Leishmaniasis, caused by protozoan parasites of the Leishmania genus, represents an important health problem in many regions of the world. Lack of effective point-of-care (POC) diagnostic tests ...applicable in resources-limited endemic areas is a critical barrier to effective treatment and control of leishmaniasis. The development of the loop-mediated isothermal amplification (LAMP) assay has provided a new tool towards the development of a POC diagnostic test based on the amplification of pathogen DNA. LAMP does not require a thermocycler, is relatively inexpensive, and is simple to perform with high amplification sensitivity and specificity. In this review, we discuss the current technical developments, applications, diagnostic performance, challenges, and future of LAMP for molecular diagnosis and surveillance of Leishmania parasites. Studies employing the LAMP assay to diagnose human leishmaniasis have reported sensitivities of 80% to 100% and specificities of 94% to 100%. These observations suggest that LAMP offers a good molecular POC technique for the diagnosis of leishmaniasis and is also readily applicable to screening at-risk populations and vector sand flies for Leishmania infection in endemic areas.
Quantitation of the nonlinear heterogeneities in Leishmania parasites, sand fly vectors, and mammalian host relationships provides insights to better understand leishmanial transmission epidemiology ...towards improving its control. The parasite manipulates the sand fly via production of promastigote secretory gel (PSG), leading to the "blocked sand fly" phenotype, persistent feeding attempts, and feeding on multiple hosts. PSG is injected into the mammalian host with the parasite and promotes the establishment of infection. Animal models demonstrate that sand flies with the highest parasite loads and percent metacyclic promastigotes transmit more parasites with greater frequency, resulting in higher load infections that are more likely to be both symptomatic and efficient reservoirs. The existence of mammalian and sand fly "super-spreaders" provides a biological basis for the spatial and temporal clustering of clinical leishmanial disease. Sand fly blood-feeding behavior will determine the efficacies of indoor residual spraying, topical insecticides, and bed nets. Interventions need to have sufficient coverage to include transmission hot spots, especially in the absence of field tools to assess infectiousness. Interventions that reduce sand fly densities in the absence of elimination could have negative consequences, for example, by interfering with partial immunity conferred by exposure to sand fly saliva. A deeper understanding of both sand fly and host biology and behavior is essential to ensuring effectiveness of vector interventions.
There is substantial experimental evidence to indicate that Leishmania infections that are transmitted naturally by the bites of infected sand flies differ in fundamental ways from those initiated by ...needle inocula. We have used flow cytometry and intravital microscopy (IVM) to reveal the heterogeneity of sand fly transmission sites with respect to the subsets of phagocytes in the skin that harbor L. major within the first hours and days after infection. By flow cytometry analysis, dermis resident macrophages (TRMs) were on average the predominant infected cell type at 1 hr and 24 hr. By confocal IVM, the co-localization of L. major and neutrophils varied depending on the proximity of deposited parasites to the presumed site of vascular damage, defined by the highly localized swarming of neutrophils. Some of the dermal TRMs could be visualized acquiring their infections via transfer from or efferocytosis of parasitized neutrophils, providing direct evidence for the "Trojan Horse" model. The role of neutrophil engulfment by dermal TRMs and the involvement of the Tyro3/Axl/Mertk family of receptor tyrosine kinases in these interactions and in sustaining the anti-inflammatory program of dermal TRMs was supported by the effects observed in neutrophil depleted and in Axl-/-Mertk-/- mice. The Axl-/-Mertk-/- mice also displayed reduced parasite burdens but more severe pathology following L. major infection transmitted by sand fly bite.
Neutrophils and dendritic cells (DCs) converge at localized sites of acute inflammation in the skin following pathogen deposition by the bites of arthropod vectors or by needle injection. Prior ...studies in mice have shown that neutrophils are the predominant recruited and infected cells during the earliest stage of Leishmania major infection in the skin, and that neutrophil depletion promotes host resistance to sand fly transmitted infection. How the massive influx of neutrophils aimed at wound repair and sterilization might modulate the function of DCs in the skin has not been previously addressed. The infected neutrophils recovered from the skin expressed elevated apoptotic markers compared to uninfected neutrophils, and were preferentially captured by dermal DCs when injected back into the mouse ear dermis. Following challenge with L. major directly, the majority of the infected DCs recovered from the skin at 24 hr stained positive for neutrophil markers, indicating that they acquired their parasites via uptake of infected neutrophils. When infected, dermal DCs were recovered from neutrophil depleted mice, their expression of activation markers was markedly enhanced, as was their capacity to present Leishmania antigens ex vivo. Neutrophil depletion also enhanced the priming of L. major specific CD4(+) T cells in vivo. The findings suggest that following their rapid uptake by neutrophils in the skin, L. major exploits the immunosuppressive effects associated with the apoptotic cell clearance function of DCs to inhibit the development of acquired resistance until the acute neutrophilic response is resolved.
The generation of an efficacious vaccine that elicits protective CD4+ T cell-mediated immunity has been elusive. The lack of a vaccine against the Leishmania parasite is particularly perplexing as ...infected individuals acquire life-long immunity to reinfection. Experimental observations suggest that the relationship between immunological memory and protection against Leishmania is not straightforward and that a new paradigm is required to inform vaccine design. These observations include: (i) induction of Th1 memory is a component of protective immunity, but is not sufficient; (ii) memory T cells may be protective only if they generate circulating effector cells prior to, not after, challenge; and (iii) the low-dose/high-inflammation conditions of physiological vector transmission compromises vaccine efficacy. Understanding the implications of these observations is likely key to efficacious vaccination.
Th1 memory is a component of protective immunity against phagosomal infections, but it is not sufficient.
Protection via CD4-mediated concomitant immunity is the gold standard of protective immunity against phagosomal infections.
Chronic primary infection is required for protection against secondary infection due to the requirement for persistent antigen exposure to maintain protective TEFF cells.
Memory T cells may mediate protection against infection only if they generate circulating effector cells prior prior to, not after, challenge.
Modeling the low-dose/high-inflammation conditions of physiological vector transmission of Leishmania has implications for the interpretation of vaccine efficacy.
Infection with the obligate intracellular protozoan Leishmania is thought to be initiated by direct parasitization of macrophages, but the early events following transmission to the skin by vector ...sand flies have been difficult to examine directly. Using dynamic intravital microscopy and flow cytometry, we observed a rapid and sustained neutrophilic infiltrate at localized sand fly bite sites. Invading neutrophils efficiently captured Leishmania major (L.m.) parasites early after sand fly transmission or needle inoculation, but phagocytosed L.m. remained viable and infected neutrophils efficiently initiated infection. Furthermore, neutrophil depletion reduced, rather than enhanced, the ability of parasites to establish productive infections. Thus, L.m. appears to have evolved to both evade and exploit the innate host response to sand fly bite in order to establish and promote disease.
•Novel immune-based intervention strategies might be developed which, instead of targeting a class of pathogen (bacteria, virus, parasite, or fungus), target a shared infectious process. We suggest ...three novel approaches to activating phagocytic cells prior to or at the time of infection by phagosomal pathogens. Preventing the formation of the pathogen niche within the phagolysosome might be crucial for preventing infectious disease; a greater emphasis on the timing of phagocyte activation might generate more effective prophylactic treatment options.
Prophylactic vaccination strategies designed to prevent diseases caused by phagosomal pathogens have been largely unsuccessful.We propose that trained immunity, natural killer cell activation, and maintenance of circulating T helper 1 effector cells may represent three novel intervention strategies to target a shared infectious process rather than a specific pathogen.These novel approaches might be effective based on the inherent cellular ability to activate phagocytes prior to, or at the time of, infection.Failure to activate phagocytes prior to, or at the time of, infection may not provide protection due to the establishment of the phagosomal pathogen niche.
Prophylactic vaccination strategies designed to prevent diseases caused by pathogens using the phagolysosome of innate immune cells as a site of intracellular replication and survival have been largely ineffective. These include Mycobacterium tuberculosis (Mtb), Leishmania spp., and Cryptococcus spp. These failed strategies have traditionally targeted CD4+ T helper (Th) 1 cell-mediated immune memory, deeming it crucial for vaccine efficacy. This failure warrants an investigation of alternative mediators of protection. Here, we suggest three novel approaches to activate phagocytic cells prior to or at the time of infection. We hypothesize that preventing the formation of the pathogen niche within the phagolysosome is essential for preventing disease, and a greater emphasis on the timing of phagocyte activation should generate more effective prophylactic treatment options.
Intracellular phagosomal pathogens represent a formidable challenge for innate immune cells, as, paradoxically, these phagocytic cells can act as both host cells that support pathogen replication ...and, when properly activated, are the critical cells that mediate pathogen elimination. Infection by parasites of the
genus provides an excellent model organism to investigate this complex host-pathogen interaction. In this review we focus on the dynamics of
infection and the host innate immune response, including the impact of the adaptive immune response on phagocytic host cell recruitment and activation.
infection represents an important public health problem in South America where, distinct from other
parasites, it has been associated with all three clinical forms of leishmaniasis in humans: cutaneous, muco-cutaneous and visceral. Experimental observations demonstrate that most experimental mouse strains are susceptible to
infection, including the C57BL/6 mouse, which is resistant to other species such as
,
and
. In general, the CD4
T helper (Th)1/Th2 paradigm does not sufficiently explain the progressive chronic disease established by
, as strong cell-mediated Th1 immunity, or a lack of Th2 immunity, does not provide protection as would be predicted. Recent findings in which the balance between Th1/Th2 immunity was found to influence permissive host cell availability
recruitment of inflammatory monocytes has also added to the complexity of the Th1/Th2 paradigm. In this review we discuss the roles played by innate cells starting from parasite recognition through to priming of the adaptive immune response. We highlight the relative importance of neutrophils, monocytes, dendritic cells and resident macrophages for the establishment and progressive nature of disease following
infection.
Numerous experimental vaccines have been developed to protect against the cutaneous and visceral forms of leishmaniasis caused by infection with the obligate intracellular protozoan Leishmania, but a ...human vaccine still does not exist. Remarkably, the efficacy of anti-Leishmania vaccines has never been fully evaluated under experimental conditions following natural vector transmission by infected sand fly bite. The only immunization strategy known to protect humans against natural exposure is "leishmanization," in which viable L. major parasites are intentionally inoculated into a selected site in the skin. We employed mice with healed L. major infections to mimic leishmanization, and found tissue-seeking, cytokine-producing CD4+ T cells specific for Leishmania at the site of challenge by infected sand fly bite within 24 hours, and these mice were highly resistant to sand fly transmitted infection. In contrast, mice vaccinated with a killed vaccine comprised of autoclaved L. major antigen (ALM)+CpG oligodeoxynucleotides that protected against needle inoculation of parasites, showed delayed expression of protective immunity and failed to protect against infected sand fly challenge. Two-photon intra-vital microscopy and flow cytometric analysis revealed that sand fly, but not needle challenge, resulted in the maintenance of a localized neutrophilic response at the inoculation site, and removal of neutrophils following vector transmission led to increased parasite-specific immune responses and promoted the efficacy of the killed vaccine. These observations identify the critical immunological factors influencing vaccine efficacy following natural transmission of Leishmania.
Intracellular infection with the parasite Leishmania major features a state of concomitant immunity in which CD4+ T helper 1 (Th1) cell-mediated immunity against reinfection coincides with a chronic ...but sub-clinical primary infection. In this setting, the rapidity of the Th1 response at a secondary site of challenge in the skin represents the best correlate of parasite elimination and has been associated with a reversal in Leishmania-mediated modulation of monocytic host cells. Remarkably, the degree to which Th1 cells are absolutely reliant upon the time at which they interact with infected monocytes to mediate their protective effect has not been defined. In the present work, we report that CXCR3-dependent recruitment of Ly6C+ Th1 effector (Th1EFF) cells is indispensable for concomitant immunity and acute (<4 days post-infection) Th1EFF cell-phagocyte interactions are critical to prevent the establishment of a permissive pathogen niche, as evidenced by altered recruitment, gene expression and functional capacity of innate and adaptive immune cells at the site of secondary challenge. Surprisingly, provision of Th1EFF cells after establishment of the pathogen niche, even when Th1 cells were provided in large quantities, abrogated protection, Th1EFF cell accumulation and IFN-γ production, and iNOS production by inflammatory monocytes. These findings indicate that protective Th1 immunity is critically dependent on activation of permissive phagocytic host cells by preactivated Th1EFF cells at the time of infection.
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