Leishmania (L.) infantum causes visceral leishmaniasis (VL) in humans across the Mediterranean basin and in Central and South America. VL is a zoonotic disease, with dogs as the predominant domestic reservoir. Traditionally, this disease is transmitted via sand flies of the genus Phlebotomus or Lutzomyia as vectors. Case reports of transmission in non-endemic areas have increased, where transmission is predominantly vertical, sexual, or bloodborne. L. infantum has been shown to be enzootic in hunting hounds within the United States (U.S.), with no indication of vector borne transmission. In this population, there is very high risk of parasite spread if the dam's diagnostic status is positive. In comparison, very little is known about the risk if the sire not the dam is positive for L. infantum. This is a retrospective cohort study of 24 U.S. hunting hound sires' L. infantum exposure between 2013 and 2022 and the resultant evidence of infection in their 183 pups over that time. Offspring from sires who tested serologically positive for L. infantum during the year of birth had 1.59 times the risk of becoming diagnostically positive via serology or molecular detection for L. infantum during their lifetime (RR: 1.59 95% CI: 1.15-2.20 p-value: 0.0046) when compared to outcomes in pups from diagnostically negative sires. The basic reproductive number (R0) for the risk of the sire to indirectly transmit L. infantum to the pup within this cohort was 3.71. These results demonstrate the impact of sire's infection on transmission of L. infantum to offspring. There is a need for control efforts that address non-vectorial transmission from both parents. Control efforts targeting vector borne transmission of canine leishmaniosis in endemic countries are also needed.

Dogs play a major role in sustaining the transmission of Leishmania infantum to people, making prevention and treatment of canine leishmaniosis (CanL) public health priorities. However, immune mechanisms underlying progression from subclinical stages to terminal disease in dogs remain ill-defined. To address this gap, we generated a single-cell RNA sequencing map of peripheral immune cells from uninfected and naturally infected dogs across well-defined stages of L. infantum infection. Disease progression was marked by a shift from a lymphoid-to a myeloid-dominated immune landscape. CD4+ T cells transition from naive to effector states, with TH1 cells showing progressive exhaustion signatures paralleling disease progression, while CD8+ T cells exhibited TPEX-like phenotypes with differentiation toward effector and proliferative programs during severe L. infantum infection. Monocytes showed inflammatory remodeling across clinical stages. The LeishDog Atlas provides a framework for understanding immune dysregulation in CanL and a resource for comparative and translational studies of its immunopathology.

Leishmania infantum causes human visceral leishmaniasis and leishmaniosis (CanL) in reservoir host, dogs. As infection progresses to disease in both humans and dogs, there is a shift from controlling type 1 immunity to a regulatory, exhausted T cell phenotype. In endemic areas, the association between tick-borne coinfections (TBCs) and Leishmania diagnosis and/or clinical severity has been demonstrated. To identify immune factors correlating with disease progression, we prospectively evaluated a cohort of L. infantum-infected dogs from 2019 to 2022. The cohort was TBC-negative with asymptomatic leishmaniosis at the time of enrollment. We measured TBC serology, anti-Leishmania antigen T cell immunity, CanL serological response, parasitemia, and disease severity to probe how nascent TBC perturbs the immune state. At the conclusion, TBC+ dogs with CanL experienced greater increases in anti-Leishmania antibody reactivity and parasite burden compared to dogs that did not have incident TBC during the study. TBC+ dogs were twice as likely to experience moderate (LeishVet stage 2) or severe/terminal disease (LeishVet stage 3/4). Prolonged exposure to TBC was associated with a shift in Leishmania antigen-induced interferon gamma (IFN-γ)/interleukin-10 (IL-10) and enhanced CD8 T cell proliferation. Frequency of proliferating CD8 T cells significantly correlated with parasitemia and antibody reactivity. TBC exacerbated parasite burden and immune exhaustion. These findings highlight the need for combined vector control efforts as prevention programs for dogs in Leishmania endemic areas to reduce transmission to humans. Public health education efforts should aim to increase awareness of the connection between TBC and leishmaniosis.

Canine leishmaniasis (CanL) and canine monocytic ehrlichiosis (CME) are enzootic in Brazil. Although studies have identified risk factors associated with Leishmania seropositivity, very few have combined environmental, social, and dog characteristics, including exposure to other vector-borne pathogens such as Ehrlichia canis, the primary agent of CME in Brazil. In this cross-sectional study, we assessed risk factors associated with Leishmania seropositivity in Fortaleza, Brazil, including dog characteristics, exposure to E. canis, family socioeconomic status, and environmental factors, using univariate chi-squared tests for independence and multivariable random forest models. Antibodies to Leishmania and Ehrlichia were assessed using a chromatographic immunoassay and a rapid ELISA, respectively. In total, 656 dogs were included, with 21.6 % of them being positive for Leishmania. Large dogs and dogs from families in social stratum 7 were more likely to be exposed to Leishmania. Random forest classification models with acceptable discrimination (∼0.65) consistently showed that sleeping outside and household social class had the highest variable importance, regardless of the methods used to impute missing explanatory data. Ehrlichia exposure status, along with environmental (backyard type and presence of cats) and dog variables (fur length, deworming frequency, and tick infestation), ranked among the top ten variables in all models. In conclusion, sleeping outdoors and lower social strata were the most relevant risk factors for Leishmania seropositivity in dogs from Fortaleza. Large-scale studies are needed to evaluate whether these variables are risk factors for L. infantum exposure in other areas where CanL is endemic, possibly also incorporating more human and vector data.

Resident synovial macrophages (RSMs) are anti-inflammatory, self-renewing macrophages that provide physical immune sequestration of the joint space from the peripheral immune system. Increased permeability of this structure is associated with peripheral immune cells in the synovial fluid (SF). Direct measures of synovial barrier integrity are possible with tissue histology, but after barrier breakdown, if these cells perpetuate or initiate chronic inflammation in SF remains unknown. We sought to identify RSM in human SF as an indirect measure of synovial barrier integrity. To validate findings, we created a novel ex vivo explant model using human synovium. scRNA-seq revealed these SF RSMs upregulated pro-fibrotic and pro-osteoclastic pathways in inflammatory arthritis, but not septic arthritis. Increased frequencies of RSMs in SF was associated with increased sRANKL regardless of underlying pathology. These findings suggest the frequency of RSMs in SF may correlate with synovial barrier damage and in turn, potential damage to joint structures.

Visceral leishmaniasis (VL) is a vector-borne disease caused by Leishmania infantum infections in Brazil, and Lutzomyia longipalpis is its main vector. It is not well understood how L. infantum circulates between three hosts of public health importance: dogs, humans, and sand flies. The focus of this study was to determine the detection rate of L. infantum DNA in sand flies and evaluate the relative contribution of the three hosts as sand fly blood sources. Insects were collected using light traps in households with recent history of human VL, close neighbors of VL cases, and households with no recent history of human VL. DNA was extracted from single sand fly guts and L. infantum DNA, and sources of blood were identified by quantitative polymerase chain reaction (qPCR) using specific cytochrome B. Among 248 female sand flies captured, qPCR detected L. infantum DNA in 56% of them. Sand flies captured in households with recent history of human VL or VL endemic neighbors were, respectively, 3.73 (95% CI: 1.64-8.48; P = 4.92e-04) or 6.38 (95% CI: 3.37-12.09; P = 4.62e-10) times more likely to carry L. infantum DNA compared with sand flies captured in houses with no history of VL. Dog blood was most common, but multiple blood sources were detected in individual flies. Our findings reaffirm the role of dogs as L. infantum reservoirs in northeast Brazil. These findings highlight the importance of topical insecticides as measures to prevent canine L. infantum infection and limit the risk of human infection.

International pet travel and commercial operations have increased animal disease importation risks, including for Leishmania infantum, a deadly parasite of humans and domestic dogs. Collaborating as an interdisciplinary working group, we developed an operational tool for veterinary and public health practitioners to assess and manage L. infantum risk in dogs imported to the United States. Overall risk varies by dog, human, and geographic factors but could be high without proper controls. We determined dog risk management strategies should include application of sand fly insecticides and repellents, sterilization, and treatment. US public health authorities can use a One Health approach to manage L. infantum importation risks via infected dogs.

Dogs represent the primary reservoir for Leishmania infantum human visceral leishmaniasis (VL) transmitted through phlebotomine sand flies. Public health initiatives targeting zoonotic VL commonly focus on dogs with severe clinical disease, often in renal failure, as they have previously been considered the most infectious to sand flies. However, more recent studies suggest that dogs with mild to moderate clinical disease may be more infectious than dogs with severe disease. The mechanisms of infectiousness from the skin and how this relates to transmissibility as clinical disease progresses is largely unknown. We evaluated dermal parasitism in dogs naturally infected with L. infantum across the four LeishVet clinical stages of disease. We establish the relationship between dermal parasitism, critical, frequently observed, clinical parameters such as anemia and creatinine, and infectiousness. Using RNAscope and confocal microscopy, we found notable variation in dermal parasitism between dogs, particularly within LeishVet II. Dogs with mild disease had significantly less dermal inflammation and parasitism than dogs with moderate or severe disease. We found significant correlations between anemia, dermal parasitism, and infectiousness (p = 0.0098, r = -0.4798; p = 0.0022, r = -0.8364). In contrast, we did not observe significant correlation between creatinine, a measure of renal function, and dermal parasitism or infectiousness. Host blood cell abnormalities, including anemia, correlate with infectiousness to sand flies. As these signs of disease often appear earlier in the course of disease, this indicates that classical measures of disease severity do not necessarily correlate with infectiousness or epidemiological importance. Public health initiatives attempting to break the zoonotic cycle of L. infantum infection should therefore focus on preventing transmission from infectious, anemic dogs, and not those with the most severe disease.

Abstract Background Visceral leishmaniosis (VL) is the most severe form of human leishmaniosis, with an estimated 95% case fatality if left untreated. Dogs act as peridomestic reservoir hosts for the protozoan parasite Leishmania infantum, a causative agent for human leishmaniosis, endemic throughout the Mediterranean basin. To assure consistent and accurate surveillance of canine infection and prevent transmission to people, consistent diagnosis of canine L. infantum infection across this region is essential for protecting both human and animal health. Our goal was to compare the accuracy, sensitivity and specificity of enzyme-linked immunosorbent assays (ELISA) and immunofluorescence antibody tests (IFAT), performed at seven academic veterinary diagnostic centres across southern Europe and Israel. Methods We performed a known sample “ring” trial to compare L. infantum quantitative serological tests. Two hundred seventy-two (n = 272) canine serum samples of known serological status were chosen from these sites, representative of the region. In-house or commercial ELISA and IFAT were performed according to each laboratory’s specifications. Latent Class Analysis (LCA) was used to determine sensitivity and specificity of each test. True and false positives were calculated to determine the probability of identifying samples. Results Sensitivity and specificity for ELISA ranged from 95 to 99% and 92% to 97%, respectively, with moderate variability from one site. Sensitivity and specificity for IFAT ranged from 89 to 99% and 83% to 94%, respectively, with increased variability compared to ELISA. Overall test agreement was 78% with a pair-wise agreement between 65 and 89%. Conclusions All sites demonstrated substantial comparative diagnostic accuracy, with good agreement based on known seropositive and seronegative samples. Studies and interventional trials that use these tests will remain valid because of high diagnostic agreement between sites. Graphical Abstract

Visceral leishmaniasis derived from Leishmania donovani is transmitted by sand flies (Phlebotomus argentipes) throughout the Indian subcontinent. Although considered anthroponotic, L. donovani infects other mammals susceptible to sand fly bites, including dogs. Aggressive strategies to reduce sand fly populations in India have led to flies seeking nonhuman hosts, so understanding the role of dogs in L. donovani transmission has become critical. Our study investigated L. donovani infection in dogs and the potential for such infections to be transmitted back to sand flies. We performed xenodiagnosis by using P. argentipes on dogs (n = 73) with quantitative PCR-detectible parasitemia in both endemic and outbreak villages. We found that 12% (9/73) of dogs were infectious to sand flies during winter and rainy seasons. Patients with visceral leishmaniasis remain primary sources of L. donovani transmission, but our findings suggest a possible link between canine infection and human exposure.

Visceral leishmaniasis is a deadly infectious disease and is one of the world's major neglected health problems. Because the symptoms of infection are similar to other endemic diseases, accurate diagnosis is crucial for appropriate treatment. Definitive diagnosis using splenic or bone marrow aspirates is highly invasive, and so, serological assays are preferred, including the direct agglutination test (DAT) or rK39 strip test. These tests, however, are either difficult to perform in the field (DAT) or lack specificity in some endemic regions (rK39), making the development of new tests a research priority. The availability of Leishmania spp. genomes presents an opportunity to identify new diagnostic targets. Here, we use genome data and a mammalian protein expression system to create a panel of 93 proteins consisting of the extracellular ectodomains of the Leishmania donovani cell surface and secreted proteins. We use these panel and sera from murine experimental infection models and natural human and canine infections to identify new candidates for serological diagnosis. We observed a concordance between the most immunoreactive antigens in different host species and transmission settings. The antigen encoded by the LdBPK_323600.1 gene can diagnose Leishmania infections with high sensitivity and specificity in patient cohorts from different endemic regions including Bangladesh and Ethiopia. In longitudinal sampling of treated patients, we observed reductions in immunoreactivity to LdBPK_323600.1 suggesting it could be used to diagnose treatment success. In summary, we have identified new antigens that could contribute to improved serological diagnostic tests to help control the impact of this deadly tropical infectious disease. IMPORTANCE:Visceral leishmaniasis is fatal if left untreated with patients often displaying mild and non-specific symptoms during the early stages of infection making accurate diagnosis important. Current methods for diagnosis require highly trained medical staff to perform highly invasive biopsies of the liver or bone marrow which pose risks to the patient. Less invasive molecular tests are available but can suffer from regional variations in their ability to accurately diagnose an infection. To identify new diagnostic markers of visceral leishmaniasis, we produced and tested a panel of 93 proteins identified from the genome of the parasite responsible for this disease. We found that the pattern of host antibody reactivity to these proteins was broadly consistent across naturally acquired infections in both human patients and dogs, as well as experimental rodent infections. We identified a new protein called LdBPK_323600.1 that could accurately diagnose visceral leishmaniasis infections in humans.

Abstract Background Visceral leishmaniasis (VL), known as Kala-azar on the Indian subcontinent, is a parasitic disease caused by the flagellated protozoa Leishmania donovani and can be fatal if left untreated. The sand fly Phlebotomus argentipes is the only proven vector of VL in the Southeast Asia region, and VL control in this region has relied on the use of synthetic insecticides for indoor residual spraying (IRS). The use of DDT in VL control programmes has led to the development of resistance to this insecticide in sand flies, resulting in DDT being replaced with the insecticide alpha-cypermethrin. However, alpha-cypermethrin has a similar mode of action as DDT and, therefore, the risk of resistance development in sand flies increases under the pressure of regular exposure to this insecticide. In the present study we assessed the susceptibility status of wild-caught sand flies and F1 progeny using the CDC bottle bioassay. Methods Sand flies were collected from 10 villages in Muzaffarpur District, Bihar, India. Eight of these villages are receiving continuous IRS with alpha-cypermethrin, one village had discontinued IRS with alpha-cypermethrin and one village had never received IRS with alpha-cypermethrin. The collected sand flies were exposed to a pre-determined diagnostic dose for a specific time duration (3 µg/ml for 40 min), and knockdown and mortality at 24 h post-exposure were recorded. Results Knockdown ranged from 91.19% to 99.47% for wild-caught sand flies and from 91.70% to 98.89% for their F1 progeny. At 24 h post-exposure, mortality ranged from 89.34% to 98.93% for wild-caught sand flies and from 90.16% to 98.33% for F1 progeny. Conclusions The results of this study showed that P. argentipes is potentially developing resistance, signalling the need for continuous monitoring and vigilance to sustain the validation of elimination once achieved. Graphical Abstract

Visceral leishmaniasis (VL) is a vector-borne protozoan disease, which can be fatal if left untreated. Synthetic chemical insecticides are very effective tools for controlling of insect vectors, including the sand fly Phlebotomus argentipes, the vector of VL in the Indian subcontinent. However, repeated use of the same insecticide with increasing doses potentially can create high selection pressure and lead to tolerance and resistance development. The objective of this study was to determine the lethal concentrations and assess levels of susceptibility, diagnostic doses and times to death of laboratory-reared P. argentipes to five insecticides that are used worldwide to control vectors. Using the Center for Disease Control and Prevention (CDC) bottle bioassay, 20–30 sand flies were exposed in insecticide- coated 500-ml glass bottles. Flies were then observed for 24 hours and mortality was recorded. Dose-response survival curves were generated for each insecticide using QCal software and lethal concentrations causing 50%, 90% and 95% mortality were determined. A bioassay was also conducted to determine diagnostic doses and diagnostic times by exposing 20–30 flies in each bottle containing set concentrations of insecticide. Mortality was recorded at 10-minute intervals for 120 minutes to generate the survival curve. Phlebotomus argentipes are highly susceptible to alpha-cypermethrin, followed by deltamethrin, malathion, chlorpyrifos, and least susceptible to DDT. Also, the lowest diagnostic doses and diagnostic times were established for alpha-cypermethrin (3μg/ml for 40 minutes) to kill 100% of the flies. The susceptibility data, diagnostic doses and diagnostic times presented here will be useful as baseline reference points for future studies to assess insecticide susceptibility and resistance monitoring of field caught sand flies and to assist in surveillance as VL elimination is achieved in the region.

Visceral leishmaniasis (VL) is a neglected tropical disease that is globally distributed and has the potential to cause very serious illness. Prior literature highlights the emergence and spread of VL is influenced by multiple factors, such as socioeconomic status, sanitation levels or animal and human reservoirs. The study aimed to retrospectively investigate the presence and infectiousness of VL in Rio Grande do Norte (RN), Brazil between 2007 and 2020. We applied a hierarchical Bayesian approach to estimate municipality-specific relative risk of VL across space and time. The results show evidence that lower socioeconomic status is connected to higher municipality-specific VL risk. Overall, estimates reveal spatially heterogeneous VL risks in RN, with a high probability that VL risk for municipalities within the West Potiguar mesoregion are more than double the expected VL risk. Additionally, given the data available, results indicate there is a high probability of increasing VL risk in the municipalities of Natal, Patu and Pau dos Ferros. These findings demonstrate opportunities for municipality-specific public health policy interventions and warrant future research on identifying epidemiological drivers in at-risk regions.

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.

Purpose: Vaccinating adolescents against COVID-19 while avoiding delays in other routine vaccination is paramount to protecting their health. Our objective was to assess parental prefer-ences to have their adolescents aged 12-17 years receive COVID-19 and other routine vaccines at the same time.Methods: An online survey with a national, quota-based cross-sectional sample of United States parents of youth aged 12-17 years was fielded in April 2021 ahead of FDA's Emergency Use Authorization of COVID-19 vaccine for age 12-15 years. Parents were asked about their willingness to have their adolescents aged 12-17 years receive both COVID-19 and routine vaccines at the same visit and/or to follow their provider's recommendation. Predictors included demographic characteristics, being behind on routine vaccines, and perceived risks and benefits.Results: Few parents were willing to have their adolescent receive COVID-19 and routine vaccines at the same visit (10.6%) or follow the healthcare provider's recommendation (18.5%). In multi-variate analyses, demographic characteristics had no effect on willingness, reporting that the adolescent was behind on routine vaccines correlated with decreased willingness (p = .004). Greater concern about the adolescent getting COVID-19 (p = .001), lower concern about the adolescent having side effects from the COVID-19 vaccine (p = .013), and more positive feelings about vaccines in general (p = .002) were associated with higher willingness.Discussion: Few parents would prefer to have their adolescents receive COVID-19 and routine vaccines at the same visit. Understanding what drives willingness to receive all recommended vaccines in the context of the COVID-19 pandemic could inform policies to optimize adolescent vaccination.(c) 2022 Society for Adolescent Health and Medicine. All rights reserved.

The impact of continued COVID-19 sequelae on singers' vocal function has yet to be determined. An online survey of singers who have contracted SARS-CoV-2 infection was designed and administered globally. Participants (n = 1,153) were recruited in Africa, the Americas, Asia, Australia, and Europe. Survey questions included demographics, peri- and post-SARS-CoV-2 infection symptoms, and self-reported sequelae attributed to long-COVID. The survey was made available in English, Portuguese, Spanish, and Traditional and Simplified Mandarin Chinese. Data were statistically analyzed to provide a useful summary of the sample and to evaluate associations between long-COVID and singers' vocal function. We found that age, gender, and vaccination status were not significantly correlated to a change in singing voice in our sample. However, severity of infection was statistically correlated with a change in singing voice. Of the 34 signs and symptoms presented, lingering cough, shortness of breath, and chronic fatigue were significantly correlated with a change in singing voice. These data and their analyses have added to our understanding of this growing population's unique vocal needs, and may inform strategies for singing voice habilitation in COVID-19 survivors.

Background: The Advisory Committee on Immunization Practices (ACIP) recommends shared clinical decision-making (SCDM) regarding HPV vaccination for adults aged 27-45 years who are not adequately vaccinated. The objective of this survey was to understand physician knowledge, attitudes, and practices regarding HPV vaccination in this age group.Methods: An online survey was administered in June 2021 to physicians who reported practicing internal medicine, family medicine, or obstetrics and gynecology (targeted N = 250 in each practice specialty), selected randomly from potentially eligible physicians from a panel of 2 million U.S. health care providers. Results: In total, 753 physicians participated in the survey: 33.3% practiced internal medicine, 33.1% practiced family medicine, and 33.6% practiced obstetrics/gynecology; 62.5% were male and mean physician age was 52.7 years. Despite the COVID-19 pandemic, at least a third of participating physicians in each practice specialty reported having more HPV vaccine SCDM discussions with patients aged 27-45 years in the past 12 months. While a majority of physicians (79.7%) reported being aware of the SCDM recommendation for adults in this age group, only half of physicians answered an objective knowledge question about SCDM recommendations correctly.Conclusions: Findings suggest that there are physician knowledge gaps related to SCDM for HPV vaccination. To improve access to HPV vaccination for people most likely to benefit, increasing availability and use of decision aids to support SCDM discussions might help healthcare providers and patients jointly make the most informed decisions about HPV vaccination.