Summarizing, the kDNA target may be used for diagnosis due to its sensitivity [78, 79, 87, 103C105], whereas ITS-1 is more specific and may be used for species identification [78]. Regarding the advances in molecular biology, the choice of the target region to be imaged is the key point for the successful use of diagnostic PCR-based technologies. Proposal of gold-standard diagnostic strategies for leishmnaniases detection in humans Technological advances have contributed to raise new diagnostic guidelines not only in the parasitological field, but also in all areas regarding pathology. the detection of leishmaniases. Classical immunological tools Montenegro skin test (MST) has been successfully used in the diagnosis of cutaneous forms, but is negative for recent lesions, in the diffuse form, and also in immunosuppressed patients [13]. The test is commonly positive in endemic areas due to the occurrence of subclinical infections. Furthermore, other characteristics may hamper the applicability of the method: cross-reactions; long time to retest, if necessary (at least two years); false-positive results caused by lack of patient cooperation, which itches the application site; subjectivity of the reading (especially when the diameter of the induration has between 4 and 5?mm) and so on [14]. In the diagnosis of CL the use of indirect immunofluorescence assay (IFA) associated with MST or a parasitological technique is recommended to provide a differential diagnosis. The limitation of IFA lies in the fact that it does not correlate the levels of circulating antibodies with disease staging. In addition, there is the possibility of cross reactivity with other trypanosomatids and fungi [15C17]. IFA is based on the detection of anti-antibodies by employing specific antigens (promastigote form, normally) and secondary antibodies (anti-immunoglobulin antibody) conjugated with a fluorescent dye [16, 17]. The technique is becoming less explored in routine not only for CL diagnosis, but also for canine VL (CVL) diagnosis mainly because of its low specificity, in contrast with the high sensitivity. Incompatibility or poor reaction between the secondary and primary antibodies or the antigen are also constraints associated with this indirect immunological assay. The majority of the immunological techniques for detection of anti-antibodies has been based on reactions like Enzyme-Linked Immunosorbent Assay (ELISA) [11, 18]. The sensitivity and specificity of ELISA depends on the antigen used [11]. In this context, several antigens with different molecular weights have been identified for potential use in the diagnosis. Recombinant protein K39 (rK39), a very important and broadly employed antigen, showed 100?% specificity and 96?% sensitivity for the diagnosis of VL [19]. An interesting feature of this antigen is that it can SAR405 be used in patients co-infected with HIV, in which anti-K39 antibody levels decline rapidly with the treatment success [11, 20, 21]. Other Rabbit Polyclonal to UBE3B candidates for the diagnosis of several forms of leishmaniases are recombinant or purified membrane glycoproteins (gp): gp63, gp70, and gp72, and A2 protein, all of which are specific for the genus antibodies has been increasingly explored [25]Researchers demonstrated an excellent performance of an FC prototype in canine VL diagnostic, with high specificity, sensitivity, predictive values and accuracy, even when animals were infected with other pathogens (such as and recombinant antigens associated with FC as a viable tool for a highly sensitive laboratorial serodiagnosis of both clinical and subclinical forms of canine disease [27]. For human form, it was shown the detection of specific IgG antibodies against using FC for cure assessment [28]. FC to detect anti-live antibodies has been first described by Rocha [29], in which they demonstrated SAR405 93.6?% sensitivity in patients with active disease. Researchers working with live and fixed showed that FC can be a useful serological technique to detect anti- IgG antibodies, with the antigens displaying an 86 and 90?% sensitivity, respectively [30]. A good performance using fixed promastigotes was also demonstrated [31]. Oliveira [32] showed that FC had a better performance compared to IFA in the monitoring of specific post therapeutic cure of CL. Therefore, FC is becoming an increasingly useful tool in health care and research laboratories, due to SAR405 its accuracy and reproducibility. Although there is still a substantial cost regarding the operational support in experiments involving FC, Shared Resource Laboratory models are enhancing the scope and quality of scientific research that applies the FC based methodologies [26, 33, 34]. The Table?1 summarizes the main aspects addressed in the immunological methods used in leishmaniases diagnosis. Table 1 Advantages and limitations of immunological methods used in leishmaniases diagnosis is also an important application of the PCR, and.