Development of loop-mediated isothermal amplification (LAMP) assay using SYBR safe and gold-nanoparticle probe for detection of Leishmania in HIV patients.

Asymptomatic leishmaniasis cases have continuously increased, especially among patients with HIV who are at risk to develop further symptoms of cutaneous and visceral leishmaniasis. Thus, early diagnosis using a simple, sensitive and reliable diagnostic assay is important because populations at risk mostly reside in rural communities where laboratory equipment is limited. In this study, the highly sensitive and selective determination of Leishmania infection in asymptomatic HIV patients was achieved using dual indicators (SYBR safe and gold-nanoparticle probe; AuNP-probe) in one-step LAMP method based on basic instruments. The assay can be simply evaluated under the naked eye due to clear interpretation of fluorescent emission of LAMP-SYBR safe dye-complex and colorimetric precipitate of specific AuNP-probes. The sensitivities and specificities of fluorescent SYBR safe dye and AuNP-probe indicators were equal, which were as high as 94.1 and 97.1%, respectively. Additionally, detection limits were 102 parasites/mL (0.0147 ng/µL), ten times more sensitivity than other related studies. To empower leishmaniasis surveillance, this inexpensive one-step SYBR safe and AuNP-LAMP assay is reliably fast and simple for field diagnostics to point-of-care settings, which can be set up in all levels of health care facilities including resource limited areas, especially in low to middle income countries.

Unraveling the Role of Immune Checkpoints in Leishmaniasis.

Leishmaniasis are Neglected Tropical Diseases affecting millions of people every year in at least 98 countries and is one of the major unsolved world health issues. Leishmania is a parasitic protozoa which are transmitted by infected sandflies and in the host they mainly infect macrophages. Immunity elicited against those parasites is complex and immune checkpoints play a key role regulating its function. T cell receptors and their respective ligands, such as PD-1, CTLA-4, CD200, CD40, OX40, HVEM, LIGHT, 2B4 and TIM-3 have been characterized for their role in regulating adaptive immunity against different pathogens. However, the exact role those receptors perform during Leishmania infections remains to be better determined. This article addresses the key role immune checkpoints play during Leishmania infections, the limiting factors and translational implications.

Case Report: Hyper IgM Syndrome Identified by Whole Genome Sequencing in a Young Syrian Man Presenting With Atypical, Severe and Recurrent Mucosal Leishmaniasis.

A previously healthy 19-year-old Syrian man presented with atypical and severe mucosal leishmaniasis caused by Leishmania tropica. During a 2-year period, he had three severe relapses despite various treatment strategies, including liposomal amphotericin B and Miltefosine. Because of the unusual clinical presentation, potential underlying immunodeficiency was investigated. Normal T and NK cell counts were found. The B cell count was slightly elevated at 0.7 × 109 cells/L (0.09 × 109 to 0.57 × 109 cells/L), but the proportions of memory and isotype switched memory B cells were severely diminished IgG levels were low, at 309 mg/dL (610-1490 mg/dL). The initial IgM and IgA levels were within normal range, but the IgA levels decreased to 57 mg/dL (70-430 mg/dL) during follow up. Common variable immunodeficiency (CVID) was initially suspected, because the immunological results of low IgG and IgA, low switched memory B cells, no profound T cell deficiency found and absence of secondary cause of hypogammaglobulinemia were compatible with this diagnosis (ESID 2019). However, the highly unusual and severe clinical presentation of L. tropica is not suggestive of B-cell deficiency or CVID. Eventually a pathogenic nonsense variant in the CD40 ligand gene [p.(Arg11∗)] was identified by whole genome sequencing, thus enabling the diagnosis of X-linked hyper IgM syndrome. This case illustrates and supports the potential for the use of whole genome sequencing in accurate diagnosis of primary immunodeficiencies.

Interleukin-27 Functional Duality Balances Leishmania Infectivity and Pathogenesis.

IL-27 is a cytokine that exerts diverse effects on the cells of innate and adaptive immune systems. Chiefly expressed in macrophages and dendritic cells during the early phase of Leishmania infection, IL-27 contributes to the protection against L. major infection but suppresses the protective Th1 response against L. donovani, L. infantum, L. amazonensis and L. braziliensis infections, suggesting its functional duality. During the late stage of Leishmania infection, IL-27 limits the immunopathogenic reactions and tissue damages. Herein, we analyze the mechanism of the functional duality of IL-27 in the resistance or susceptibility to Leishmania infection, prompting IL-27 for anti-Leishmanial therapy.

Environmental risk factors of leishmaniasis in Bahia State, Brazil using NASA Earth observation satellites / Fatores de risco ambientais para leishmanioses no estado da Bahia, Brasil, utilizando satélites da NASA de observação da Terra

NASA’s Earth Observing Satellites (EOS) were used to calculate three vegetation indices, extract precipitation and elevation data, and then evaluate their applicability for assessing risk of visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL) in Bahia State, Brazil. Regression models showed that either form of leishmaniasis can be predicted by NDVI, NDMI, NDWI data products and TRMM) precipitation data (R2 = 0.370; p<0.001). Elevation was not significantly associated with the distribution of either VL or CL. In areas of high annual precipitation, CL was 3.6 times more likely to occur than VL. For vegetative moisture (NDMI), CL was 2.11 times more likely to occur than VL. Odds of CL occurrence increased to 5.5 times when vegetation (NDVI) and 13.5 times when liquid water content of vegetation canopies (NDWI) was considered. Areas at risk of CL and VL were mapped based on the selected explanatory variables. Accuracy of models were assessed using area under the receiver operating characteristic curve (AUC=0.72). We propose that statewide scale risk models based on use of EOS products will be a useful tool at 1 km2 spatial resolution to enable health workers to identify and target high risk areas to prevent transmission of leishmaniasis.

Os satélites de observação da Terra (SOT) da NASA foram usados para calcular três índices de vegetação, extrair dados de precipitação e elevação e avaliar sua aplicabilidade para identificar o risco para leishmaniose visceral (LV) e leishmaniose tegumentar (LT) no Estado da Bahia, Brasil. Modelos de regressão mostraram que ambas as formas de leishmaniose podem ser preditas pelos NDVI, NDMI, NDWI e precipitação TRMM (R2 = 0,370; p<0,001). A elevação não foi significativamente associada à distribuição de LV ou LT. Em áreas de alta precipitação anual, a LT foi 3,6 vezes mais provável de ocorrer do que a LV. Para a umidade vegetativa (NDMI), a LT apresentou 2,11 maior probabilidade de ocorrer do que a LV. As chances de ocorrência de LT aumentaram para 5,5 vezes em relação com a vegetação (NDVI) e 13,5 vezes quando o conteúdo de água líquida dos dosséis da vegetação (NDWI) foi considerado. Áreas em risco de LT e LV foram mapeadas com base nas variáveis explicativas selecionadas. A precisão dos modelos foi avaliada usando a área sob curva característica de operação do receptor (Curva COR=0,72). Propusemos que os modelos de risco em escala estadual baseados no uso de produtos SOT são uma ferramenta útil na resolução espacial de 1 km2 por permitir que profissionais de saúde identifiquem e direcionem áreas de alto risco para evitar a transmissão da leishmaniose.

Tissue and blood protozoa including toxoplasmosis, Chagas disease, leishmaniasis, Babesia, Acanthamoeba, Balamuthia, and Naegleria in solid organ transplant recipients- Guidelines from the American Society of Transplantation Infectious Diseases Community of Practice.

These updated guidelines from the Infectious Diseases Community of Practice of the American Society of Transplantation review the diagnosis, prevention, and management of tissue and blood protozoal infections in the pre- and post-transplant period. Significant new developments in the field have made it necessary to divide the previous single guideline published in 2013 into two sections, with the intestinal parasites separated from this guideline devoted to tissue and blood protozoa. The current update reflects the increased focus on donor screening and risk-based recipient monitoring for parasitic infections. Increased donor testing has led to new recommendations for recipient management of Toxoplasma gondii and Trypanosoma cruzi. Molecular diagnostics have impacted the field, with access to rapid diagnostic testing for malaria and polymerase chain reaction testing for Leishmania. Changes in Babesia treatment regimens in the immunocompromised host are outlined. The risk of donor transmission of free-living amebae infection is reviewed. Changing immigration patterns and the expansion of transplant medicine in developing countries has contributed to the recognition of parasitic infections as an important threat to transplant outcomes. Medications such as benznidazole and miltefosine are now available to US prescribers as access to treatment of tissue and blood protozoa is increasingly prioritized.

What's eating you? sand flies.

As thousands of Americans descended upon Brazil for the Olympic games in the summer of 2016, the mosquito-borne Zika virus became a source of great concern among the countless athletes and travelers in Rio. As is often the case, the media frenzy that ensued drew travelers' attention away from a lesser known flying vector that often carries with it grave consequences. The Phlebotominae, commonly known as sand flies, are biting insects known for their ability to transmit the protozoa Leishmania as well as a number of other viruses and bacteria. As the impact of sand flies continues to grow in the United States and worldwide, knowledge of the vector is important for proper treatment and prevention of the diseases they carry.

Leishmaniasis in solid organ and hematopoietic stem cell transplant recipients.

Leishmaniasis occurs in <1% of solid organ and hematopoietic stem cell transplant recipients in endemic countries in which transplants are performed. Visceral leishmaniasis (VL) makes up the bulk of reported cases. The onset generally occurs months after transplantation and the mode of acquisition is often impossible to determine, but de novo vector-borne infection and reactivation of inapparent infection are thought to be the principal means. The potential role of clinically inapparent donor infection is uncertain and screening is not currently recommended, nor is it recommended for recipients from endemic areas, some of whom may have detectable circulating protozoan nucleic acid. While transplant recipients with VL often present with the non-specific findings of fever and cytopenia, the additional presence of hepatosplenomegaly in patients from endemic areas should lead to a directed diagnostic evaluation with bone marrow examination and PCR testing of marrow and peripheral blood having a high yield. Management may often be complicated by the presence of concomitant infections. A lipid formulation of amphotericin B is the preferred treatment, especially for VL, but the relapse rate in transplant recipients is approximately 25%. PCR monitoring of blood for either secondary prophylaxis or preemptive therapy requires further study.

Are Aedes aegypti mosquitoes potential vectors for leishmaniasis? ­ Case report / Mosquitos Aedes aegypti são vetores potenciais de leishmaniose? ­ Relato de caso

In Brazil dipters of the Lutzomyia genus are the main vectors of leishmaniasis for humans and animals. However, other hematophagous insects such as ticks, fleas, and horse flies may also be considered potential vectors of this protozoon. This paper, regarding an endemic area for visceral leishmaniasis, is the the first description of the Leishmania spp. presence in Aedes aegypti mosquitoes. Two A. aegypti mosquitoes were captured: one of them was feeding on a polysymptomatic dog with leishmaniasis, confirmed by parasitic demonstration and positive PCR for Leishmania spp., and the other was collected in the environment where the dog was isolated. The mosquito engorged with dog's blood was crushed between two microscopic slides and the other one was processed by the polymerase chain reaction assay (PCR) searching for the presence of Leishmania spp. DNA. Amastigote forms of Leishmania sp, were observed in the smear prepared from one mosquito by microscopic examination, as well as other protozoa's flagellated forms. In the other insect it was observed Leishmania DNA amplification. This observation reinforces the role of dogs as sources of infection of Leishmania spp. even to other potential vector species.(AU)

No Brasil, os dípteros do gênero Lutzomyia são os principais vetores da leishmaniose para humanos e animais. No entanto, tem sido constatado que outras espécies de invertebrados hematófagos, como carrapatos, pulgas e mutucas, também podem ser vetores desse protozoário. Este trabalho, realizado em uma área endêmica de leishmaniose visceral, é a primeira descrição da presença de Leishmania spp. em mosquitos da espécie A. aegypti. Dois mosquitos A. aegypti foram capturados no local onde estava isolado um cão polissintomático acometido por leishmaniose visceral, confirmada pela demonstração do parasita em biópsias de órgãos e por resultado positivo na prova de PCR para Leishmania spp. Um dos mosquitos estava sugando o sangue do cão e o outro estava livre no ambiente. O mosquito ingurgitado com o sangue do animal foi esmagado entre duas lâminas de microscopia e o outro foi processado por meio da reação em cadeia pela polimerase (PCR) aplicada à pesquisa do ADN de Leishmania spp. Ao exame microscópico do esfregaço preparado com o mosquito que estava parasitando o cão foram observadas formas amastigotas de Leishmania spp., bem como formas flageladas de outra espécie de protozoário. No outro inseto foi detectada amplificação de ADN do gênero Leishmania. Esta constatação reforça o papel dos cães como fontes de infecção de Leishmania spp. até mesmo para outras espécies de vetores potenciais.(AU)

Efficacy of Permethrin Treated Bed Nets Against Leishmania major Infected Sand Flies.

Insecticide treated nets (ITNs) are a potential tool to help control sand flies and prevent Leishmaniasis. However, little is currently known about the response of Leishmania infected sand flies to ITNs. In this study, Phlebotomus duboscqi sand flies were infected with the parasite Leishmania major. Infected and noninfected sand flies were then evaluated against permethrin treated and untreated bed nets in a laboratory assay that required sand flies to pass through suspended netting material to feed on a mouse serving as an attractive host. The number of sand flies passing through the nets and blood feeding was recorded. There was not a significant difference in the ability of infected or noninfected sand flies to move through treated or untreated nets. Fewer sand flies entered the permethrin treated nets compared to the untreated nets, indicating that permethrin creates an effective barrier. The results show that in addition to reducing the nuisance bites of noninfected sand flies, ITNs also protect against Leishmania infected sand flies and therefore can play in key role in reducing the rates of Leishmaniasis. This study is important to the Department of Defense as it continues to develop and field new bed nets to protect service members.

Is leishmaniasis an "unusual suspect" of infection in allogeneic transplantation?

Leishmaniasis is a disease of the immunocompetent population, more often affecting infants and young children. However, the number of leishmaniasis cases associated with immunosuppression has increased over the last 20 years. The visceral form of the disease, visceral leishmaniasis (VL), is identified as an opportunistic infection in immunosuppressed individuals, occurring mainly after solid organ transplantation, especially in renal transplant recipients. Limited data are available about VL after hematopoietic stem cell transplantation (HSCT). We report the cases of 3 patients with late VL after allogeneic HSCT, and review the literature.

[Severe oral mucositis in a patient with HIV infection]. / Schwere orale Mukositis bei einem Patienten mit HIV-Infektion.

HISTORY AND CLINICAL FINDINGS: A 50-year-old man with HIV infection (first diagnosed > 20 years ago) presented at our hospital with fulminant oral mucositis. Antiretroviral therapy (tenofovir, emtricitabine, raltegravir) had been started 2 months ago. Previously he had no opportunistic infections and no other pre-existing illnesses. He had not travelled outside Europe but stayed in Spain for several weeks during summer. INVESTIGATIONS: Physical examination revealed swelling of the lips and severe ulcerative mucositis of the gums and pharynx. The patient complained of painful swallowing. The blood-chemistry showed no abnormalities. The microscopical analysis of a smear and a biopsy of the buccal mucosa revealed amastigotes of leishmania. By means of PCR technique, Leishmania donovani complex was specified. TREATMENT AND COURSE: The patient was treated with liposomal amphotericin B (1 mg/kg) for 21 days. Because of the immunosuppression he was put on maintenance therapy afterwards (liposomal amphotericin B every 3 weeks). However, 4 months later there was a clinical relapse of the mucositis and a new cultural and PCR detection of leishmania in a buccal biopsy. After another course of 21 days with liposomal amphotericin B (3 mg/kg) and miltefosine (150 mg/d), the mucositis subsided. Therapy with liposomal amphotericin B (3 mg/kg single dose every 3 weeks) has since been maintained. The antiretroviral therapy was changed meanwhile to lamivudin, abacavir and raltegravir because of kidney failure with elevated urea and creatinine. The patient has been clinically stable ever since without any other HIV-related problems. The latest CD4 count was 456/µl and the HIV load 340 copies/ml. CONCLUSION: Leishmaniasis is a severe infection in HIV-positive patients. Clinical manifestations can be atypical in immunosuppressed patients and the treatment is complicated with HIV coinfection. This is also due to a lifelong persistence of the parasite with potential reactivation especially in patients with suppressed CD4 cells. Therefore maintenance therapy after standard therapy of leishmaniasis is mandatory at least for a CD4 count below 350/µl. Especially in HIV patients with a leishmaniasis relapse lifelong maintenance therapy should be considered.

Mucosal leishmaniasis: an underestimated presentation of a neglected disease.

We present a review of current knowledge about mucosal leishmaniasis (ML). Although involvement of mucous membranes is classically admitted in New World leishmaniasis, particularly occurring in infection by Leishmania (L.) braziliensis species complex, ML is also a possible presentation of Old World leishmaniasis, in either L. donovani or L. major species complex infections. Thus, ML has to be considered not only as a Latin American disease but as an Old and New World disease. We describe ML epidemiology, pathogenesis, clinics, diagnosis, and therapy. Considering both its highly disfiguring lesions and its possible lethal outcome, ML should not be underestimated by physicians. Moreover, leishmaniasis is expected to increase its burden in many countries as sandfly vector distribution is widespreading towards non-endemic areas. Finally, the lack of clear understanding of ML pathogenesis and the absence of effective human vaccines strongly claim for more research.

Leishmaniasis worldwide and global estimates of its incidence.

As part of a World Health Organization-led effort to update the empirical evidence base for the leishmaniases, national experts provided leishmaniasis case data for the last 5 years and information regarding treatment and control in their respective countries and a comprehensive literature review was conducted covering publications on leishmaniasis in 98 countries and three territories (see 'Leishmaniasis Country Profiles Text S1, S2, S3, S4, S5, S6, S7, S8, S9, S10, S11, S12, S13, S14, S15, S16, S17, S18, S19, S20, S21, S22, S23, S24, S25, S26, S27, S28, S29, S30, S31, S32, S33, S34, S35, S36, S37, S38, S39, S40, S41, S42, S43, S44, S45, S46, S47, S48, S49, S50, S51, S52, S53, S54, S55, S56, S57, S58, S59, S60, S61, S62, S63, S64, S65, S66, S67, S68, S69, S70, S71, S72, S73, S74, S75, S76, S77, S78, S79, S80, S81, S82, S83, S84, S85, S86, S87, S88, S89, S90, S91, S92, S93, S94, S95, S96, S97, S98, S99, S100, S101'). Additional information was collated during meetings conducted at WHO regional level between 2007 and 2011. Two questionnaires regarding epidemiology and drug access were completed by experts and national program managers. Visceral and cutaneous leishmaniasis incidence ranges were estimated by country and epidemiological region based on reported incidence, underreporting rates if available, and the judgment of national and international experts. Based on these estimates, approximately 0.2 to 0.4 cases and 0.7 to 1.2 million VL and CL cases, respectively, occur each year. More than 90% of global VL cases occur in six countries: India, Bangladesh, Sudan, South Sudan, Ethiopia and Brazil. Cutaneous leishmaniasis is more widely distributed, with about one-third of cases occurring in each of three epidemiological regions, the Americas, the Mediterranean basin, and western Asia from the Middle East to Central Asia. The ten countries with the highest estimated case counts, Afghanistan, Algeria, Colombia, Brazil, Iran, Syria, Ethiopia, North Sudan, Costa Rica and Peru, together account for 70 to 75% of global estimated CL incidence. Mortality data were extremely sparse and generally represent hospital-based deaths only. Using an overall case-fatality rate of 10%, we reach a tentative estimate of 20,000 to 40,000 leishmaniasis deaths per year. Although the information is very poor in a number of countries, this is the first in-depth exercise to better estimate the real impact of leishmaniasis. These data should help to define control strategies and reinforce leishmaniasis advocacy.