmaniasis in Ecuador.

Improved method for extraction and detection of Helicobacter pylori DNA in formalin-fixed paraffin embedded gastric biopsies using laser micro-dissection.

To assess the molecular events exerted by Helicobacter pylori interacting directly with gastric epithelial cells, an improved procedure for microbial DNA isolation from stained hematoxilin-eosin gastric biopsies was developed based on laser micro-dissection (LM) [1]. Few articles have described the use of LM to select and detect H. pylori genome from formalin-fixed paraffin embedded gastric tissue [2]. To improve the yield and quality of DNA isolated from H. pylori contacting intestinal epithelial cells, the following conditions were established after modification of the QIAamp DNA Micro kit. •Use of at least 25 cut sections of 10-20 µm of diameter and 3 µm thick with more than 10 bacteria in each cut.•Lysis with 30 µL of tissue lysis buffer and 20 µL of proteinase K (PK) with the tube in an upside-down position.•The use of thin purification columns with 35 µL of elution buffer. The mean of DNA concentration obtained from 25 LM cut sections was 1.94± 0 .16 ng/µL, and it was efficiently amplified with qPCR in a Bio Rad iCycler instrument. The LM can improve the sample selection and DNA extraction for molecular analysis of H. pylori associated with human gastric epithelium.