Advances in the understanding of leishmaniasis progression indicate that cellular interactions more complex than the Th1/Th2 paradigmdefine the course of infection. Th17 cells are a crucial modulator of adaptive immunity against Leishmania parasites acting mainly on neutrophil recruitment and playing a dual role at the site of infection. This review describes the roles of both these cell typesin linking innate defenseresponses to the establishment of specific immunity. We focus on the Th17–neutrophil interaction as a crucial component of anti-Leishmania immunity, and the clinical evolution of cutaneous or visceral leishmaniasis.To date, information obtained through experimental models and patient evaluations suggests that the influence of the presence of interleukin (IL)-17 (the main cytokine produced by Th17 cells) and neutrophilsduring Leishmania infections is strictly dependent on the tissue (skin or liver/spleen) and parasite species. Also, the time at which neutrophils are recruited, and the persistence of IL-17 in the infection microenvironment, may also be significant. A clearer understanding of these interactionswill enable better measurement ofthe influence of IL-17 and its regulators,and contribute to the identification of disease/resistance biomarkers.

Leishmaniases are caused by obligate intracellular protozoan parasites of the genus Leishmania. They cause a spectrum of diseases, most notably visceral (VL), cutaneous (CL), and mucosal (ML) leishmaniasis, which affect millions of people around the world, each year. Despite scientific advances, leishmaniases cases are expanding, constituting an important public health problem. Immunological and molecular diagnostic tools have been increasingly applied for the early detection of these parasitic infections, since the existence of limitations in clinical and parasitological examinations may provide false results, thus interfering in epidemiological research and diseases control. Although there is a great diversity of available immunological assays, important common deficiencies persist, which explains the current exploration of the molecular biology in research fields, especially the Polymerase Chain Reaction (PCR) and its variants, such as real-time quantitative PCR. However, in the last years, significant results have also been reached inside of immunological context (especially by Flow Cytometry), for humans and dogs, demonstrated by research works of the New and Old worlds. In spite of their potential to clarify and minimize the present global situation of the diseases, the implementation of molecular or immunological innovative reference assays for VL and CL at health services is still a challenge due to several reasons, including lack of standardization among laboratories and structural concerns. In this article we bring classical and current information about technological advances for the immunological and molecular leishmaniases diagnosis, their features, and applications.

Phlebotomine sand flies are blood-feeding insects of great medical and veterinary significance acting as vectors of Leishmania parasites. Studying the blood-feeding pattern of these insects may help in the understanding of their interactions with potential reservoir hosts of Leishmania parasites. In this study, we developed real time PCR assays for the identification of sand fly blood meal. Six pairs of primers were designed based on cytochrome b gene sequences available in GenBank of the following potential hosts: dog, cat, horse, chicken, black rat, and human. Firstly, SYBR Green-based real time PCR assays were conducted using a standard curve with eight different concentrations (i.e., 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg and 1 fg per 2 μl) of DNA samples extracted from EDTA blood samples from each target animal. Then, DNA samples extracted from field-collected engorged female sand flies belonging to three species (i.e., Lutzomyia longipalpis, L. migonei and L. lenti) were tested by the protocols standardized herein. Additionally, female sand flies were experimentally fed on a black rat (Rattus rattus) and used for evaluating the time course of the detection of the protocol targeting this species. The protocols performed well with detection limits of 10 pg to 100 fg. Field-collected female sand flies were fed on blood from humans (73%), chickens (23%), dogs (22%), horses (15%), black rats (11%) and cats (2%). Interestingly, 76.1% of the L. longipalpis females were positive for human blood. In total, 48% of the tested females were fed on single sources, 31% on two and 12% on three. The analysis of the time course showed that the real time PCR protocol targeting the black rat DNA was able to detect small amounts of the host DNA up to 5 days after the blood meal. The real time PCR assays standardized herein successfully detected small amounts of host DNA in female sand flies fed on different vertebrate species and, specifically for the black rats, up to 5 days after the blood meal. These assays represent promising tools for the identification of blood meal in field-collected female sand flies.

In recent years, the polymerase chain reaction (PCR) technique has significantly advanced towards expanding its use and versatility by working with quantitative real-time PCR (qPCR). Data from the literature show that both methods present interesting characteristics for the diagnosis of visceral leishmaniasis. The benefits of qPCR in relation to conventional PCR include speed, reproducibility and quantitative ability. In addition to operational advantages, qPCR is more sensitive and reproducible and may replace conventional PCR in diagnostic routines. Regarding visceral leishmaniasis, the possibility of deployment of real-time PCR in highly complex diagnoses (reference services) in endemic areas will facilitate a swift and safe return for patients. Moreover, the use of a technique that possesses elevated diagnostic sensitivity, and can monitor therapy and prevent relapses promotes broader prospects for the disease control