Advances in the understanding of leishmaniasis progression indicate that cellular interactions more complex than the Th1/Th2 paradigm define the course of infection. Th17 cells are a crucial ...modulator of adaptive immunity against
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 types in linking innate defense responses to the establishment of specific immunity. We focus on the Th17-neutrophil interaction as a crucial component of anti-
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 neutrophils during
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 interactions will enable better measurement of the influence of IL-17 and its regulators, and contribute to the identification of disease/resistance biomarkers.
•The association between canine leishmaniasis and IL-17 polymorphisms was assessed.•Healthy dogs showed higher frequency of IL-17 promoter major/wild alleles.•Results suggest that IL-17 polymorphisms ...may affect canine immunity to L. infantum.•IL-17 polymorphisms do not appear to be to useful prognostic markers for CanL.
Proinflammatory cytokine secretion determines the infection course in leishmaniasis. The immunopathology of canine leishmaniasis (CanL) caused by Leishmania infantum is characterized by low Leishmania-specific IFN-γ and IL-17 production. Mutations in the human IL-17 gene promoter alter cytokine expression and may increase the susceptibility of humans to some infectious diseases. In this study, we correlated canine IL-17 single nucleotide polymorphisms (SNPs) with anti-Leishmania IgG levels, parasite load and external clinical signs in dogs naturally exposed to L. infantum in Brazil. A higher frequency (Chi-square test: X2= 5.378, df= 1, P= 0.020) of major alleles was observed among dogs showing no external clinical signs attributable to Leishmania infection. A high proportion of A allele carriers (mutant) were observed among dogs with high antibody levels, although differences were not statistically significant (Chi-square test: X2= 4.410, df= 4, P= 0.353), as compared to dogs with low antibody levels. In general, the association of canine IL-17 SNPs with disease expression or disease exasperation did not reach enough statistical power to allow the use of these mutations as prognostic markers. This knowledge may pave the way for further investigations on the genetic aspects of CanL and its immunotherapy.
Molecular biological methods have become increasingly relevant to the diagnosis and control of infectious diseases, such as leishmaniasis. Since various factors may affect the sensitivity of PCR ...assays, including DNA yield and purity, an optimal extraction method is pivotal. Losses of a parasite's DNA during extraction may significantly impair its detection by PCR and lead to false-negative results. This study proposes a triplex PCR assay targeting the parasite's DNA, an external control (pUC18) and an internal control (G3PD) for accurate diagnosis of leishmaniasis.
Two primer pairs were designed to detect the plasmid pUC18 and a triplex PCR assay targeting the Leishmania braziliensis kinetoplast DNA, the external control and the internal control was standardized. The triplex PCR assay was assessed for its ability to detect the three target DNA fragments simultaneously. PCR products from pUC18 DNA resulted in bands of 368 (P1) and 316 (P2) base pairs (bp). The triplex PCR optimized with the chosen external control system (P1) allowed the simultaneous detection of the internal control (G3PD - 567 bp) as well as of small quantities (10 pg) of the target parasite's DNA, detected by amplification of a 138 bp product.
The new tool standardized herein enables a more reliable interpretation of PCR results, mainly by contributing to quality assurance of leishmaniasis diagnosis. Furthermore, after simple standardization steps, this protocol could be applied to the diagnosis of other infectious diseases in reference laboratories. This triplex PCR enables the assessment of small losses during the DNA extraction process, problems concerning DNA degradation (sample quality) and the detection of L. braziliensis kDNA.
Early detection of leishmaniases and prompt institution of treatment are paramount for individuals and communities affected by these diseases. To overcome the remaining limitations inherent to ...molecular methods currently used and to ensure the accuracy of results in leishmaniases diagnosis, two triplex polymerase chain reaction (PCR) assays with quality controls for the reactions were developed. Validity indicators were assessed in 186 dog blood samples from endemic areas in Brazil. The level of agreement between the new tools and their singleplex protocols was assessed by kappa analysis. The triplex PCR for visceral leishmaniasis showed sensitivity (
S
) = 78.68 %, specificity (
E
) = 85.29 %, and efficiency (
e
) = 81.05 %. The cutaneous leishmaniasis protocol showed
S
= 97.29 %,
E
= 79.16 %, and
e
= 90.16 %. Both protocols showed good agreement with gold standards. These new tools enable, in a single reaction, the diagnosis of the diseases and the evaluation of the sample quality and DNA extraction process, thus reducing the cost of reagents and avoiding the eventual need for collecting a second sample.
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