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.
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.
Developing molecular strategies to manipulate gene expression in trypanosomatids is challenging, particularly with respect to the unique gene expression mechanisms adopted by these unicellular ...parasites, such as polycistronic mRNA transcription and multi-gene families. In the case of Trypanosoma cruzi (T. cruzi), the causative agent of Chagas Disease, the lack of RNA interference machinery further complicated functional genetic studies important for understanding parasitic biology and developing biomarkers and potential therapeutic targets. Therefore, alternative methods of performing knockout and/or endogenous labelling experiments were developed to identify and understand the function of proteins for survival and interaction with the host. In this review, we present the main tools for the genetic manipulation of T. cruzi, focusing on the Clustered Regularly Interspaced Short Palindromic Repeats Cas9-associated system technique widely used in this organism. Moreover, we highlight the importance of using these tools to elucidate the function of uncharacterized and glycosylated proteins. Further developments of these technologies will allow the identification of new biomarkers, therapeutic targets and potential vaccines against Chagas disease with greater efficiency and speed.
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.
The Trypanosomatidae family is composed by protozoan parasites
responsible for causing a variety of human and non-human diseases, of varying
gravities, in both new and old worlds. The distribution ...and adaptation of the
trypanosomatids in tropical and subtropical countries, as well as the usual habitats of
vectors commonly favour contamination of poor neglected populations, despite the
increasing expansion to peri-urban and urban areas, thus reaching other social and
economic realities. Illnesses like leishmaniasis and American trypanosomiasis (or
Chagas disease) share the fact that both have a great diversity of reservoirs associated
to the cycle of the parasites, which include synanthropic mammalians and the Homo
sapiens itself. Therefore, to an efficient epidemiological control, the continuous search
for new human cases, as well as the monitoring and controlling of infected animals is
fundamental. The laboratorial techniques routinely used for detection of these diseases
may vary from simple parasitological analysis to cutting-edge molecular technology.
Due to its easiness and diagnostic sensitivity, the immunology/serology is broadly
applied in the laboratories and also in field, even with its limitations. The molecular
biology and its tools are emerging as sensitive and specific alternative strategies for the
trypanosomiasis diagnosis, but some challenges still remain, especially concerning the
standardization of protocols and the establishment of gold-standard procedures. New
serological and molecular methods arise annually aiming to overlap deficiencies that
may reduce the feasibility for applying in routine and in epidemiological researches. In
this chapter, the past and current diagnostic situation of leishmaniases, Chagas disease
and Human African trypanosomiasis (HAT) or "sleeping sickness" will be described,
highlighting the technological advances and the difficulties to be faced for the next
years, mainly regarding the applicability for public health.
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