Multiplex Polymerase Chain Reaction for Detection of Leishmania Sp. in Sandflies of the Paraná River Islands, Southern Brazil.

Leishmaniases are classified as tegumentary leishmaniasis (TL) and visceral leishmaniasis (VL). Brazil is among the countries with the highest number of TL and VL cases. This study was undertaken to standardize the multiplex polymerase chain reaction (PCR) for the detection of the genus Leishmania in sandflies of endemic regions, on islands in the Upper Paraná River, northwestern Paraná. The sandflies were collected on 10 islands, from November 2012 to November 2014, with Falcão light traps, identified and conserved in tubes containing isopropanol, for subsequent DNA extraction. Two pairs of primers were used for multiplex PCR: A1/A2 and 5Llcac/3Llcac. Nyssomyia neivai was the predominant species of the collected specimens. A total of 3870 samples of female sandflies were analyzed and submitted to multiplex PCR, for the validation of the technique. All pools showed the 220 bp fragment for sandfly DNA detection, but no ∼120 bp fragment of Leishmania DNA was found. Although no natural infection of Ny. neivai by Leishmania was found in this study, the interaction of sandflies with Leishmania and its natural reservoirs continues in these Paraná River islands, despite the low diversity of the sandfly fauna. Some of these islands have permanent residents and are frequented by tourists.

Major globally disseminated clonal complexes of antimicrobial resistant enterococci associated with infections in cancer patients in Brazil.

Cancer and hematological malignancies constitute major comorbidities in enterococcal infections, but little is known about the characteristics of enterococci affecting cancer patients. The aim of this study was to characterize 132 enterococcal clinical isolates obtained from cancer patients attending a Cancer Reference Center in Brazil between April 2013 and March 2014. Susceptibility to 17 antimicrobial agents was assessed by disk diffusion method. Resistance and virulence genes were investigated by PCR. Multilocus sequence typing (MLST) was performed for selected Enterococcus faecalis and Enterococcus faecium isolates. The predominant species was E. faecalis (108 isolates), followed by E. faecium (18), Enterococcus gallinarum (3), Enterococcus avium (2) and Enterococcus durans (1). Multidrug-resistant (MDR) isolates made up 44.7%, but all isolates were susceptible to fosfomycin, linezolid and glycopeptides. The most prevalent genes associated with erythromycin- and tetracycline-non susceptible isolates were erm(B) (47/71; 66.2%) and tet(M) (24/68; 35.3%), respectively. High-level resistance (HLR) to gentamicin was found in 22 (16.7%) isolates and 13 (59.1%) of them carried the aac(6')-Ie-aph(2″)-Ia gene. HLR to streptomycin was detected in 34 (25.8%) isolates, of which 15 (44.1%) isolates had the ant(6')-Ia gene. The most common virulence genes were gelE (48.9%), esp (30.5%) and asa1 (29.8%). MLST performed for 26 E. faecalis isolates revealed 18 different sequence-types (STs), with seven corresponding to novel STs (625, 626, 627, 628, 629, 630, and 635). On the other hand, nine of 10 E. faecium isolates analyzed by MLST belonged to a single clonal complex, comprised of mostly ST412, which emerged worldwide after mid-2000s, but also two novel STs (963 and 964). We detected major globally disseminated E. faecalis and E. faecium clonal complexes along with novel closely related STs, indicating the fitness and continuous evolution of these hospital-adapted lineages.

Potency determination of recombinant IFN-alpha based on phosphorylated STAT1 using flow cytometry.

The interferon (IFN) family of cytokines is recognized as a key component of the innate immune response and the first line of defense against viral infection. The usage of the IFN-alpha as a biopharmaceutical has been mainly applied in the treatment of chronic hepatitis C. In the literature it is possible to find a great variety of methods to determine the potency of these cytokines, and many efforts have been made in order to develop practical bioassays to study the biological activity of IFNs. In this technical note, we present a different approach to determine the potency of a recombinant IFN-alpha preparation based on the activation of the signal transducers and activators of transcription 1 (STAT1) using flow cytometry technique. Under the conditions of this study, this new approach proved to be useful and promising to assess the potency of these biopharmaceuticals and may also be used as an important tool in the quality control of such biological products.