Within-host bayesian joint modeling of longitudinal and time-to-event data of Leishmania infection.

The host immune system plays a significant role in managing and clearing pathogen material during an infection, but this complex process presents numerous challenges from a modeling perspective. There are many mathematical and statistical models for these kinds of processes that take into account a wide range of events that happen within the host. In this work, we present a Bayesian joint model of longitudinal and time-to-event data of Leishmania infection that considers the interplay between key drivers of the disease process: pathogen load, antibody level, and disease. The longitudinal model also considers approximate inflammatory and regulatory immune factors. In addition to measuring antibody levels produced by the immune system, we adapt data from CD4+ and CD8+ T cell proliferation, and expression of interleukin 10, interferon-gamma, and programmed cell death 1 as inflammatory or regulatory factors mediating the disease process. The model is developed using data collected from a cohort of dogs naturally exposed to Leishmania infantum. The cohort was chosen to start with healthy infected animals, and this is the majority of the data. The model also characterizes the relationship features of the longitudinal outcomes and time-to-death due to progressive Leishmania infection. In addition to describing the mechanisms causing disease progression and impacting the risk of death, we also present the model's ability to predict individual trajectories of Canine Leishmaniosis (CanL) progression. The within-host model structure we present here provides a way forward to address vital research questions regarding the understanding of the progression of complex chronic diseases such as Visceral Leishmaniasis, a parasitic disease causing significant morbidity worldwide.

Resident Synovial Macrophages in Synovial Fluid: Implications for Immunoregulation in Infectious and Inflammatory Arthritis.

Objectives: Resident synovial macrophages (RSM) provide immune sequestration of the joint space and are likely involved in initiation and perpetuation of the joint-specific immune response. We sought to identify RSM in synovial fluid (SF) and demonstrate migratory ability, in additional to functional changes that may perpetuate a chronic inflammatory response within joint spaces. Methods: We recruited human patients presenting with undifferentiated arthritis in multiple clinical settings. We used flow cytometry to identify mononuclear cells in peripheral blood and SF. We used a novel transwell migration assay with human ex-vivo synovium obtained intra-operatively to validate flow cytometry findings. We used single cell RNA-sequencing (scRNA-seq) to further identify macrophage/monocyte subsets. ELISA was used to evaluate the bone-resorption potential of SF. Results: We were able to identify a rare population of CD14dim, OPG+, ZO-1+ cells consistent with RSM in SF via flow cytometry. These cells were relatively enriched in the SF during infectious processes, but absolutely decreased compared to healthy controls. Similar putative RSM were identified using ex vivo migration assays when MCP-1 and LPS were used as migratory stimulus. scRNA-seq revealed a population consistent with RSM transcriptionally related to CD56+ cytotoxic dendritic cells and IDO+ M2 macrophages. Conclusion: We identified a rare cell population consistent with RSM, indicating these cells are likely migratory and able to initiate or coordinate both acute (septic) or chronic (autoimmune or inflammatory) arthritis. RSM analysis via scRNA-seq indicated these cells are M2 skewed, capable of antigen presentation, and have consistent functions in both septic and inflammatory arthritis.

Within-Host Bayesian Joint Modeling of Longitudinal and Time-to-Event Data of Leishmania Infection.

The host immune system plays a significant role in managing and clearing pathogen material during an infection, but this complex process presents numerous challenges from a modeling perspective. There are many mathematical and statistical models for these kinds of processes that take into account a wide range of events that happen within the host. In this work, we present a Bayesian joint model of longitudinal and time-to-event data of Leishmania infection that considers the interplay between key drivers of the disease process: pathogen load, antibody level, and disease. The longitudinal model also considers approximate inflammatory and regulatory immune factors. In addition to measuring antibody levels produced by the immune system, we adapt data from CD4+ and CD8+ T cell proliferation, and expression of interleukin 10, interferon-gamma, and programmed cell death 1 as inflammatory or regulatory factors mediating the disease process. The model is developed using data collected from a cohort of dogs naturally exposed to Leishmania infantum. The cohort was chosen to start with healthy infected animals, and this is the majority of the data. The model also characterizes the relationship features of the longitudinal outcomes and time of death due to progressive Leishmania infection. In addition to describing the mechanisms causing disease progression and impacting the risk of death, we also present the model's ability to predict individual trajectories of Canine Leishmaniosis (CanL) progression. The within-host model structure we present here provides a way forward to address vital research questions regarding the understanding progression of complex chronic diseases such as Visceral Leishmaniasis, a parasitic disease causing significant morbidity worldwide.

Modulation of Macrophage Redox and Apoptotic Processes to Leishmania infantum during Coinfection with the Tick-Borne Bacteria Borrelia burgdorferi.

Canine leishmaniosis (CanL) is a zoonotic disease caused by protozoan Leishmania infantum. Dogs with CanL are often coinfected with tick-borne bacterial pathogens, including Borrelia burgdorferi in the United States. These coinfections have been causally associated with hastened disease progression and mortality. However, the specific cellular mechanisms of how coinfections affect microbicidal responses against L. infantum are unknown. We hypothesized that B. burgdorferi coinfection impacts host macrophage effector functions, prompting L. infantum intracellular survival. In vitro experiments demonstrated that exposure to B. burgdorferi spirochetes significantly increased L. infantum parasite burden and pro-inflammatory responses in DH82 canine macrophage cells. Induction of cell death and generation of mitochondrial ROS were significantly decreased in coinfected DH82 cells compared to uninfected and L. infantum-infected cells. Ex vivo stimulation of PBMCs from L. infantum-seronegative and -seropositive subclinical dogs with spirochetes and/or total Leishmania antigens promoted limited induction of IFNγ. Coexposure significantly induced expression of pro-inflammatory cytokines and chemokines associated with Th17 differentiation and neutrophilic and monocytic recruitment in PBMCs from L. infantum-seropositive dogs. Excessive pro-inflammatory responses have previously been shown to cause CanL pathology. This work supports effective tick prevention and risk management of coinfections as critical strategies to prevent and control L. infantum progression in dogs.

Bayesian multivariate longitudinal model for immune responses to Leishmania: A tick-borne co-infection study.

While many Bayesian state-space models for infectious disease processes focus on population infection dynamics (eg, compartmental models), in this work we examine the evolution of infection processes and the complexities of the immune responses within the host using these techniques. We present a joint Bayesian state-space model to better understand how the immune system contributes to the control of Leishmania infantum infections over the disease course. We use longitudinal molecular diagnostic and clinical data of a cohort of dogs to describe population progression rates and present evidence for important drivers of clinical disease. Among these results, we find evidence for the importance of co-infection in disease progression. We also show that as dogs progress through the infection, parasite load is influenced by their age, ectoparasiticide treatment status, and serology. Furthermore, we present evidence that pathogen load information from an earlier point in time influences its future value and that the size of this effect varies depending on the clinical stage of the dog. In addition to characterizing the processes driving disease progression, we predict individual and aggregate patterns of Canine Leishmaniasis progression. Both our findings and the application to individual-level predictions are of direct clinical relevance, presenting possible opportunities for application in veterinary practice and motivating lines of additional investigation to better understand and predict disease progression. Finally, as an important zoonotic human pathogen, these results may support future efforts to prevent and treat human Leishmaniosis.

Livestock and rodents within an endemic focus of Visceral Leishmaniasis are not reservoir hosts for Leishmania donovani.

Leishmaniasis on the Indian subcontinent is thought to have an anthroponotic transmission cycle. There is no direct evidence that a mammalian host other than humans can be infected with Leishmania donovani and transmit infection to the sand fly vector. The aim of the present study was to evaluate the impact of sand fly feeding on other domestic species and provide clinical evidence regarding possible non-human reservoirs through experimental sand fly feeding on cows, water buffalo goats and rodents. We performed xenodiagnosis using colonized Phlebotomus argentipes sand flies to feed on animals residing in villages with active Leishmania transmission based on current human cases. Xenodiagnoses on mammals within the endemic area were performed and blood-fed flies were analyzed for the presence of Leishmania via qPCR 48hrs after feeding. Blood samples were also collected from these mammals for qPCR and serology. Although we found evidence of Leishmania infection within some domestic mammals, they were not infectious to vector sand flies. Monitoring infection in sand flies and non-human blood meal sources in endemic villages leads to scientific proof of exposure and parasitemia in resident mammals. Lack of infectiousness of these domestic mammals to vector sand flies indicates that they likely play no role, or a very limited role in Leishmania donovani transmission to people in Bihar. Therefore, a surveillance system in the peri-/post-elimination phase of visceral leishmaniasis (VL) must monitor absence of transmission. Continued surveillance of domestic mammals in outbreak villages is necessary to ensure that a non-human reservoir is not established, including domestic mammals not present in this study, specifically dogs.

Visceral Leishmaniasis and the Skin: Dermal Parasite Transmission to Sand Flies.

Visceral leishmaniasis is a parasitic disease with significant dermal tropism. The skin is an important site of infection contributing to parasite transmission to naïve sand flies, but understanding how parasitism of host skin and the related immune microenvironment supports or prevents skin parasite replication is now the focus of major investigation in the field of leishmaniasis research. Here, we review dermatoimmunology during visceral leishmaniasis (VL), dermal Leishmania parasite burden, and the role of skin parasitism in transmissibility to sand fly vectors. First, we discuss the epidemiology of VL amongst dogs, the primary zoonotic reservoir for human infection. We explore the association between spatial distribution and the burden of parasites in the skin in driving outward transmission. Factors associated with parasite persistence in the skin are examined. We discuss systemic immunity during VL and what is known about immunological correlates in the skin microenvironment. Finally, we touch on factors egested into the skin during Leishmania inoculation by sand flies. Throughout, we discuss factors associated with the early and chronic establishment of Leishmania parasites in the skin and the role of the dermal immune response.

Role of NK-Like CD8+ T Cells during Asymptomatic Borrelia burgdorferi Infection.

Lyme disease (LD) due to Borrelia burgdorferi is the most prevalent vector-borne disease in the United States. There is a poor understanding of how immunity contributes to bacterial control, pathology, or both during LD. Dogs in an area of endemicity were screened for B. burgdorferi and Anaplasma exposure and stratified according to seropositivity, presence of LD symptoms, and doxycycline treatment. Significantly elevated serum interleukin-21 (IL-21) and increased circulating CD3+ CD94+ lymphocytes with an NK-like CD8+ T cell phenotype were predominant in asymptomatic dogs exposed to B. burgdorferi. Both CD94+ T cells and CD3- CD94+ lymphocytes, corresponding to NK cells, from symptomatic dogs expressed gamma interferon (IFN-γ) at a 3-fold-higher frequency upon stimulation with B. burgdorferi than the same subset among endemic controls. Surface expression of activating receptor NKp46 was reduced on CD94+ T cells from LD, compared to cells after doxycycline treatment. A higher frequency of NKp46-expressing CD94+ T cells correlated with significantly increased peripheral blood mononuclear cell (PBMC) cytotoxic activity via calcein release assay. PBMCs from dogs with symptomatic LD showed significantly reduced killing ability compared with endemic control PBMCs. An elevated NK-like CD8+ T cell response was associated with protection against development of clinical LD, while excess IFN-γ was associated with clinical disease.

Bayesian latent class models for identifying canine visceral leishmaniosis using diagnostic tests in the absence of a gold standard.

BACKGROUND: Like many infectious diseases, there is no practical gold standard for diagnosing clinical visceral leishmaniasis (VL). Latent class modeling has been proposed to estimate a latent gold standard for identifying disease. These proposed models for VL have leveraged information from diagnostic tests with dichotomous serological and PCR assays, but have not employed continuous diagnostic test information. METHODS/PRINCIPAL FINDINGS: In this paper, we employ Bayesian latent class models to improve the identification of canine visceral leishmaniasis using the dichotomous PCR assay and the Dual Path Platform (DPP) serology test. The DPP test has historically been used as a dichotomous assay, but can also yield numerical information via the DPP reader. Using data collected from a cohort of hunting dogs across the United States, which were identified as having either negative or symptomatic disease, we evaluate the impact of including numerical DPP reader information as a proxy for immune response. We find that inclusion of DPP reader information allows us to illustrate changes in immune response as a function of age. CONCLUSIONS/SIGNIFICANCE: Utilization of continuous DPP reader information can improve the correct discrimination between individuals that are negative for disease and those with clinical VL. These models provide a promising avenue for diagnostic testing in contexts with multiple, imperfect diagnostic tests. Specifically, they can easily be applied to human visceral leishmaniasis when diagnostic test results are available. Also, appropriate diagnosis of canine visceral leishmaniasis has important consequences for curtailing spread of disease to humans.

Domestic mammals as reservoirs for Leishmania donovani on the Indian subcontinent: Possibility and consequences on elimination.

Leishmania donovani is the causative agent of historically anthroponotic visceral leishmaniasis (VL) on the Indian subcontinent (ISC). L. donovani is transmitted by the sand fly species Phlebotomus argentipes. Our collaborative group and others have shown that sand flies trapped outside in endemic villages have fed on cattle and dogs in addition to people. Domestic animals are reservoirs for L. donovani complex spp., particularly L. infantum, in other endemic areas. Multiple studies using quantitative PCR or serological detection methods have demonstrated that goats, cattle, rats and dogs were diagnostically positive for L. donovani infection or exposure in eastern Africa, Bangladesh, Nepal and India. There is a limited understanding of the extent to which L. donovani infection of domestic animals drives transmission to other animals or humans on the ISC. Evidence from other vector-borne disease elimination strategies indicated that emerging infections in domestic species hindered eradication. The predominant lesson learned from these other situations is that non-human reservoirs must be identified, controlled and/or prevented. Massive efforts are underway for VL elimination on the Indian subcontinent. Despite these herculean efforts, residual VL incidence persists. The spectre of an animal reservoir complicating elimination efforts haunts the final push towards full VL control. Better understanding of L. donovani transmission on the Indian subcontinent and rigorous consideration of how non-human reservoirs alter VL ecology are critical to sustain elimination goals.

Epidemiologic, Clinical and Immunological Consequences of Co-Infections during Canine Leishmaniosis.

Canine leishmaniosis (CanL) is a vector-borne, parasitic disease. CanL is endemic in the Mediterranean basin and South America but also found in Northern Africa, Asia, and the U.S. Regions with both competent sand fly vectors and L. infantum parasites are also endemic for additional infectious diseases that could cause co-infections in dogs. Growing evidence indicates that co-infections can impact immunologic responses and thus the clinical course of both CanL and the comorbid disease(s). The aim for this review is to summarize epidemiologic, clinical, and immunologic factors contributing to eight primary co-infections reported with CanL: Ehrlichia spp., Anaplasma spp., Borrelia spp., Babesia spp., Trypanosoma cruzi, Toxoplasma gondii, Dirofilaria immitis, Paracoccidioides braziliensis. Co-infection causes mechanistic differences in immunity which can alter diagnostics, therapeutic management, and prognosis of dogs with CanL. More research is needed to further explore immunomodulation during CanL co-infection(s) and their clinical impact.

Leishmania infantum xenodiagnosis from vertically infected dogs reveals significant skin tropism.

BACKGROUND: Dogs are the primary reservoir for human visceral leishmaniasis due to Leishmania infantum. Phlebotomine sand flies maintain zoonotic transmission of parasites between dogs and humans. A subset of dogs is infected transplacentally during gestation, but at what stage of the clinical spectrum vertically infected dogs contribute to the infected sand fly pool is unknown. METHODOLOGY/PRINCIPAL FINDINGS: We examined infectiousness of dogs vertically infected with L. infantum from multiple clinical states to the vector Lutzomyia longipalpis using xenodiagnosis and found that vertically infected dogs were infectious to sand flies at differing rates. Dogs with mild to moderate disease showed significantly higher transmission to the vector than dogs with subclinical or severe disease. We documented a substantial parasite burden in the skin of vertically infected dogs by RT-qPCR, despite these dogs not having received intradermal parasites via sand flies. There was a highly significant correlation between skin parasite burden at the feeding site and sand fly parasite uptake. This suggests dogs with high skin parasite burden contribute the most to the infected sand fly pool. Although skin parasite load and parasitemia correlated with one another, the average parasite number detected in skin was significantly higher compared to blood in matched subjects. Thus, dermal resident parasites were infectious to sand flies from dogs without detectable parasitemia. CONCLUSIONS/SIGNIFICANCE: Together, our data implicate skin parasite burden and earlier clinical status as stronger indicators of outward transmission potential than blood parasite burden. Our studies of a population of dogs without vector transmission highlights the need to consider canine vertical transmission in surveillance and prevention strategies.

Canine leishmaniasis in Northern California-A case report.

A 3-year-old dog was referred to the Veterinary Medical Teaching Hospital of the University of California-Davis for further evaluation of episodes of epistaxis of 1-year duration and peripheral lymphadenopathy. The patient had a history of atopic dermatitis with no travel history outside of California. Hyperglobulinemia with a polyclonal gammopathy was noted on serum protein electrophoresis. Microscopic evaluation of a bone marrow aspirate sample revealed many free and intra-cellular amastigotes of Leishmania sp. that was further confirmed by qPCR as L infantum. This is, to the best of our knowledge, the first reported case of canine leishmaniasis in the state of California. The patient is believed to have been vertically infected from the dam who is from Serbia and remained subclinical until presentation. Because the clinical progression of leishmaniasis is variable, it is important that precautions be discussed with owners acquiring puppies with dams from endemic regions of leishmaniasis to prevent zoonotic exposure in states where competent vectors are present.

Bayesian compartmental models and associated reproductive numbers for an infection with multiple transmission modes.

Zoonotic visceral leishmaniasis (ZVL) is a serious neglected tropical disease that is endemic in 98 countries. ZVL is primarily transmitted via a sand fly vector. In the United States, it is enzootic in some canine populations; it is transmitted from infectious mother to pup transplacentally, and vector-borne transmission is absent. This absence affords a unique opportunity to study (1) vertical transmission dynamics in dogs and (2) the importance of vertical transmission in maintaining an infectious reservoir in the presence of a vector. In this paper, we present Bayesian compartmental models and reproductive number formulations to examine (1) and (2), providing a mechanism to plan and evaluate interventions in regions where both transmission modes are present. First, we propose an individual-level susceptible, infectious, removed (SIR) model to study the effect of maternal infection status during pregnancy on pup infection progression. We provide evidence that pups born to diagnostically positive mothers during pregnancy are more likely to become diagnostically positive both earlier in life, and at some point during their lifetime, than those born to diagnostically negative mothers. Second, we propose a population-level SIR model to study the impact of a vertically maintained reservoir on propagating infection in a naive canine population through emergent vector transmission using simulation studies. We also present reproductive numbers to quantify contributions of vertically infected and vector-infected dogs to maintaining infection in the population. We show that a vertically maintained canine reservoir can propagate infection in a theoretical naive population in the presence of a vector.

Coinfection with Leishmania major and Staphylococcus aureus enhances the pathologic responses to both microbes through a pathway involving IL-17A.

Cutaneous leishmaniasis (CL) is a parasitic disease causing chronic, ulcerating skin lesions. Most humans infected with the causative Leishmania protozoa are asymptomatic. Leishmania spp. are usually introduced by sand flies into the dermis of mammalian hosts in the presence of bacteria from either the host skin, sand fly gut or both. We hypothesized that bacteria at the dermal inoculation site of Leishmania major will influence the severity of infection that ensues. A C57BL/6 mouse ear model of single or coinfection with Leishmania major, Staphylococcus aureus, or both showed that single pathogen infections caused localized lesions that peaked after 2-3 days for S. aureus and 3 weeks for L. major infection, but that coinfection produced lesions that were two-fold larger than single infection throughout 4 weeks after coinfection. Coinfection increased S. aureus burdens over 7 days, whereas L. major burdens (3, 7, 28 days) were the same in singly and coinfected ears. Inflammatory lesions throughout the first 4 weeks of coinfection had more neutrophils than did singly infected lesions, and the recruited neutrophils from early (day 1) lesions had similar phagocytic and NADPH oxidase capacities. However, most neutrophils were apoptotic, and transcription of immunomodulatory genes that promote efferocytosis was not upregulated, suggesting that the increased numbers of neutrophils may, in part, reflect defective clearance and resolution of the inflammatory response. In addition, the presence of more IL-17A-producing γδ and non-γδ T cells in early lesions (1-7 days), and L. major antigen-responsive Th17 cells after 28 days of coinfection, with a corresponding increase in IL-1ß, may recruit more naïve neutrophils into the inflammatory site. Neutralization studies suggest that IL-17A contributed to an enhanced inflammatory response, whereas IL-1ß has an important role in controlling bacterial replication. Taken together, these data suggest that coinfection of L. major infection with S. aureus exacerbates disease, both by promoting more inflammation and neutrophil recruitment and by increasing neutrophil apoptosis and delaying resolution of the inflammatory response. These data illustrate the profound impact that coinfecting microorganisms can exert on inflammatory lesion pathology and host adaptive immune responses.

An Anti-Inflammatory Role for NLRP10 in Murine Cutaneous Leishmaniasis.

The role of the nucleotide-binding domain and leucine-rich repeat containing receptor NLRP10 in disease is incompletely understood. Using three mouse strains lacking the gene encoding NLRP10, only one of which had a coincidental mutation in DOCK8, we documented a role for NLRP10 as a suppressor of the cutaneous inflammatory response to Leishmania major infection. There was no evidence that the enhanced local inflammation was due to enhanced inflammasome activity. NLRP10/DOCK8-deficient mice harbored lower parasite burdens at the cutaneous site of inoculation compared with wild-type controls, whereas NLRP10-deficient mice and controls had similar parasite loads, suggesting that DOCK8 promotes local growth of parasites in the skin, whereas NLRP10 does not. NLRP10-deficient mice developed vigorous adaptive immune responses, indicating that there was not a global defect in the development of Ag-specific cytokine production. Bone marrow chimeras showed that the anti-inflammatory role of NLRP10 was mediated by NLRP10 expressed in resident cells in the skin rather than by bone marrow-derived cells. These data suggest a novel role for NLRP10 in the resolution of local inflammatory responses during L. major infection.

Differential Activation of Human Keratinocytes by Leishmania Species Causing Localized or Disseminated Disease.

All Leishmania species parasites are introduced into mammalian skin through a sand fly bite, but different species cause distinct clinical outcomes. Mouse studies suggest that early responses are critical determinants of subsequent adaptive immunity in leishmaniasis, yet few studies address the role of keratinocytes, the most abundant cell in the epidermis. We hypothesized that Leishmania infection causes keratinocytes to produce immunomodulatory factors that influence the outcome of infection. Incubation of primary or immortalized human keratinocytes with Leishmania infantum or Leishmania major, which cause visceral or cutaneous leishmaniasis, respectively, elicited dramatically different responses. Keratinocytes incubated with L. infantum significantly increased expression of proinflammatory genes for IL-6, IL-8, tumor necrosis factor, and IL-1B, whereas keratinocytes exposed to several L. major isolates did not. Furthermore, keratinocyte-monocyte co-incubation studies across a 4 µM semipermeable membrane suggested that L. infantum-exposed keratinocytes release soluble factors that enhance monocyte control of intracellular L. infantum replication (P < 0.01). L. major-exposed keratinocytes had no comparable effect. These data suggest that L. infantum and L. major differentially activate keratinocytes to release factors that limit infection in monocytes. We propose that keratinocytes initiate or withhold a proinflammatory response at the site of infection, generating a microenvironment uniquely tailored to each Leishmania species that may affect the course of disease.

Cutaneous Manifestations of Human and Murine Leishmaniasis.

The leishmaniases are diseases caused by pathogenic protozoan parasites of the genus Leishmania. Infections are initiated when a sand fly vector inoculates Leishmania parasites into the skin of a mammalian host. Leishmania causes a spectrum of inflammatory cutaneous disease manifestations. The type of cutaneous pathology is determined in part by the infecting Leishmania species, but also by a combination of inflammatory and anti-inflammatory host immune response factors resulting in different clinical outcomes. This review discusses the distinct cutaneous syndromes described in humans, and current knowledge of the inflammatory responses associated with divergent cutaneous pathologic responses to different Leishmania species. The contribution of key hematopoietic cells in experimental cutaneous leishmaniasis in mouse models are also reviewed and compared with those observed during human infection. We hypothesize that local skin events influence the ensuing adaptive immune response to Leishmania spp. infections, and that the balance between inflammatory and regulatory factors induced by infection are critical for determining cutaneous pathology and outcome of infection.