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Background: Visceral leishmaniasis (VL), or kala-azar, is a common comorbidity in patients with AIDS in endemic areas. Many patients continue to experiences relapses of VL despite virological control, but with immunological failure. These patients remain chronically symptomatic with hypersplenism, for example with anemia, leukopenia, and thrombocytopenia, and are at risk of severe co-infection due to low CD4+ count. Therefore, in this study, splenectomized patients with VL and HIV infection were investigated to understand why the CD4+ count fails to recover in these patients, evaluating the importance of spleen mass for hypersplenism and immunological failure. Methods: From a retrospective open cohort of 13 patients who had previously undergone splenectomy as salvage therapy for relapsing VL, 11 patients with HIV infection were investigated. This study compared the patients' complete blood cell count (CBC) and CD4+ and CD8+ cell counts before and after splenectomy with respect to spleen weight. Results: CBC was substantially improved after splenectomy, indicating hypersplenism. However, to the best of our knowledge, this is the first study to show that spleen mass is strongly and negatively correlated with CD4+ cell count (ρ = −0.71, P = 0.015). Conclusions: This finding was unexpected, as the spleen is the most extensive lymphoid tissue and T-lymphocyte source. After reviewing the literature and reasoning, we hypothesized that the immunological failure was secondary to CD4+ loss initially by apoptosis in the spleen induced by productive HIV infection and, subsequently, by pyroptosis sustained by parasitic infection in spleen macrophages. [ABSTRACT FROM AUTHOR]

Background: Iran is a country with a high prevalence of visceral leishmaniasis (VL) and seven endemic provinces. In this study, we tried to identify unobserved classes of knowledge among Iranians toward VL and assess the predictors of each latent class. Methods: This cross-sectional study was conducted among randomly selected participants from endemic and non-endemic areas of VL in Iran in 2020 and 2021. The collected data included demographic characteristics and questions about knowledge, attitude, and practice toward VL. We performed latent class analysis using a procedure for latent class analysis (PROC LCA) in SAS to identify the class membership of knowledge of participants toward VL. Results: Five latent classes were identified: very low (38.9%), low (15.5%), moderate (6.2%), high (14.1%), and very high (25.2%) knowledge about VL. Living in endemic areas significantly increased the odds of belonging to the low (adjusted OR (AOR = 7.23; 95% confidence interval (CI):4.52–11.58), high (AOR = 2.71; 95%CI: 1.73–4.23), and very high (AOR = 8.47; 95%CI: 5.78–12.41) classes compared to the very low class. Also, having academic education increased the odds of membership in the very high class (AOR = 2.36; 95%CI: 1.61–3.47) compared to the very low class. Conclusion: This study revealed that more than 50% of the participants fell into the latent classes of very low and low knowledge toward VL. Some educational workshops in the endemic areas could be effective in enhancing knowledge about VL. [ABSTRACT FROM AUTHOR]

Background: Leishmaniasis is mainly prevalent in tropical and subtropical developing countries, where chronic undernutrition often co-exists. Undernutrition is reported to promote the progression of leishmaniasis, but its immune mechanisms have not been fully elucidated. Methods: To simulate chronic undernutrition of patients in epidemic areas and explore the immune mechanism of undernutrition promoting leishmaniasis, BALB/c mouse models with different nutritional imbalances were established, including undernutrition 75%, undernutrition 65% and obesity mouse models. After infection with Leishmania donovani in these model mice, we focused on evaluating the progress of leishmaniasis in the spleen and liver, the expression of important immunosuppressive and immunoactivation molecules, and changes of spleen transcriptome. The immune signaling pathways enriched by differentially expressed genes and hub genes were analyzed. Results: The results showed that among the mouse infection models, undernutrition 75% + infection group had the highest parasite load in the spleen and liver at the 8th week post-infection, possibly due to the continuous increase of PD-1, PD-L1 and TCR. Spleen RNA-seq results suggested that some immune signaling pathways were downregulated in undernutrition 75% + infection group, including neutrophil extracellular trap formation, IL-17 signaling pathway, natural killer cell-mediated cytotoxicity, etc. Among them, neutrophil extracellular trap formation pathway had the largest number of downregulated genes. This also explained why undernutrition 75% + infection group had the highest parasite load. Through PPI network analysis, hub genes such as Lcn2, Ltf, Mpo, Dnaja1, Hspa1a, Hspa1b and Hsph1 were screened out and might play important roles in the process of undernutrition promoting leishmaniasis. Conclusions: Undernutrition upregulated PD-1 and PD-L1 expression and downregulated immune signaling pathways in mice with visceral leishmaniasis. The signaling pathways and hub genes may serve as drug targets or intervention targets for the treatment of leishmaniasis patients with undernutrition. [ABSTRACT FROM AUTHOR]

Background: Leishmaniasis is a parasitic disease caused by obligate intracellular protozoa of the genus Leishmania. This infection is characterized by a wide range of clinical manifestations, with symptoms greatly dependent on the causal parasitic species. Here we present the design and application of a new 70-kDa heat shock protein gene (hsp70)-based marker of 771 bp (HSP70-Long). We evaluated its sensitivity, specificity and diagnostic performance employing an amplicon-based MinION™ DNA sequencing assay to identify different Leishmania species in clinical samples from humans and reservoirs with cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL). We also conducted a comparative analysis between our novel marker and a previously published HSP70 marker known as HSP70-Short, which spans 330 bp. Methods: A dataset of 27 samples from Colombia, Venezuela and the USA was assembled, of which 26 samples were collected from humans, dogs and cats affected by CL and one sample was collected from a dog with VL in the USA (but originally from Greece). DNA was extracted from each sample and underwent conventional PCR amplification utilizing two distinct HSP70 markers: HSP70-Short and HSP70-Long. The subsequent products were then sequenced using the MinION™ sequencing platform. Results: The results highlight the distinct characteristics of the newly devised HSP70-Long primer, showcasing the notable specificity of this primer, although its sensitivity is lower than that of the HSP70-Short marker. Notably, both markers demonstrated strong discriminatory capabilities, not only in distinguishing between different species within the Leishmania genus but also in identifying instances of coinfection. Conclusions: This study underscores the outstanding specificity and effectiveness of HSP70-based MinION™ sequencing, in successfully discriminating between diverse Leishmania species and identifying coinfection events within samples sourced from leishmaniasis cases. [ABSTRACT FROM AUTHOR]

Background: Post-kala-azar dermal leishmaniasis (PKDL) is a dermatosis that occurs 2–3 years after an apparently successful treatment of visceral leishmaniasis (VL). In rare cases, PKDL occurs concurrently with VL and is characterized by fever, splenomegaly, hepatomegaly or lymphadenopathy, and poor nutritional status and is known as Para-kala-azar dermal leishmaniasis (Para-KDL). Co-association of active VL in PKDL patients is documented in Africa, but very few case reports are found in South Asia. Case summary: We present a case of Para-kala-azar Dermal Leishmaniasis (Para-KDL) in a 50-year-old male patient with a history of one primary Visceral Leishmaniasis (VL) and 2 times relapse of Visceral Leishmaniasis (VL). The patient presented with fever, skin lesions, and hepatosplenomegaly. Laboratory tests revealed LD bodies in the slit skin smear and splenic biopsy. The patient was treated with two cycles of Amphotericin B with Miltefosine in between cycles for 12 weeks to obtain full recovery. Conclusion: This case report serves as a reminder that Para-kala-azar dermal leishmaniasis can develop as a consequence of prior visceral leishmaniasis episodes, even after apparently effective therapy. Since para-kala-azar is a source of infectious spread, endemics cannot be avoided unless it is effectively recognized and treated. [ABSTRACT FROM AUTHOR]

Background: Leishmaniasis is a common neglected tropical disease in Ethiopia. Visceral leishmaniasis (VL) caused by Leishmania donovani presents in the lowlands, while cutaneous leishmaniasis (CL) affects people living in the highlands. Although CL is described as being caused by Leishmania aethiopica, there is also evidence of L. tropica and L. major isolated from a patient, sand flies and potential reservoirs. Information on species causing CL in Ethiopia is patchy, and no nation-wide study has ever been done. Understanding which species are causing CL in Ethiopia can have important implications for patient management and disease prevention. Methods: We analyzed stored routine samples and biobanked DNA isolates from previously conducted studies of CL patients from different centers in the north, center and south of Ethiopia. Species typing was performed using ITS-1 PCR with high-resolution melt (HRM) analysis, followed by HSP70 amplicon sequencing on a selection of the samples. Additionally, sociodemographic, clinical and laboratory data of patients were analyzed. Results: Of the 226 CL samples collected, the Leishmania species could be determined for 105 (45.5%). Leishmania aethiopica was identified in 101 (96.2%) samples from across the country. In four samples originating from Amhara region, northwestern Ethiopia, L. donovani was identified by ITS-1 HRM PCR, of which two were confirmed with HSP70 sequences. While none of these four patients had symptoms of VL, two originated from known VL endemic areas. Conclusions: The majority of CL was caused by L. aethiopica, but CL due to L. tropica and L. major cannot be ruled out. Our study is the first to our knowledge to demonstrate CL patients caused by L. donovani in Ethiopia. This should spark future research to investigate where, how and to which extent such transmission takes place, how it differs genetically from L. donovani causing VL and whether such patients can be diagnosed and treated successfully with the currently available tools and drugs. [ABSTRACT FROM AUTHOR]

Background: Parasitological investigation of bone marrow, splenic or lymph node aspirations is the gold standard for the diagnosis of visceral leishmaniasis (VL). However, this invasive test requires skilled clinical and laboratory staff and adequate facilities, and sensitivity varies depending on the tissue used. The direct agglutination test (DAT) is a serological test that does not need specialised staff, with just minimal training required. While previous meta-analysis has shown DAT to have high sensitivity and specificity when using parasitology as the reference test for diagnosis, meta-analysis of DAT compared to other diagnostic techniques, such as PCR and ELISA, that are increasingly used in clinical and research settings, has not been done. Methods: We conducted a systematic review to determine the diagnostic performance of DAT compared to all available tests for the laboratory diagnosis of human VL. We searched electronic databases including Medline, Embase, Global Health, Scopus, WoS Science Citation Index, Wiley Cochrane Central Register of Controlled Trials, Africa-Wide Information, LILACS and WHO Global Index. Three independent reviewers screened reports and extracted data from eligible studies. A meta-analysis estimated the diagnostic sensitivity and specificity of DAT. Results: Of 987 titles screened, 358 were selected for full data extraction and 78 were included in the analysis, reporting on 32,822 participants from 19 countries. Studies included were conducted between 1987–2020. Meta-analysis of studies using serum and DAT compared to any other test showed pooled sensitivity of 95% (95%CrI 90–98%) and pooled specificity of 95% (95%CrI 88–98%). Results were similar for freeze-dried DAT and liquid DAT when analysed separately. Sensitivity was lower for HIV-positive patients (90%, CrI 59–98%) and specificity was lower for symptomatic patients (70%, CrI 43–89%). When comparing different geographical regions, the lowest median sensitivity (89%, CrI 67–97%) was in Western Asia (five studies). Conclusions: This systematic review and meta-analysis demonstrates high estimated pooled sensitivity and specificity of DAT for diagnosis of VL, although sensitivity and specificity were lower for different patient groups and geographical locations. This review highlights the lack of standardisation of DAT methods and preparations, and the lack of data from some important geographical locations. Future well-reported studies could provide better evidence to inform test implementation for different patient populations and use cases. PROSPERO registration: CRD42021240830 [ABSTRACT FROM AUTHOR]

Background: Visceral leishmaniasis (VL), a life-threatening neglected tropical disease, is targeted for elimination from Nepal by the year 2026. The national VL elimination program is still confronted with many challenges including the increasingly widespread distribution of the disease over the country, local resurgence and the questionable efficacy of the key vector control activities. In this study, we assessed the status and risk of Leishmania donovani transmission based on entomological indicators including seasonality, natural Leishmania infection rate and feeding behavior of vector sand flies, Phlebotomus argentipes, in three districts that had received disease control interventions in the past several years in the context of the disease elimination effort. Methods: We selected two epidemiologically contrasting settings in each survey district, one village with and one without reported VL cases in recent years. Adult sand flies were collected using CDC light traps and mouth aspirators in each village for 12 consecutive months from July 2017 to June 2018. Leishmania infection was assessed in gravid sand flies targeting the small-subunit ribosomal RNA gene of the parasite (SSU-rRNA) and further sequenced for species identification. A segment (~ 350 bp) of the vertebrate cytochrome b (cytb) gene was amplified from blood-fed P. argentipes from dwellings shared by both humans and cattle and sequenced to identify the preferred host. Results: Vector abundance varied among districts and village types and peaks were observed in June, July and September to November. The estimated Leishmania infection rate in vector sand flies was 2.2% (1.1%–3.7% at 95% credible interval) and 0.6% (0.2%–1.3% at 95% credible interval) in VL and non-VL villages respectively. The common source of blood meal was humans in both VL (52.7%) and non-VL (74.2%) villages followed by cattle. Conclusions: Our findings highlight the risk of ongoing L. donovani transmission not only in villages with VL cases but also in villages not reporting the presence of the disease over the past several years within the districts having disease elimination efforts, emphasize the remaining threats of VL re-emergence and inform the national program for critical evaluation of disease elimination strategies in Nepal. [ABSTRACT FROM AUTHOR]

Background: Follow-up assessment of visceral leishmaniasis (VL) treated cases is important to monitor the long term effectiveness of treatment regimens. The main objective of this study was to identify the gaps and challenges in the follow-up of treated VL cases, to monitor treatment outcome and to assess the impact of COVID-19 on VL elimination services and activities. Methods: Clinicians treating VL patients, district focal persons for VL, and patients treated for VL in seven high endemic districts in Nepal during 2019–2022 were interviewed to collect data on challenges in the follow-up of VL treated patients as per national strategy. Results: Follow up status was poor in two districts with the largest number of reported cases. The majority of cases were children under 10 years of age (44.2%). Among 104 VL treated cases interviewed, 60.6% mentioned that clinicians had called them for follow-up but only 37.5% had complied. Among 112 VL treated cases followed up, 8 (7.14%) had relapse and 2 (1.8%) had PKDL. Among 66 cases who had VL during the COVID-19 lock down period, 32 (48.5%) were diagnosed within 1 week; however, 10 (15.1%) were diagnosed only after 4 weeks or more. During the COVID-19 pandemic, there was no active search for VL because of budget constraints and lack of diagnostic tests, and no insecticide spraying was done. Conclusion: Relapses and PKDL are challenges for VL elimination and a matter of concern. Successful implementation of the national strategy for follow up of treated VL cases requires addressing elements related to patients (awareness, transport, communication) clinicians (compliance) and organization of service delivery (local health worker training and deployment). COVID-19 did not have much impact on VL diagnosis and treatment; however, public health programmes including active case detection and insecticide spraying for vector control were severely reduced. [ABSTRACT FROM AUTHOR]

Background: Leishmaniasis is one of the most neglected tropical diseases and is spread mainly in impoverished regions of the world. Although many studies have focused on the host's response to Leishmania invasion, relatively less is known about the complex processes at the metabolic level, especially the metabolic alterations in the infected hosts. Methods: In this study, we conducted metabolomics analysis on the urine of golden hamsters in the presence or absence of visceral leishmaniasis (VL) using the ultra-performance liquid chromatography (UPLC) system tandem high-resolution mass spectrometer (HRMS). The metabolic characteristics of urine samples, along with the histopathological change and the parasite burden of liver and spleen tissues, were detected at 4 and 12 weeks post infection (WPI), respectively. Results: Amino acid metabolism was extensively affected at both stages of VL progression. Meanwhile, there were also distinct metabolic features at different stages. At 4 WPI, the significantly affected metabolic pathways involved alanine, aspartate and glutamate metabolism, the pentose phosphate pathway (PPP), histidine metabolism, tryptophan metabolism and tyrosine metabolism. At 12 WPI, the markedly enriched metabolic pathways were almost concentrated on amino acid metabolism, including tyrosine metabolism, taurine and hypotaurine metabolism and tryptophan metabolism. The dysregulated metabolites and metabolic pathways at 12 WPI were obviously less than those at 4 WPI. In addition, seven metabolites that were dysregulated at both stages through partial least squares-discriminant analysis (PLS-DA) and receiver-operating characteristic (ROC) tests were screened to be of diagnostic potential. The combination of these metabolites as a potential biomarker panel showed satisfactory performance in distinguishing infection groups from control groups as well as among different stages of infection. Conclusion: Our findings could provide valuable information for further understanding of the host response to Leishmania infection from the aspect of the urine metabolome. The proposed urine biomarker panel could help in the development of a novel approach for the diagnosis and prognosis of VL. [ABSTRACT FROM AUTHOR]

Background: Leishmaniasis is a rare infectious disease observed in subtropical and tropical areas. This disease that demonstrates different clinical characteristics is caused by intracellular Leishmania protozoan. One of the important countries facing the incidence of this infectious disease is Iran. Recently, human immunodeficiency virus–Leishmania coinfection has been indicated in Iran. Case presentation: In the present case report, we show an atypical case of severe visceral leishmaniasis in a 52-year-old Iranian-Arab male with positive human immunodeficiency virus status. Leishmaniasis was detected by node biopsy and subsequently histopathology evaluations and confirmed by molecular methods. Conclusions: The current study was the first report of an atypical case of a patient with Leishmania–human immunodeficiency virus coinfection in southwestern Iran, which was not responsive to the treatment. Therefore, the health authorities should be aware of these reports, which require permanent clinical follow-up of the patients as well as effective treatments. [ABSTRACT FROM AUTHOR]

Objective: Visceral leishmaniasis (VL) is an endemic parasitic disease in Latin America, and its clinical picture is aggravated in coinfections with the human immunodeficiency virus (HIV). The objective of this study was to investigate clinical factors and laboratory variables associated with VL relapse and death in VL/HIV coinfected patients. Methods: A prospective longitudinal study was conducted from January 2013 to July 2020 among 169 patients coinfected with VL and HIV. The outcomes investigated were the occurrence of VL relapse and death. Chi-square test, Mann–Whitney test and logistic regression models were used for statistical analysis. Results: The occurrence rates were 41.4% for VL relapse and 11.2% for death. Splenomegaly and adenomegaly were associated with the increased risk of VL relapse. Patients with VL relapse had higher levels of urea (p =.005) and creatinine (p <.001). Patients who died had lower red blood cell counts (p =.012), hemoglobin (p =.017) and platelets (p <.001). The adjusted model showed that antiretroviral therapy for more than 6 months was associated with a decrease in VL relapse, and adenomegaly was associated with an increase in VL relapse. In addition, edema, dehydration, poor general health status, and paleness were associated with an increase in hospital death. Conclusion: The findings suggest that adenomegaly, antiretroviral therapy, and renal abnormalities can be associated with VL relapse, while hematological abnormalities, and clinical manifestations like paleness, and edema can be associated with an increased odds of hospital death. Trial registration number: The study was submitted to the Ethics and Research Committee of the Federal University of Maranhão (Protocol: 409.351). [ABSTRACT FROM AUTHOR]

Background: Visceral leishmaniasis is caused by the Leishmania donovani species complex that can spread to internal organs and leading to death if not treated on time. Diagnosis of leishmaniasis is based on clinical signs and symptoms, microscopy, serological and molecular techniques. Because of a broad spectrum of diverse clinical manifestations and similarities of the responses to different species, identification to the species level is often difficult for the proper patient treatment and management. Therefore, the objective of this study was to evaluate the PCR- RFLP assay of the ITS1 region for identification of L. donovani species from clinical smear slide patient samples. Method: DNA extraction was performed on a total of 90 smear slide samples using phenol—chloroform method. The PCR detection limit was determined by L. donovani reference strain DNA. The ITS1 region was amplified at 320 bp using LITSR/L5.8S genus specific primers and then the ITS1-PCR products were subjected to RFLP assay for confirmation of L. donovani species using HaeIII restriction enzyme. Results: Of the total samples ITS1-PCR revealed the true positive, false positive, true negative, and false negative results of 42 (46.7%), 6 (6.7%), 37 (41.1%) and 5 (5.6%), respectively. Considering microscopy as the gold standard, the sensitivity, specificity, positive predictive values, and negative predictive values of the ITS1- PCR technique was 89.4%, 86.0%, 87.5%, and 88.1% respectively. All ITS1-PCR positive clinical samples were confirmed as L. donovani species by PCR–RFLP patterns. Conclusion: In conclusion, the ITS1- RFLP method is highly sensitive and more specific for identification of L. donovani species in the smear negative clinical samples of visceral leishmaniasis patients. There is also significant association and degree of agreement between the two methods. For direct identification of L. donovani species from clinical samples, irrespective of genus and species level, PCR–RFLP is more recommendable than a microscope. [ABSTRACT FROM AUTHOR]

Background: Visceral leishmaniasis (VL) is the most severe form of all leishmanial infections and is caused by infection with protozoa of Leishmania donovani and Leishmania infantum. This parasitic disease occurs in over 80 countries and its geographic distribution is on the rise. Although the interaction between the intestinal microbiome and the immune response has been established in several pathologies, it has not been widely studied in leishmaniasis. The Syrian hamster is the most advanced laboratory model for developing vaccines and new drugs against VL. In the study reported here, we explored the relationship between the intestinal microbiome and infection with L. infantum in this surrogate host. Methods: Male Syrian hamsters (120–140 g) were inoculated with 108 promastigotes of a canine-derived L. infantum strain or left as uninfected control animals. Infection was maintained for 19 weeks (endpoint) and monitored by an immunoglobulin G (IgG) enyzme-linked immunosorbent assay throughout the experiment. Individual faecal samples, obtained at weeks 16, 18 and 19 post-inoculation, were analysed to determine the 16S metagenomic composition (the operational taxonomic units [OTUs] of the intestinal microbiome and the comparison between groups were FDR (false discovery rate)-adjusted). Results: Leishmania infantum infection elicited moderate clinical signs and lesions and a steady increase in specific anti-Leishmania serum IgG. The predominant phyla (Firmicutes + Bacteriodetes: > 90%), families (Muribaculaceae + Lachnospiraceae + Ruminococcaceae: 70–80%) and genera found in the uninfected hamsters showed no significant variations throughout the experiment. Leishmania infantum infection provoked a slightly higher—albeit non-significant—value for the Firmicutes/Bacteriodetes ratio but no notable differences were found in the relative abundance or diversity of phyla and families. The microbiome of the infected hamsters was enriched in CAG-352, whereas Lachnospiraceae UCG-004, the [Eubacterium] ventriosum group and Allobaculum were less abundant. Conclusions: The lack of extensive significant differences between hamsters infected and uninfected with L. infantum in the higher taxa (phyla, families) and the scarce variation found, which was restricted to genera with a low relative abundance, suggest that there is no clear VL infection-intestinal microbiome axis in hamsters. Further studies are needed (chronic infections, co-abundance analyses, intestinal sampling, functional analysis) to confirm these findings and to determine more precisely the possible relationship between microbiome composition and VL infection. [ABSTRACT FROM AUTHOR]

Background: Visceral leishmaniasis (VL) is one of the most important neglected tropical diseases. Although VL was controlled in several regions of China during the last century, the mountain-type zoonotic visceral leishmaniasis (MT-ZVL) has reemerged in the hilly areas of China in recent decades. The purpose of this study was to construct an indicator framework for assessing the risk of the MT-ZVL in China, and to provide guidance for preventing disease. Methods: Based on a literature review and expert interview, a 3-level indicator framework was initially established in November 2021, and 28 experts were selected to perform two rounds of consultation using the Delphi method. The comprehensive weight of the tertiary indicators was determined by the Delphi and the entropy weight methods. Results: Two rounds of Delphi consultation were conducted. Four primary indicators, 11 secondary indicators, and 35 tertiary indicators were identified. The Delphi-entropy weight method was performed to calculate the comprehensive weight of the tertiary indicators. The normalized weights of the primary indicators were 0.268, 0.261, 0.242, and 0.229, respectively, for biological factors, interventions, environmental factors, and social factors. The normalized weights of the top four secondary indicators were 0.122, 0.120, 0.098, and 0.096, respectively, for climatic features, geographical features, sandflies, and dogs. Among the tertiary indicators, the top four normalized comprehensive weights were the population density of sandflies (0.076), topography (0.057), the population density of dogs, including tethering (0.056), and use of bed nets or other protective measures (0.056). Conclusions: An indicator framework of transmission risk assessment for MT-ZVL was established using the Delphi-entropy weight method. The framework provides a practical tool to evaluate transmission risk in endemic areas. [ABSTRACT FROM AUTHOR]

Nanobiosensor platforms have emerged as convenient and promising approaches with remarkable efficacy for the diagnosis of infectious diseases. Gold nanoparticles (AuNPs) have been widely used due to numerous advantageous properties such as optical, electrical, physicochemical and great biomolecules binding capabilities. This study aimed to apply AuNP-Probe Conjugate for the detection of Leishmania spp., using colorimetric and amplification methods targeting parasitic ITS2 fragment. The first method was carried out by hybridization of 10µL of DNA with 4 µL of probe and addition of 5 µL of 0.2 N HCl (non-amplification method). Second method was followed by polymerase chain reaction (PCR) amplification using thiolated primer, 5 µL of AuNP and 5 µL of 0.2 N HCl. The appearance of red and purple colors indicated positive and negative results, respectively. The minimum of detection for non-amplification and amplification methods for three strains of Leishmania namely L. major, L. tropica and L. infantum were determined to be 32 fg/µL and 16 fg/µL, respectively. Sensitivity for detection of visceral leishmaniasis (VL) for non-amplification and amplification methods included 96% and 100%, respectively and for cutaneous leishmaniasis (CL) included 98% and 100%, respectively. The results of this investigation revealed that sensitivity of amplification method was the same as RT-qPCR, while that of non-amplification method was lower. However, this method was promising because of no need for any equipment, high specificity, enough sensitivity, low cost and rapidity (less than 30 min) to complete after genomic DNA extraction. [ABSTRACT FROM AUTHOR]

Background: Recombinant antigens rK39 (based on kinesin sequence) and rK28 (comprising kinesin and HASPB sequences) are a mainstay of serological diagnosis for visceral leishmaniasis (VL). However, their key epitopes and the significance of their structural conformation are not clearly defined, particularly in relation to reported cross-reactivity with sera from patients with malaria, schistosomiasis, and tuberculosis. Methods: To assess the effect of conformation on antigenicity with Sudanese VL sera, antigens rK39 and rK28 were heat-denatured at 95 °C for 10 min and then assayed by enzyme-linked immunosorbent assay (ELISA). Amino acid sequences of rK39 and rK28 were submitted to NCBI BLASTp to assess homology with Plasmodium, Schistosoma, and Mycobacterium. Results: Heat denaturation significantly diminished the antigenicity of rK39 compared to non-denatured antigen (P = 0.001), but not for rK28 (P = 0.275). In BLASTp searches, HASPB sequences from rK28 had similarities with sequences from Plasmodium, encompassing software-predicted B-cell epitopes. Conclusions: The antigenicity of rK39 appears to be dependent on structural conformation, whereas that of rK28 depends on linear sequence. HASPB sequence homology with Plasmodium may be responsible for the reported cross-reactivity of rK28 with malaria sera. Further work is warranted to refine the specificity of these antigens. [ABSTRACT FROM AUTHOR]

Background: Most sand fly species are located in the Americas; some act as vectors of leishmaniasis and other human diseases. In Bolivia, about 25% of Neotropical species have been identified, and only a few have been implicated as vectors of cutaneous and visceral leishmaniasis. A new species of anthropophilic sand fly from the sub-Andean region of Alto Beni is described herein. Methods: A large systematic entomological survey was carried out in a subtropical humid forest located in the Marimonos mountain range, at around 900 m altitude, in the municipality of Palos Blancos, Sud Yungas Province, Department of La Paz, Bolivia. Sand flies were captured over a period of 26 months between January 1982 and February 1984, at the ground and canopy level, using both CDC light traps and protected human bait. A total of 24,730 sand flies were collected on the ground, distributed in 16 species, and 3259 in the canopy, with eight species. One of these species was labeled as Pintomia (Pifanomyia) nevesi, although certain morphological features allowed us to doubt that it was that taxon. To define the identity of this sand fly, a re-evaluation (this work) was recently carried out through morphological analyses and measurements of the available specimens mounted on Euparal, previously labeled as Pi. (Pif.) nevesi. Results: Based on the morphological traits and measurements, the re-evaluated specimens were definitively identified as a new sand fly species, Pintomyia (Pifanomyia) veintemillasi, closely related to Pi. (Pif.) nevesi and Pintomyia (Pifanomyia) maranonensis within the Evansi series. This new sand fly was the third most numerous anthropophilic species at the floor (6.2%) and the second most numerous anthropophilic at the canopy (35.1%). Conclusions: A new anthropophilic sand fly species is described as Pi. (Pif.) veintemillasi n. sp. This sand fly species was caught at about 900 m altitude in the Marimonos mountain range, a highly endemic area for cutaneous and mucosal leishmaniasis due to Leishmania (Viannia) braziliensis. Therefore, this species could be involved in the leishmaniasis transmission in the sub-Andean foothills of Alto Beni, Department of La Paz, Bolivia. [ABSTRACT FROM AUTHOR]

Background: Polyarthritis has been associated with canine visceral leishmaniasis (CanVL), and co-infection with Ehrlichia canis is common and may alter clinical manifestations. Methods: A total of 89 dogs presenting CanVL were subdivided into two groups: (1) G1, consisting of 46 dogs seronegative to Ehrlichia spp., and (ii) G2, consisting of 43 dogs seropositive to Ehrlichia spp. Eight joints (carpal, tarsal, stifles and elbows) from each dog were evaluated by radiography and synovial fluid (SF) cytologic analysis. Results: Overall, 74 of the 89 (83.1%) dogs presented joint abnormalities suggestive of osteoarthritis by radiography (G1: 40/46 [86.9%]; G2: 34/43 [79.0%]), with no statistically significant between-group difference. All dogs with abnormal joint X-ray images presented radiographic lesions bilaterally, independent of the characteristics of the lesion. Soft tissue swelling around the joint and joint space narrowing were more commonly observed in G1 than in G2 dogs. There was no significant between-group difference in terms of other radiographic abnormalities suggestive of osteoarthritis (evident trabecular pattern, subchondral bone sclerosis, osteolysis, osteolytic–proliferative lesions or bone proliferation). SF from 174/315 (55.2%) and 152/307 (49.5%) joints from G1 and G2 dogs, respectively, presented an inflammatory infiltrate, but there was no significant association between the presence of inflammatory infiltrate and group. There was also no statistical difference between groups in either of the evaluated joints in terms of the percentage of neutrophils or mononuclear cells. Leishmania spp. amastigotes were found in 69/315 (21.9%) joints from G1 dogs and in 100/307 (32.5%) joints from G2 dogs (Fisherʼs exact test, P = 0.002, odds ratio = 0.5, 95% confidence interval = 0.4–0.8). The neutrophilic infiltrate was significantly higher in joints with amastigote forms in both G1 (Mann–Whitney U-test, U(18) = 817, Z = -3.76, P = 0.0001) and G2 dogs (Mann–Whitney U-test, U(18) = 6543, Z = − 5.06, P < 0.0001). Conclusions: A high prevalence of arthritis in dogs with CanVL was found, and all dogs presented involvement in multiple joints. Although no difference was observed between groups in terms of the number of dogs with polyarthritis and the presence of an inflammatory infiltrate in SF, Leishmania spp. amastigotes were found more frequently in joints from G2 dogs. Further studies evaluating SF in dogs co-infected with L. infantum and E. canis should be performed to evaluate this finding. [ABSTRACT FROM AUTHOR]

Background: Visceral Leishmaniasis (VL) is a fatal vector-borne parasitic disorder occurring mainly in tropical and subtropical regions. VL falls under the category of neglected tropical diseases with growing drug resistance and lacking a licensed vaccine. Conventional vaccine synthesis techniques are often very laborious and challenging. With the advancement of bioinformatics and its application in immunology, it is now more convenient to design multi-epitope vaccines comprising predicted immuno-dominant epitopes of multiple antigenic proteins. We have chosen four antigenic proteins of Leishmania donovani and identified their T-cell and B-cell epitopes, utilizing those for in-silico chimeric vaccine designing. The various physicochemical characteristics of the vaccine have been explored and the tertiary structure of the chimeric construct is predicted to perform docking studies and molecular dynamics simulations. Results: The vaccine construct is generated by joining the epitopes with specific linkers. The predicted tertiary structure of the vaccine has been found to be valid and docking studies reveal the construct shows a high affinity towards the TLR-4 receptor. Population coverage analysis shows the vaccine can be effective on the majority of the world population. In-silico immune simulation studies confirms the vaccine to raise a pro-inflammatory response with the proliferation of activated T and B cells. In-silico codon optimization and cloning of the vaccine nucleic acid sequence have also been achieved in the pET28a vector. Conclusion: The above bioinformatics data support that the construct may act as a potential vaccine. Further wet lab synthesis of the vaccine and in vivo works has to be undertaken in animal model to confirm vaccine potency. [ABSTRACT FROM AUTHOR]

Background: In the State of Mato Grosso do Sul, Brazil, sand flies and cases of visceral (VL) and cutaneous (CL) leishmaniases have been reported in almost all municipalities. The aim of this study was to analyze the geographic distribution of VL and CL in relation the sand fly species found in the municipalities of Mato Grosso do Sul. Methods: We analyzed VL and CL cases from 2001 to 2018 using data from the Notifiable Diseases Information System (SINAN). Data collected since 2003 on the presence of sand fly vectors (proven or suspected) were provided by the State Health Secretariat. Results: A total of 3566 and 3030 cases of VL and CL, respectively, were reported from 2001 to 2018. The municipalities with the most reported cases of VL were Campo Grande (2495), Três Lagoas (442), Corumbá (140) and Aquidauana (136); and those for CL were Campo Grande (635) and Bodoquena (197). The following sand fly species with vector potential were found in 59 municipalities (74.7%): Lutzomyia longipalpis, Lutzomyia cruzi, Nyssomyia whitmani, Migonemyia migonei, Nyssomyia neivai, Pintomyia pessoai, Bichromomyia flaviscutellata and Pintomyia fischeri. Sand flies were present in six municipalities where no cases of VL were reported and in two municipalities where no cases of CL were reported. Conclusions: Our results indicate that the geographical distribution of VL and CL in Mato Grosso do Sul expanded during the study period, and highlight the presence of sand fly vectors in municipalities where these diseases are currently considered to be non-endemic. [ABSTRACT FROM AUTHOR]

Background: The magnitude of the health problems caused by leishmaniasis has been a major driving factor behind the development and implementation of leishmaniasis control programs by the national authorities in Iran, with a priority for health and environmental management. Such programs are not achievable unless all of the factors leading to the infection, including the parasite's life-cycle, vectors and reservoirs, are recognized. So far in Iran, humans and rodents have been considered the principal reservoirs of Leishmania tropica and Leishmania major, respectively, both associated with cutaneous leishmaniasis (CL), with domestic dogs considered to be the main reservoir for Leishmania infantum, associated with visceral leishmaniasis (VL). The role of other mammals in maintaining the Leishmania parasite has remained unclear. This study aimed to investigate Leishmania infection among livestock in endemic areas of VL and CL in Fars province, southern Iran, using serological and molecular methods. Methods: Blood samples from 181 clinically healthy livestock, including 49 sheep, 114 goats, 16 cattle and two donkeys, were screened to detect Leishmania DNA and anti-Leishmania antibodies using qPCR (quantitative PCR) and the direct agglutination test (DAT), respectively. Four qPCR-positive samples were amplified using the internal transcribed spacer one (ITS1) primers in conventional PCR and sent for directional sequencing. Results: Of the 181 livestock tested, 51 (28.2%) were infected with Leishmania, using serological and molecular methods. Anti-Leishmania antibodies were detected in 70 (38.7%) (95% confidence interval [CI]: 31.5–46.2) and Leishmania DNA in 93 (51.4%) (95% CI: 43.9–58.9) livestock. The identified Leishmania spp. were L. infantum and L. major. Conclusions: The findings of the present study show a relatively high prevalence of Leishmania infection among livestock in endemic areas of the disease, in Fars province, southern Iran. Given the large population of this group of animals and the fact that they live in the vicinity of the main reservoirs of the disease and vectors, it seems that sand flies regularly bite these animals. Further studies are needed to determine the role of livestock in the parasite's life-cycle and the epidemiology of Leishmania infection. [ABSTRACT FROM AUTHOR]

Background: Query ID="Q1" Text="Graphical abstract: As per journal requirements, graphical abstract is necessary. Kindly check and provide the same."The magnitude of the health problems caused by leishmaniasis has been a major driving factor behind the development and implementation of leishmaniasis control programs by the national authorities in Iran, with a priority for health and environmental management. Such programs are not achievable unless all of the factors leading to the infection, including the parasite's life-cycle, vectors and reservoirs, are recognized. So far in Iran, humans and rodents have been considered the principal reservoirs of Leishmania tropica and Leishmania major, respectively, both associated with cutaneous leishmaniasis (CL), with domestic dogs considered to be the main reservoir for Leishmania infantum, associated with visceral leishmaniasis (VL). The role of other mammals in maintaining the Leishmania parasite has remained unclear. This study aimed to investigate Leishmania infection among livestock in endemic areas of VL and CL in Fars province, southern Iran, using serological and molecular methods. Methods: Blood samples from 181 clinically healthy livestock, including 49 sheep, 114 goats, 16 cattle and two donkeys, were screened to detect Leishmania DNA and anti-Leishmania antibodies using qPCR (quantitative PCR) and the direct agglutination test (DAT), respectively. Four qPCR-positive samples were amplified using the internal transcribed spacer one (ITS1) primers in conventional PCR and sent for directional sequencing. Results: Of the 181 livestock tested, 51 (28.2%) were infected with Leishmania, using serological and molecular methods. Anti-Leishmania antibodies were detected in 70 (38.7%) (95% confidence interval [CI]: 31.5–46.2) and Leishmania DNA in 93 (51.4%) (95% CI: 43.9–58.9) livestock. The identified Leishmania spp. were L. infantum and L. major. Conclusions: The findings of the present study show a relatively high prevalence of Leishmania infection among livestock in endemic areas of the disease, in Fars province, southern Iran. Given the large population of this group of animals and the fact that they live in the vicinity of the main reservoirs of the disease and vectors, it seems that sand flies regularly bite these animals. Further studies are needed to determine the role of livestock in the parasite's life-cycle and the epidemiology of Leishmania infection. [ABSTRACT FROM AUTHOR]

Background: Leishmaniasis is a serious neglected tropical disease that may lead to life-threatening outcome, which species are closely related to clinical diagnosis and patient management. The current Leishmania species determination method is not appropriate for clinical application. New Leishmania species identification tool is needed using clinical samples directly without isolation and cultivation of parasites. Methods: A probe-based allele-specific real-time PCR assay was established for Leishmania species identification between Leishmania donovani and L. infantum for visceral leishmaniasis (VL) and among L. major, L. tropica and L. donovani/L. infantum for cutaneous leishmaniasis (CL), targeting hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and spermidine synthase (SPDSYN) gene with their species-specific single nucleotide polymorphisms (SNPs). The limit of detection of this assay was evaluated based on 8 repeated tests with intra-assay standard deviation < 0.5 and inter-assay coefficients of variability < 5%. The specificity of this assay was tested with DNA samples obtained from Plasmodium falciparum, Toxoplasma gondii, Brucella melitensis and Orientia tsutsugamushi. Total 42 clinical specimens were used to evaluate the ability of this assay for Leishmania species identification. The phylogenetic tree was constructed using HGPRT and SPDSYN gene fragments to validate the performance of this assay. Results: This new method was able to detect 3 and 12 parasites/reaction for VL and CL respectively, and exhibited no cross-reaction with P. falciparum, T. gondii, B. melitensis, O. tsutsugamushi and non-target species of Leishmania. Twenty-two samples from VL patients were identified as L. donovani (n = 3) and L. infantum (n = 19), and 20 specimens from CL patients were identified as L. major (n = 20), providing an agreement of 100% compared with sequencing results. For further validation, 29 sequences of HGPRT fragment from nine Leishmania species and 22 sequences from VL patients were used for phylogenetic analysis, which agreed with the results of this new method. Similar results were obtained with 43 sequences of SPDSYN fragment from 18 Leishmania species and 20 sequences from CL patients. Conclusions: Our assay provides a rapid and accurate tool for Leishmania species identification which is applicable for species-adapted therapeutic schedule and patient management. [ABSTRACT FROM AUTHOR]

Background: Responsible companion animal guardianship (RCAG) comprises a set of concepts involving activities, behavior and care that guardians must provide to ensure the welfare of their animals. When such principles are disregarded, the risk of animals developing zoonotic diseases, such as canine visceral leishmaniasis (CVL), increases. This disease is a public health problem in many urban settings in Brazil because dogs are the main reservoirs of Leishmania and are involved in the transmission of the parasites to humans. Our analytical cross-sectional epidemiological survey aimed to investigate the prevalence of CVL in a city in southeastern Brazil and to establish the association between the disease and a number of predictor variables including dog traits, socioeconomic status of guardians, ecological features of the domicile and RCAG. Results: Our study showed that the global prevalence of CVL in the sample canine population was 6.7% (47/704). All variables related to better dog care were associated with lower chances of infection. Multiple regression analysis revealed that the chances of animals being seropositive for CVL were significantly (p < 0.05) higher when guardians had no formal education or possessed a university degree (vs. those with complete primary or secondary schooling) and when dogs were sheltered outside the house and had free access to the streets. An additional novel finding was that dogs that were acquired as puppies presented half of the chance of developing the disease in comparison with those acquired at the adult stage. Geographically weighted logistic regression coefficients showed that the strengths of the predictor/CVL associations varied depending on the studied geographical space. Both models demonstrated that the associations were always in the same directions. Conclusions: Our findings indicate that regardless of age and mode of acquisition, adult dogs should be submitted to clinical evaluation and tests for CVL. RCAG can exert positive effects on the control of CVL. [ABSTRACT FROM AUTHOR]

Sensitive, reliable and fast diagnostic tools that are applicable in low-resource settings, at the point of care (PoC), are seen as crucial in the fight against visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Addressing the need for a PoC test, several diagnostic tests, including serological and molecular methods, have been developed and evaluated in the past. One promising molecular method, already implemented for diagnosis of a range of diseases, is the loop-mediated isothermal amplification (LAMP) protocol. In this systematic review and meta-analysis, using a comprehensive search strategy, we focus on studies evaluating the performance of LAMP for the diagnosis of leishmaniasis in humans and other mammals such as dogs, compared with microscopy and/or any other molecular diagnostic method. A meta-analysis, pooling sensitivity and specificity rates and calculating areas under the curve (AUCs) in summary receiver operating characteristic (SROC) plots, was conducted on datasets extracted from studies, grouped by clinical condition and sample type. We found high sensitivity and specificity for LAMP when compared with microscopy and PCR using blood samples, with pooled estimate values of > 90% for all subgroups, corresponding to calculated AUC values > 0.96, except for LAMP compared to microscopy for diagnosis of CL. However, only a limited number of studies were truly comparable. Most of the observed heterogeneity is likely based on true differences between the studies rather than sampling error only. Due to simple readout methods and low laboratory equipment requirements for sample preparation compared to other molecular methods, LAMP is a promising candidate for a molecular (near-)PoC diagnostic method for VL and CL. [ABSTRACT FROM AUTHOR]

Background: Dogs are the reservoir host of Leishmania infantum, the agent of zoonotic visceral leishmaniasis (VL), which is transmitted by the bite of phlebotomine sand flies. The sand fly Phlebotomus perniciosus is the main vector of zoonotic VL in the western Mediterranean region. Fluralaner has been shown to effectively kill this vector. The aim of this study was to evaluate the insecticidal efficacy of oral fluralaner in dogs bitten by P. perniciosus. Methods: Two parallel-group, negative-controlled, randomized, masked laboratory trials with equivalent designs were performed in two different locations using two different pathogen-free laboratory-bred P. perniciosus strains for the challenge. In each trial, 12 purpose-bred beagles, initially ranked on natural attractiveness to sand flies, were randomly allocated to two groups (6 animals/group). Dogs in one group received fluralaner orally at the approved dose on day 0, and dogs in the control group were not treated. Each dog was subsequently exposed to an average of 70 unfed live sand fly females on days 1, 28, 56 and 84. Viability of blood-fed females was then evaluated for up to 96 h after exposure, and insecticidal efficacy was measured as the survival rate of flies fed on the fluralaner-treated dogs versus that of dogs in the control group. Significance was calculated for the proportion of live fed sand fly counts from treated versus control group dogs. Results: Comparison of the survival proportions between treated and control groups showed that fluralaner insecticidal efficacy was highly significant in both trials (P < 0.001 or P < 0.01 in different assessments) through to day 56. In the first trial, efficacy reached 100% on days 1 and 28, and 99.1% on day 56; in the second trial, the insecticidal efficacy was 98.5, 100 and 85.9%, respectively on the same days. On day 84, efficacy was in the range of 53–57% (P < 0.05) in the first trial and 0% in the second trial. Conclusion: A single oral fluralaner administration to dogs under laboratory conditions results in strong and reproducible insecticidal efficacy against P. perniciosus for at least 8 weeks. [ABSTRACT FROM AUTHOR]

Background: Leishmania infantum is the most important etiological agent of visceral leishmaniasis in the Americas and Mediterranean region, and the dog is the main host. Miltefosine was authorized to treat canine leishmaniasis (CanL) in Brazil in 2017, but there is a persistent fear of the emergence of parasites resistant not only to this drug but, through cross-resistance mechanisms, also to meglumine antimoniate and amphotericin B. Additionally, the literature shows that acquisition of resistance is followed by increased parasite fitness, with higher rates of proliferation, infectivity and metacyclogenesis, which are drivers of parasite virulence. In this context, the aim of this study was to analyze the impact of treating a dog with miltefosine and allopurinol on the generation of parasites resistant to miltefosine, amphotericin B and meglumine antimoniate. Methods: In vitro susceptibility tests were conducted against miltefosine, amphotericin B and meglumine antimoniate with T0 (parasites isolated from a dog before treatment with miltefosine plus allopurinol), T1 (after 1 course of treatment) and T2 (after 2 courses of treatment) isolates. The rates of cell proliferation, infectivity and metacyclogenesis of the isolates were also evaluated. Results: The results indicate a gradual increase in parasite resistance to miltefosine and amphotericin B with increasing the number of treatment courses. An increasing trend in the metacyclogenesis rate of the parasites was also observed as drug resistance increased. Conclusion: The data indicates an increased L. infantum resistance to miltefosine and amphotericin B after the treatment of a dog with miltefosine plus allopurinol. Further studies with a larger number of L. infantum strains isolated from dogs with varied immune response profiles and undergoing different treatment regimes, are advocated. [ABSTRACT FROM AUTHOR]

Background: Malaria and visceral leishmaniasis (VL) co-infection can occur due to the overlapping geographical distributions of these diseases; however, only limited data of this co-infection have been reported and reviewed. This study aimed to explore the pooled prevalence and characteristics of this co-infection using a systematic review approach. Methods: The PubMed, Web of Science and Scopus databases were searched for relevant studies. The quality of these studies was assessed in accordance with strengthening the reporting of observational studies in epidemiology (STROBE) guidelines. The numbers of individuals co-infected with Plasmodium and VL and the total numbers of individuals with VL were used to estimate the pooled prevalence using random-effects models. Differences in age, sex and the presence of anemia and malnutrition on admission were compared between co-infected individuals and individuals with VL using a random-effects model; the results are presented as odds ratios (ORs) and 95% confidence intervals (CIs). Heterogeneity among the included studies was assessed and quantified using Cochrane Q and I2 statistics. Results: Of the 3075 studies identified, 12 met the eligibility criteria and were included in this systematic review. The pooled prevalence of Plasmodium infection among the 6453 individuals with VL was 13%, with substantial heterogeneity of the data (95% CI 7–18%, I2 97.9%). Subgroup analysis demonstrated that the highest prevalence of co-infection occurred in African countries, whereas the lowest prevalence occurred in Asian countries. Patients aged < 5 years had higher odds of having co-infection than having VL (co-infection, n = 202; VL, n = 410) (OR 1.66, 95% CI 1.37–2.01, I2 0%; P < 0.0001), whereas patients aged 20–29 years had lower odds of having co-infection than having VL (co-infection, n = 170; VL, n = 699) (OR 0.75, 95% CI 0.60–0.93, I2 18%; P = 0.01). Male patients had equivalent odds of having co-infection and having VL (co-infection, n = 525; VL, n = 2232) (OR 0.92, 95% CI 0.078–1.08, I2 0%; P = 0.29). Patients with co-infection had lower odds of having anemia at admission than those with VL (co-infection, n = 902; VL, n = 2939) (OR 0.64, 95% CI 0.44–0.93, I2 0%; P = 0.02). No difference in malnutrition at admission was found in the meta-analysis. Conclusions: The prevalence of malaria co-infection among individuals with VL was heterogeneous and ranged from 7 to 18%, depending on geographical area. Age and anemia at admission were associated with co-infection status. Further longitudinal studies are needed to determine if co-infection with malaria has an impact on the severity of VL. [ABSTRACT FROM AUTHOR]

Background: With visceral leishmaniasis (VL) incidence at its lowest level since the 1960s, increasing attention has turned to early detection and investigation of outbreaks. Methods: Outbreak investigations were triggered by recognition of case clusters in the VL surveillance system established for the elimination program. Investigations included ascertainment of all VL cases by date of fever onset, household mapping and structured collection of risk factor data. Results: VL outbreaks were investigated in 13 villages in 10 blocks of 7 districts. Data were collected for 20,670 individuals, of whom 272 were diagnosed with VL between 2012 and 2019. Risk was significantly higher among 10–19 year-olds and adults 35 or older compared to children younger than 10 years. Outbreak confirmation triggered vector control activities and heightened surveillance. VL cases strongly clustered in tolas (hamlets within villages) in which > 66% of residents self-identified as scheduled caste or scheduled tribe (SC/ST); 79.8% of VL cases occurred in SC/ST tolas whereas only 24.2% of the population resided in them. Other significant risk factors included being an unskilled non-agricultural laborer, migration for work in a brick kiln, living in a kuccha (mud brick) house, household crowding, habitually sleeping outside or on the ground, and open defecation. Conclusions: Our data highlight the importance of sensitive surveillance with triggers for case cluster detection and rapid, careful outbreak investigations to better respond to ongoing and new transmission. The strong association with SC/ST tolas suggests that efforts should focus on enhanced surveillance in these disadvantaged communities. [ABSTRACT FROM AUTHOR]

Background: Although visceral leishmaniasis (VL) was largely brought under control in most regions of China during the previous century, VL cases have rebounded in western and central China in recent decades. The aim of this study was to investigate the epidemiological features and spatial–temporal distribution of VL in mainland China from 2004 to 2019. Methods: Incidence and mortality data for VL during the period 2004–2019 were collected from the Public Health Sciences Data Center of China and annual national epidemic reports of VL, whose data source was the National Diseases Reporting Information System. Joinpoint regression analysis was performed to explore the trends of VL. Spatial autocorrelation and spatial–temporal clustering analysis were conducted to identify the distribution and risk areas of VL transmission. Results: A total of 4877 VL cases were reported in mainland China during 2004–2019, with mean annual incidence of 0.0228/100,000. VL incidence showed a decreasing trend in general during our study period (annual percentage change [APC] = −4.2564, 95% confidence interval [CI]: −8.0856 to −0.2677). Among mainly endemic provinces, VL was initially heavily epidemic in Gansu, Sichuan, and especially Xinjiang, but subsequently decreased considerably. In contrast, Shaanxi and Shanxi witnessed significantly increasing trends, especially in 2017–2019. The first-level spatial–temporal aggregation area covered two endemic provinces in northwestern China, including Gansu and Xinjiang, with the gathering time from 2004 to 2011 (relative risk [RR] = 13.91, log-likelihood ratio [LLR] = 3308.87, P < 0.001). The secondary aggregation area was detected in Shanxi province of central China, with the gathering time of 2019 (RR = 1.61, LLR = 4.88, P = 0.041). The epidemic peak of October to November disappeared in 2018–2019, leaving only one peak in March to May. Conclusions: Our findings suggest that VL is still an important endemic infectious disease in China. Epidemic trends in different provinces changed significantly and spatial–temporal aggregation areas shifted from northwestern to central China during our study period. Mitigation strategies, including large-scale screening, insecticide spraying, and health education encouraging behavioral change, in combination with other integrated approaches, are needed to decrease transmission risk in areas at risk, especially in Shanxi, Shaanxi, and Gansu provinces. [ABSTRACT FROM AUTHOR]

Background: Visceral leshimaniasis is a parasitic disease characterized by systemic infection of phagocytic cells and an intense inflammatory response. The progression of the disease or treatment may have an effect on hematological parameters of these patients'. Thus, the current study sought to compare the hematological profiles of visceral leishmaniasis patients before and after treatment with anti-leishmaniasis drugs.Method: An institutional-based retrospective cohort study was conducted among visceral leishmaniasis patients admitted to the University of Gondar comprehensive specialized referral hospital leishmaniasis research and treatment centre between September 2013 and August 2018. Hematological profiles were extracted from the laboratory registration book before and after treatment. Data were entered to Epi-info and exported to SPSS for analysis. Descriptive statistics were summarized using frequency and percentage to present with the table. The mean, standard deviation, median, and interquartile range were used to present the data. Furthermore, using the paired t-test and the Wilcoxon Signed rank test, the mean difference for normally and non-normally distributed data was compared. Spearman and Pearson correlation analysis were used to describe the relationship between hematological parameters and various variables. A P value of 0.05 was considered statistically significant.Result: With the exception of the absolute neutrophil count, all post-treatment hematological parameters show a significant increase when compared to pre-treatment levels. Prior to treatment, the prevalence of anemia, leukopenia, and thrombocytopenia was 85.5, 83.4, and 75.8%, respectively, whereas it was 58.3, 38.2, and 19.2% following treatment. Furthermore, parasite load was found to have a statistically significant negative correlation with hematological profiles, specifically with white blood cell and red blood cell parameters.Conclusion: According to our findings, patients with visceral leishmaniasis had improved hematological profiles after treatment. The effect of treatment on parasite proliferation and concentration within visceral organs, in which the parasite load could directly affect the patient's hematological profiles, may be associated with the change in hematological profiles. [ABSTRACT FROM AUTHOR]

Background: Leishmaniases are regularly seen in non-endemic areas due to the increase of international travels. They include cutaneous leishmaniases (CL) and mucocutaneous (MC) caused by different Leishmania species, and visceral leishmaniases (VL) which present with non-specific symptoms.Methods: We reviewed all consecutive leishmaniasis cases seen between September 2012 and May 2020. The diagnostic strategy included microscopy after May-Grünwald-Giemsa staining, a diagnostic quantitative PCR (qPCR) assay, and species identification based on sequencing of the cytochrome b gene.Results: Eighty-nine patients had a definitive leishmaniasis diagnosis. Nine patients had VL with Leishmania infantum. Eighty patients had CL. Twelve patients acquired CL after trips in Latin America (7 Leishmania guyanensis, 2 Leishmania braziliensis, 2 Leishmania mexicana, and 1 Leishmania panamensis). Species could be identified in 63 of the 68 CLs mainly after travel in North Africa (59%) with Leishmania major (65%), Leishmania tropica/killicki (24%), and L. infantum (11%), or in West Sub-Saharan Africa (32%), all due to L. major. The median day between appearance of the lesions and diagnosis was 90 [range 60-127].Conclusions: Our diagnostic strategy allows both positive diagnoses and species identifications. Travelers in West Sub-Saharan Africa and North Africa should be better aware of the risk of contracting leishmananiasis. [ABSTRACT FROM AUTHOR]

Background: Phlebotomines are a group of insects which include vectors of the Leishmania parasites that cause visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL), diseases primarily affecting populations of low socioeconomic status. VL in Brazil is caused by Leishmania infantum, with transmission mainly attributed to Lutzomyia longipalpis, a species complex of sand fly, and is concentrated mainly in the northeastern part of the country. CL is distributed worldwide and occurs in five regions of Brazil, at a higher incidence in the north and northeast regions, with etiological agents, vectors, reservoirs and epidemiological patterns that differ from VL. The aim of this study was to determine the composition, distribution and ecological relationships of phlebotomine species in an Atlantic Forest conservation unit and nearby residential area in northeastern Brazil. Methods: Centers for Disease Control and Shannon traps were used for collections, the former at six points inside the forest and in the peridomestic environment of surrounding residences, three times per month for 36 months, and the latter in a forest area, once a month for 3 months. The phlebotomines identified were compared with climate data using simple linear correlation, Pearson's correlation coefficient and cross-correlation. The estimate of ecological parameters was calculated according to the Shannon-Wiener diversity index, standardized index of species abundance and the dominance index. Results: A total of 75,499 phlebotomines belonging to 11 species were captured in the CDC traps, the most abundant being Evandromyia walkeri, Psychodopygus wellcomei and Lu. longipalpis. Evandromyia walkeri abundance was most influenced by temperature at collection time and during the months preceding collection and rainfall during the months preceding collection. Psychodopygus wellcomei abundance was most affected by rainfall and relative humidity during the collection month and the month immediately preceding collection time. Lutzomyia longipalpis abundance showed a correlation with temperature and the rainfall during the months preceding collection time. The Shannon trap contained a total of 3914 phlebotomines from these different species. Psychodopygus wellcomei, accounting for 91.93% of the total, was anthropophilic and active mainly at night. Conclusions: Most of the species collected in the traps were seasonal and exhibited changes in their composition and population dynamics associated with local adaptions. The presence of vectors Ps. wellcomei and Lu. longipalpis underscore the epidemiological importance of these phlebotomines in the conservation unit and surrounding anthropized areas. Neighboring residential areas should be permanently monitored to prevent VL or CL transmission and outbreaks. [ABSTRACT FROM AUTHOR]

Background: Cutaneous leishmaniasis (CL) is endemic in 70 countries worldwide. Nepal is considered non-endemic for CL and hence the control program is targeted to visceral leishmaniasis (VL) only. Here, we report the emergence of CL cases in different parts of Nepal. Methods: We analyzed the CL and VL cases reported to Epidemiology and Diseases Control Division (EDCD), Ministry of Health and Population, Nepal through District Health Information System 2 (DHIS-2) and Early Warning and Reporting System (EWRS) during the past 4 years (2016-2019). Any laboratory-confirmed case was included in the study. Demographic and clinical details of each patient were transcribed into Excel sheets, verified with the case report forms and analyzed. Results: VL has been reported in Nepal since 1980, but CL was reported very recently. From 2016 to 2019, 42 CL cases were reported from 26 different hospitals to EDCD which had been diagnosed on the basis of clinical presentation, and laboratory findings (demonstration of amastigotes in Giemsa-stained smears and rK39 test results). Majority of the patients (31.0%, 13/42) visited to the hospital within 1-6 months of onset of lesions. Facial region (38.1%, 16/42) was the common place where lesions were found ompared to other exposed parts of the body. CL was successfully treated with miltefosine for 28 days. The majority of CL patients did not have history of travel outside the endemic areas and there was no report of sandfly from these areas. Conclusion: These evidences highlight that the Government of Nepal need to pay more efforts on CL and include it in differential diagnosis by clinicians, and plan for an active surveillance when the country is targeting leishmaniasis elimination by the year 2025 with the decreasing number of VL cases. [ABSTRACT FROM AUTHOR]

Background: Post-kala-azar dermal leishmaniasis (PKDL) caused by Leishmania donovani (LD) is a skin disorder that often appears after treatment of visceral leishmaniasis (VL) patients. PKDL patients are potential reservoirs of LD parasites, which can initiate a new epidemic of anthroponotic VL. Therefore, host infectiousness to its sand fly vector is a critical factor for transmission, and its accurate estimation can facilitate control strategies. At present, conventional microscopy serves as the reference method to detect parasites in its vector. However, low sensitivity of microscopy can be a limiting factor. Methods: In this study, real-time quantitative PCR (LD-qPCR) and recombinase polymerase amplification (LD-RPA) assays were evaluated against microscopy for the detection of LD DNA extracted from live sand flies five days after controlled feeding on PKDL cases. Results: The sensitivity of LD-qPCR and LD-RPA assays were found to be 96.43 and 100%, respectively, against microscopy for the selected fed sand flies (n = 28), and an absolute specificity of both molecular tools for apparently unfed sand flies (n = 30). While the proportion of infectious cases among 47 PKDL patients was estimated as 46.81% as defined by microscopic detection of LD in at least one fed sand fly per case, LD-RPA assay evaluation of only the microscopy negative sand flies fed to those 47 PKDL cases estimated an even greater proportion of infectious cases (51.06%). In overall estimation of the infectious cases in retrospective manner, discordance in positivity rate was observed (p < 0.05) between LD-RPA (59.57%) assay and microscopy (46.81%), while LD-RPA had slightly better positivity rate than LD-qPCR (55.32%) as well. Conclusions: Considering the sensitivity, cost, detection time, and field applicability, RPA assay can be considered as a promising single molecular detection tool for investigations pertaining to LD infections in sand flies and/or host infectiousness in PKDL, while it can also be useful in confirmation of microscopy negative sand fly samples. [ABSTRACT FROM AUTHOR]

Background: With visceral leishmaniasis (VL) incidence at its lowest level since the 1960s, increasing attention has turned to early detection and investigation of outbreaks. Methods: Outbreak investigations were triggered by recognition of case clusters in the VL surveillance system established for the elimination program. Investigations included ascertainment of all VL cases by date of fever onset, household mapping and structured collection of risk factor data. Results: VL outbreaks were investigated in 13 villages in 10 blocks of 7 districts. Data were collected for 20,670 individuals, of whom 272 were diagnosed with VL between 2012 and 2019. Risk was significantly higher among 10–19 year-olds and adults 35 or older compared to children younger than 10 years. Outbreak confirmation triggered vector control activities and heightened surveillance. VL cases strongly clustered in tolas (hamlets within villages) in which > 66% of residents self-identified as scheduled caste or scheduled tribe (SC/ST); 79.8% of VL cases occurred in SC/ST tolas whereas only 24.2% of the population resided in them. Other significant risk factors included being an unskilled non-agricultural laborer, migration for work in a brick kiln, living in a kuccha (mud brick) house, household crowding, habitually sleeping outside or on the ground, and open defecation. Conclusions: Our data highlight the importance of sensitive surveillance with triggers for case cluster detection and rapid, careful outbreak investigations to better respond to ongoing and new transmission. The strong association with SC/ST tolas suggests that efforts should focus on enhanced surveillance in these disadvantaged communities. [ABSTRACT FROM AUTHOR]

Background: Visceral leishmaniasis (VL) and zoonotic cutaneous leishmaniasis (ZCL) are of public health concern in Merti sub-County, Kenya, but epidemiological data on transmission, vector abundance, distribution, and reservoir hosts remain limited. To better understand the disease and inform control measures to reduce transmission, we investigated the abundance and distribution of sand fly species responsible for Leishmania transmission in the sub-County and their blood-meal hosts. Methods: We conducted an entomological survey in five villages with reported cases of VL in Merti sub-County, Kenya, using CDC miniature light traps and castor oil sticky papers. Sand flies were dissected and identified to the species level using standard taxonomic keys and PCR analysis of the cytochrome c oxidase subunit 1 (cox1) gene. Leishmania parasites were detected and identified by PCR and sequencing of internal transcribed spacer 1 (ITS1) genes. Blood-meal sources of engorged females were identified by high-resolution melting analysis of vertebrate cytochrome b (cyt-b) gene PCR products. Results: We sampled 526 sand flies consisting of 8 species, Phlebotomus orientalis (1.52%; n = 8), and 7 Sergentomyia spp. Sergentomyia squamipleuris was the most abundant sand fly species (78.71%; n = 414) followed by Sergentomyia clydei (10.46%; n = 55). Leishmania major, Leishmania donovani, and Trypanosoma DNA were detected in S. squamipleuris specimens. Humans were the main sources of sand fly blood meals. However, we also detected mixed blood meals; one S. squamipleuris specimen had fed on both human and mouse (Mus musculus) blood, while two Ph. orientalis specimens fed on human, hyrax (Procavia capensis), and mouse (Mus musculus) blood. Conclusions: Our findings implicate the potential involvement of S. squamipleuris in the transmission of Leishmania and question the dogma that human leishmaniases in the Old World are exclusively transmitted by sand flies of the Phlebotomus genus. The presence of Trypanosoma spp. may indicate mechanical transmission, whose efficiency should be investigated. Host preference analysis revealed the possibility of zoonotic transmission of leishmaniasis and other pathogens in the sub-County. Leishmania major and L. donovani are known to cause ZCL and VL, respectively. However, the reservoir status of the parasites is not uniform. Further studies are needed to determine the reservoir hosts of Leishmania spp. in the area. [ABSTRACT FROM AUTHOR]

Background: Visceral leishmaniasis (VL) is a zoonotic disease caused by Leishmania infantum, for which dogs constitute the main urban parasite reservoir. Control measures and the treatment of canine visceral leishmaniasis (CVL) are essential to reduce VL cases. Early and accurate detection of L. infantum-infected dogs is crucial to the success of VL control. To improve the serological detection of L. infantum-exposed dogs, we evaluated the early diagnosis capacity of a recombinant protein (rLci5) in an immunosorbent assay (ELISA) to detect naturally infected dogs. Additionally, we evaluated the persistence of the positive results obtained by rLci5 ELISA in comparison to other conventional diagnostic test methods. Methods: Serum samples obtained from 48 L. infantum-infected dogs involved in a cohort study were evaluated using different diagnostic methods (qPCR, EIE-LVC, DPP-LVC and splenic culture). The results were compared to rLci5 ELISA to determine its capacity to diagnose L. infantum infection at earlier infection time points. The persistence of positive diagnostic test results was also compared for each dog evaluated. Results: rLci5 ELISA presented higher rates of positive results at early time points compared to the other diagnostic tests employed in the cohort study, as early as 24 months prior to detection by other tests. rLci5 ELISA positivity was 52.1% (25/48) at baseline, while qPCR was 35.4% (17/48), DPP-LVC 27.1% (13/48), EIE-LVC 22.9% (11/48) and culture only 4.2% (2/48). In at least one of the time points of the 24-month cohort study, rLci5 ELISA was positive in 100% (48/48) of the dogs, versus 83% (40/48) for qPCR, 75% (36/48) for DPP-LVC, 65% (31/48) for EIE-LVC and 31% (15/48) for culture. Investigating clinical signs in association with diagnostic test positivity, rLci5 ELISA successfully detected CVL in 62.9% (95/151) of the clinical evaluations with a score of 0–3, 64.3% (45/70) with scores between 4 and 7, and 73.7% (14/19) with scores > 7, providing higher rates of positivity than all other methods evaluated. Moreover, rLci5 ELISA presented the greatest persistence with respect to test positivity: 45.8% of the dogs evaluated. Conclusion: Four diagnostic tests were compared to rLci5 ELISA, which presented earlier infection diagnosis and a greater persistence of positive test results. Accordingly, the use of the rLci5 ELISA can improve CVL diagnostic performance by detecting infected dogs sooner than other testing methods, with enhanced persistence of positive results over the course of the infection. [ABSTRACT FROM AUTHOR]

Background: Hemophagocytic lymphohistiocytosis (HLH) is a syndrome of excessive inflammation. We aimed to describe the clinical and laboratory findings of HLH patients secondary to Visceral leishmaniasis (VL) and their treatment outcome during a 4-year follow-up period compared to primary HLH.Method: Forty children with primary HLH confirmed by genetic study and 20 children with HLH secondary to VL confirmed by a blood or bone marrow polymerase chain reaction from 2014 to 2018 in Shiraz, Fars province, Southern Iran, were enrolled.Results: The median age at diagnosis was 11.5 months (range 1-170), and 56.7% were male. Fever and splenomegaly were the most frequent clinical presentations. 93.3% of the subjects had an HScore > 169, which had a good correlation with HLH-2004 criteria (r = 0.371, P = 0.004). Patients with primary HLH experienced more thrombocytopenia (P = 0.012) and higher alanine transaminase (P = 0.016), while patients with VL-associated HLH had higher ferritin (P = 0.034) and erythrocyte sedimentation rate (P = 0.011). Central nervous system (CNS) involvement occurred in 38.3% of patients. The mortality rate was higher in patients with CNS disease (61% vs. 35%, P = 0.051). The 3-yr overall survival rate was 35.9%. (24% in primary HLH and 100% in VL-associated HLH, P < 0.001). In Cox regression analysis, platelet count < 100,000/ μ l (hazard ratio 4.472, 95% confidence interval 1.324-15.107, P = 0.016) correlated with increased mortality in patients with primary HLH.Conclusion: VL is a potential source of secondary HLH in regions with high endemicity. Treatment of the underlying disease in VL-associated HLH is sufficient in most cases, with no need to start etoposide-based chemotherapy. [ABSTRACT FROM AUTHOR]

Objective: Canine visceral leishmaniasis (CVL) is the main source of human visceral leishmaniosis (HVL) in Mediterranean region, including Iran and is spread from domestic dogs to Phlebotomine sand flies vectors to humans. To control the transmission of HVL, early and accurate detection of infected dogs is paramount importance despite it remains a confronting challenge. Herein, we evaluated the performance of direct agglutination test (DAT) against gold standard nested polymerase chain reaction (nested-PCR) for CVL diagnosis in symptomatic and asymptomatic domestic dogs from endemic areas of Iran. Results: Venous blood samples were collected from dogs without clinical signs (n = 30) and with clinical signs (n = 35) suggestive of Leishmania infantum infection. Among 65 samples examined, Leishmania DNA was detected by nested-PCR in 89.23% (58/65). Furthermore, 86.15% (56/65) nested-PCR positive samples were also DAT positive. The results of the DAT sensitivity test were 96.43% and 96.67% in symptomatic and asymptomatic dogs, respectively, while the specificity was 100.00% and 60.00% in symptomatic and asymptomatic dogs, respectively. The results of this study also pointed out substantial concordance between DAT test and nested-PCR method in both symptomatic dogs (Κ = 0.783; P < 0.001) and asymptomatic dogs (Κ = 0.618; P < 0.001). Thus, DAT represents as a simple and economic tool for initial diagnosis of CVL particularly in endemic areas of the disease. [ABSTRACT FROM AUTHOR]

Background: Zoonotic visceral leishmaniasis is caused by the protozoan Leishmania infantum and is highly lethal in humans and dogs if left untreated. The frequency of this parasite and associated histological changes in the pancreas of dogs are poorly studied. Therefore, the objectives of this study were to evaluate the frequency of detection and load of amastigotes in the pancreas of L. infantum-seropositive dogs and to identify the clinical signs and histological changes associated with parasitism of this organ. Methods: One hundred forty-three dogs from an endemic area in Brazil that tested seropositive for L. infantum were studied. The dogs were clinically examined, killed, and necropsied between 2013 and 2014. One fragment of the pancreas was randomly collected for histopathology and immunohistochemistry, and spleen and bone marrow were collected for culture. Results: Leishmania amastigotes were detected in the pancreas of 22 dogs (15.4%) by immunohistochemistry, all exhibiting L. infantum parasitism in the spleen and/or bone marrow. Poor body condition and cachexia were only associated with infection of the pancreas with Leishmania spp. (p = 0.021) and were found in 40.9% of dogs with pancreatic infection. Anorexia, vomiting, and/or diarrhea were observed in 9.2% of dogs with pancreatitis. The median parasite load in the pancreas was 1.4 infected macrophages/mm2. Pancreatic histological changes and their frequencies were: granulomatous pancreatitis (28.0%), lymphoplasmacytic pancreatitis (23.8%), acinar cell degeneration (6.3%), fibrosis (5.6%), hemorrhage (2.1%), eosinophilic pancreatitis (0.7%), suppurative pancreatitis (0.7%), and necrosis (0.7%). Conclusions: The present results demonstrate that L. infantum is one of the etiological agents of chronic pancreatitis in dogs; however, the frequency of detection and parasite load are low in this organ. The lack of an association of poor body condition and cachexia with pancreatitis and the low frequency of clinical signs commonly associated with pancreatitis suggest that a significant portion of the organ is not affected by this parasite. On the other hand, the association of poor body condition and cachexia with concomitant infection of the pancreas, spleen, and/or bone marrow with this parasite suggests that these manifestations are the result of a more advanced stage of canine visceral leishmaniasis. [ABSTRACT FROM AUTHOR]

Background: Leishmaniasis is one of the most neglected tropical diseases in the world and remains endemic in some underdeveloped regions, including western China. The phylogeny and classification of Chinese Leishmania has not been completely clarified to date, especially within the Leishmania (L.) donovani complex, although phylogenetic analyses based on a series of gene markers have been performed. More analytic methods and data are still needed. Random amplified polymorphic DNA (RAPD) technology can sensitively identify slight intraspecific differences, and it is a powerful tool to seek species-specific markers. This work attempted to identify Chinese Leishmania isolates from diverse geographic regions at the genomic level. Meanwhile, specific markers of the L. donovani complex were also developed by RAPD.Methods: RAPD was applied to 14 Chinese Leishmania isolates from diverse geographic regions and 3 WHO reference strains. The polymorphic sites of amplification were transformed into a data matrix, based on which genetic similarity was calculated, and a UPGMA dendrogram was constructed to analyse the genetic diversity of these Leishmania isolates. Meanwhile, the specific amplification loci of the L. donovani complex were TA-cloned, sequenced and converted into sequence characterized amplified region (SCAR) markers, which were validated preliminarily in 17 available Leishmania strains in this study and analysed by bioinformatics.Results: The cluster analyses showed that the three Leishmania sp. isolates SC10H2, SD and GL clustered together and apart from others, the strains of the L. donovani complex clearly divided into two clades, and the three isolates Cy, WenChuan and 801 formed a subclade. Three specific SCAR markers of the L. donovani complex, i.e., 1-AD17, 2-A816 and 3-O13, were successfully obtained and validated on 17 available Leishmania strains in this study. Through bioinformatic analyses, Marker 1-AD17 may have more specificity for PCR detection of VL, and Marker 3-O13 has the potential to encode a protein.Conclusions: The RAPD results verified that the undescribed Leishmania species causing visceral leishmaniasis (VL) in China was a unique clade distinguished from L. donovani and revealed that there was genetic differentiation among Chinese L. donovani. The identification of L. donovani-specific markers may help to provide a foundation for future research attempting to develop new specific diagnostic markers of VL and identify specific gene functions. [ABSTRACT FROM AUTHOR]

Background: Visceral leishmaniasis (VL) is severe and potentially fatal. Brazil is one of the countries with the greatest endemicity for the disease in the world. The reduction of CD4+ T lymphocytes, B cells activation and high levels of inflammatory cytokines (IL-6/IL-8/TNF/IL-1β), plasma LPS, soluble CD14, anti-Leishmania IgG3 and low leptin levels are involved in the immunopathogenesis of VL, most associated with severe VL. Despite relapses occurring in about 4-5% of patients with VL not associated with HIV infection, the factors underlying relapses are little known. Our aim was to identify clinical, laboratory and immunological parameters that may be associated with recurrences in VL.Methods: Fifteen VL patients recruited from Hospital Eduardo de Menezes (BH-MG) were grouped into relapsing (R-VL, n = 5) and non-relapsing (NR-VL, n = 10) and evaluated during active disease, immediately after treatment (post-treatment) and 6 months post-treatment (6mpt). Clinical and laboratory data obtained from medical records were correlated with CD4+ and CD8+ T cell counts and anti-Leishmania Igs and IL-6 plasma levels and compared to those parameters of ten healthy controls.Results: During the active phase of VL, despite similarity in the clinical symptoms, the rates of thrombocytopenia, elevated transaminases (AST and ALT) and hyperbilirubinemia were higher in the NR-VL group compared to R-VL (p < 0.05), a profile reversed during the post-treatment phase. All patients had low CD4+ T counts in active phase, however, NR-VL patients had a higher gain of this cell type than R-VL in the post-treatment (p < 0.05). There was a significant reduction in IgG3 levels during the follow-up in the NR-VL group compared to the R-VL, especially at 6mpt (p < 0.05). In addition, IgG3 levels were negatively correlated with CD4+ T counts in the R-VL group (r = - 0.52). Elevated levels of IL-6 were observed in active VL and correlated with clinical markers of severity.Conclusions: During active phase of VL, the NR-VL patients presented more severe laboratorial abnormalities compared to R-VL, probably because the latter had already received previous treatment. On the other hand, R-VL exhibited greater impairment of immune reconstitution and a high degree of B lymphocyte activation, which must be a factor that favored relapses. [ABSTRACT FROM AUTHOR]

Background: Asymptomatic Leishmania infections outnumber clinical infections on the Indian subcontinent (ISC), where disease reservoirs are anthroponotic. Diagnostics which detect active asymptomatic infection, which are suitable for monitoring and surveillance, may be of benefit to the visceral leishmaniasis (VL) elimination campaign on the ISC. Methods: Quantitative polymerase chain reaction (qPCR), loop-mediated isothermal amplification (LAMP), and the direct agglutination test (DAT) were carried out on blood samples, and the Leishmania antigen ELISA was carried out on urine samples collected from 720 household and neighbouring contacts of 276 VL and post–kala-azar dermal leishmaniasis (PKDL) index cases, with no symptoms or history of VL or PKDL, in endemic regions of Bangladesh between September 2016 and March 2018. Results: Of the 720 contacts of index cases, asymptomatic infection was detected in 69 (9.6%) participants by a combination of qPCR (1.0%), LAMP (2.1%), DAT (3.9%), and Leishmania antigen ELISA (3.3%). Only one (0.1%) participant was detected positive by all four diagnostic tests. Poor agreement between tests was calculated using Cohen's kappa (κ) statistics; however, the Leishmania antigen ELISA and DAT in combination captured all participants as positive by more than one test. We find evidence for a moderately strong association between the index case being a PKDL case (OR 1.94, p = 0.009), specifically macular PKDL (OR 2.12, p = 0.004), and being positive for at least one of the four tests. Conclusions: Leishmania antigen ELISA on urine detects active asymptomatic infection, requires a non-invasive sample, and therefore may be of benefit for monitoring transmission and surveillance in an elimination setting in combination with serology. Development of an antigen detection test in a rapid diagnostic test (RDT) format would be of benefit to the elimination campaign. [ABSTRACT FROM AUTHOR]

Background: Kidney involvement of visceral Leishmaniasis is previously reported, but knowledge is limited. Hypergammaglobulinemia is common in visceral leishmaniasis patients. Whether hypergammaglobulinemia after leishmaniasis depletion can cause kidney injury is not well reported yet.Case Presentation: We reported a patient who recovered from visceral Leishmaniasis but showed persistent hypergammaglobulinemia and elevated urinary protein. Kidney biopsy showed glomerular hypertrophy with mild segmental mesangial proliferation without tubulointerstitial involvement in light microscopy. No immune complex deposit was found in the mesangial area by neither immunofluorescent staining nor electronic microscope. Increased lysosomes were observed in proximal tubules by electronic microscope. Valsartan was administered to decrease urinary protein, and no immune-suppressive therapy was added. The urinary protein and serum IgG level gradually dropped, and serum creatinine level remained stable during three- month follow up.Conclusions: Hypergammaglobulinemia is unlikely to cause renal structural or functional damage in the short term. Angiotensin blockade significantly reduced urine protein, with a minor effect on IgG elimination. [ABSTRACT FROM AUTHOR]

Background: Visceral leishmaniasis (VL) and zoonotic cutaneous leishmaniasis (ZCL) are of public health concern in Merti sub-County, Kenya, but epidemiological data on transmission, vector abundance, distribution, and reservoir hosts remain limited. To better understand the disease and inform control measures to reduce transmission, we investigated the abundance and distribution of sand fly species responsible for Leishmania transmission in the sub-County and their blood-meal hosts. Methods: We conducted an entomological survey in five villages with reported cases of VL in Merti sub-County, Kenya, using CDC miniature light traps and castor oil sticky papers. Sand flies were dissected and identified to the species level using standard taxonomic keys and PCR analysis of the cytochrome c oxidase subunit 1 (cox1) gene. Leishmania parasites were detected and identified by PCR and sequencing of internal transcribed spacer 1 (ITS1) genes. Blood-meal sources of engorged females were identified by high-resolution melting analysis of vertebrate cytochrome b (cyt-b) gene PCR products. Results: We sampled 526 sand flies consisting of 8 species, Phlebotomus orientalis (1.52%; n = 8), and 7 Sergentomyia spp. Sergentomyia squamipleuris was the most abundant sand fly species (78.71%; n = 414) followed by Sergentomyia clydei (10.46%; n = 55). Leishmania major, Leishmania donovani, and Trypanosoma DNA were detected in S. squamipleuris specimens. Humans were the main sources of sand fly blood meals. However, we also detected mixed blood meals; one S. squamipleuris specimen had fed on both human and mouse (Mus musculus) blood, while two Ph. orientalis specimens fed on human, hyrax (Procavia capensis), and mouse (Mus musculus) blood. Conclusions: Our findings implicate the potential involvement of S. squamipleuris in the transmission of Leishmania and question the dogma that human leishmaniases in the Old World are exclusively transmitted by sand flies of the Phlebotomus genus. The presence of Trypanosoma spp. may indicate mechanical transmission, whose efficiency should be investigated. Host preference analysis revealed the possibility of zoonotic transmission of leishmaniasis and other pathogens in the sub-County. Leishmania major and L. donovani are known to cause ZCL and VL, respectively. However, the reservoir status of the parasites is not uniform. Further studies are needed to determine the reservoir hosts of Leishmania spp. in the area. [ABSTRACT FROM AUTHOR]

Background: With visceral leishmaniasis (VL) incidence at its lowest level since the 1960s, increasing attention has turned to early detection and investigation of outbreaks. Methods: Outbreak investigations were triggered by recognition of case clusters in the VL surveillance system established for the elimination program. Investigations included ascertainment of all VL cases by date of fever onset, household mapping and structured collection of risk factor data. Results: VL outbreaks were investigated in 13 villages in 10 blocks of 7 districts. Data were collected for 20,670 individuals, of whom 272 were diagnosed with VL between 2012 and 2019. Risk was significantly higher among 10–19 year-olds and adults 35 or older compared to children younger than 10 years. Outbreak confirmation triggered vector control activities and heightened surveillance. VL cases strongly clustered in tolas (hamlets within villages) in which > 66% of residents self-identified as scheduled caste or scheduled tribe (SC/ST); 79.8% of VL cases occurred in SC/ST tolas whereas only 24.2% of the population resided in them. Other significant risk factors included being an unskilled non-agricultural laborer, migration for work in a brick kiln, living in a kuccha (mud brick) house, household crowding, habitually sleeping outside or on the ground, and open defecation. Conclusions: Our data highlight the importance of sensitive surveillance with triggers for case cluster detection and rapid, careful outbreak investigations to better respond to ongoing and new transmission. The strong association with SC/ST tolas suggests that efforts should focus on enhanced surveillance in these disadvantaged communities. [ABSTRACT FROM AUTHOR]

Background: The Benishangul-Gumuz region is an important development corridor in Ethiopia. Large-scale projects such as the Great Renaissance Dam, mining and agriculture have entailed huge environmental modifications and settlement pattern changes. There is no detailed epidemiological information on visceral leishmaniasis (VL) in the region. Materials and methods: A cross-sectional study was carried out to assess the epidemiology and risk factors associated with Leishmania infection. A leishmanin skin test (LST) was done for 1342 participants, and for 253 of them rK39 and DAT were carried out. Thirty-six dogs owned by households with LST-positive member(s) were rK39 and DAT tested. A pretested questionnaire was used to capture individual and household characteristics. Results: Of the 89.2% (1197/1342) who availed themselves of the LST reading, 6.0% were positive. The rk39 and DAT positivity among the 253 tested were 3.2% and 5.9%, respectively. In dogs, positivity rates by rK39 and DAT were 13.9% and 5.6%, respectively. Of the household and individual risk factors, presence of a dog in the household (P = 0.005), male sex (0.003), residence woreda (0.000) and occupation (0.023) showed a strong positive association with LST positivity. Individuals who lived in households that had dogs were 2.6 times more likely to be LST positive (AOR = 2.6; 95% CI = 1.54, 4.40). Being female decreased the probability of being LST positive by 0.38 times (AOR = 0.38; 95% CI = 0.20, 0.72). People living in Guba and Kurmuk had 4.7 (AOR = 4.74, 95% CI 1.83, 12.31) and 5.9 (AOR = 5.85, 95% CI 2.27, 15.09) times more risk of being infected. Conclusions: We demonstrated the presence of active VL transmission in the areas. Thus, we underline the need to establish the responsible vector(s) and reservoir(s) for comprehensive early containment plans to prevent potentially harmful public health and economic consequences. [ABSTRACT FROM AUTHOR]

Background: Phlebotomines are a group of insects which include vectors of the Leishmania parasites that cause visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL), diseases primarily affecting populations of low socioeconomic status. VL in Brazil is caused by Leishmania infantum, with transmission mainly attributed to Lutzomyia longipalpis, a species complex of sand fly, and is concentrated mainly in the northeastern part of the country. CL is distributed worldwide and occurs in five regions of Brazil, at a higher incidence in the north and northeast regions, with etiological agents, vectors, reservoirs and epidemiological patterns that differ from VL. The aim of this study was to determine the composition, distribution and ecological relationships of phlebotomine species in an Atlantic Forest conservation unit and nearby residential area in northeastern Brazil. Methods: Centers for Disease Control and Shannon traps were used for collections, the former at six points inside the forest and in the peridomestic environment of surrounding residences, three times per month for 36 months, and the latter in a forest area, once a month for 3 months. The phlebotomines identified were compared with climate data using simple linear correlation, Pearson's correlation coefficient and cross-correlation. The estimate of ecological parameters was calculated according to the Shannon-Wiener diversity index, standardized index of species abundance and the dominance index. Results: A total of 75,499 phlebotomines belonging to 11 species were captured in the CDC traps, the most abundant being Evandromyia walkeri, Psychodopygus wellcomei and Lu. longipalpis. Evandromyia walkeri abundance was most influenced by temperature at collection time and during the months preceding collection and rainfall during the months preceding collection. Psychodopygus wellcomei abundance was most affected by rainfall and relative humidity during the collection month and the month immediately preceding collection time. Lutzomyia longipalpis abundance showed a correlation with temperature and the rainfall during the months preceding collection time. The Shannon trap contained a total of 3914 phlebotomines from these different species. Psychodopygus wellcomei, accounting for 91.93% of the total, was anthropophilic and active mainly at night. Conclusions: Most of the species collected in the traps were seasonal and exhibited changes in their composition and population dynamics associated with local adaptions. The presence of vectors Ps. wellcomei and Lu. longipalpis underscore the epidemiological importance of these phlebotomines in the conservation unit and surrounding anthropized areas. Neighboring residential areas should be permanently monitored to prevent VL or CL transmission and outbreaks. [ABSTRACT FROM AUTHOR]