First detection of Leishmania major in dogs living in an endemic area of zoonotic cutaneous leishmaniasis in Tunisia.

BACKGROUND: Dogs are considered the main domestic animals that may be a reservoir for Leishmania infantum, the agent of zoonotic visceral leishmaniasis (ZVL) in several countries of the world. The dog may host other Leishmania species, but its epidemiological role in the maintenance and spreading of these parasites is not completely elucidated. Zoonotic cutaneous leishmaniasis (ZCL), caused by Leishmania major, affects thousands of people every year and is particularly diffused in many countries of North Africa and Middle East Asia. In ZCL endemic countries, few reports of L. major-positive dogs have been reported, probably because most human cases occur in poor rural areas where the social role of the dog and its medical management is not well considered. The aim of the present study is to better understand the possible involvement of domestic dogs in the epidemiology of ZCL. METHODS: Our research focused on a well-established endemic focus of ZCL, in the area of Echrarda, Kairouan Governorate, central Tunisia. A total of 51 dogs with no or mild clinical signs of vector borne diseases were selected in small villages where human cases of ZCL are yearly present. All dogs were sampled for the Leishmania spp. diagnosis, by using the following procedures: blood sample for serology and buffy coat quantitative polymerase chain reaction (qPCR), popliteal fine needle aspiration, and cutaneous biopsy punch for lymph node and skin qPCR. RESULTS: The results demonstrated a high percentage (21.6%) of dogs positive at least at one or more test; the most sensitive technique was the lymph node qPCR that detected 8/11 positive dogs. Nine, out of the eleven positive dogs, resulted as infected by Leishmania infantum; ITS1-PCR-sequencing allowed Leishmania major identification in the remaining two cases, both from the popliteal lymph node samples, which can suggest a possible visceral spread of a cutaneous Leishmania species in the dog. Interestingly, one of the two L. major-positive dogs was living in the same house where 6-year-old children showed cutaneous lesions referred to as ZCL. CONCLUSIONS: To our knowledge, this is the first report of L. major-positive dogs in Tunisia, the  epidemiological role of which remains under investigation.

Emergence of imported cutaneous leishmaniasis caused by Leishmania major: a case series from Kerala, India.

Cutaneous leishmaniasis (CL) is often considered a 'great imitator' and is the most common form of leishmaniasis. The Leishmania species responsible for CL varies among countries, as these species exhibit specific distribution patterns. The increased mobility of people across countries has resulted in the imported incidences of leishmaniasis caused by non-endemic species of Leishmania. During 2023, we confirmed three CL cases caused by L. major from Kerala, India, and upon detailed investigation, these were identified to be imported from the Middle East and Kazakhstan regions. This is the first report of CL caused by L. major from Kerala. The lesion morphology, detection of anti-rK 39 antibody and Leishmania parasite DNA from the blood samples were the unique observations of these cases. Kerala, being an emerging endemic zone of visceral leishmaniasis (VL) and CL, the imported incidences of leishmaniasis by non-endemic species can pose a significant threat, potentially initiating new transmission cycles of leishmaniasis caused by non-endemic species.

Severe Inflammatory and Disseminated Cutaneous Leishmaniasis in a Pregnant Woman: A Case Report from Portoviejo, Ecuador.

BACKGROUND Leishmaniasis is a zoonosis with worldwide prevalence that causes dermal lesions and can be serious in humans. This report presents a case of cutaneous leishmaniasis (CL) that was apparently associated with a zoonotic transmission in a peri-urban area of the city of Portoviejo, Ecuador, close to mountainous and forested sites. CASE REPORT For 37 years, we have studied transmission of leishmaniasis in Ecuador, and have seen a wide variety of clinical presentations of the disease caused by different strains of the parasite Leishmania in patients, including pregnant women, without marked difference among them. CL without complications causes painless lesions of different clinical aspect. The present study reports a case of a 25-year-old woman presenting with severely inflamed, disseminated, and painful lesions of CL. The patient was not given antimonial treatment; however, local cryotherapy was given, together with topical anti-inflammatory and antibiotic ointment. All the lesions were observed to heal, and no amastigotes were found in smear stains after clinical healing. Since there was no reactivation after 1.5 years of follow-up, conventional antileishmanial treatment with meglumine antimoniate was not given to the patient. CONCLUSIONS This report shows the importance of a properly done epidemiological and clinical presumtive diagnosis, followed by parasitological confirmation, and the benefit of using an alternative treatment for vulnerable patients, such as this pregnant woman, for whom the therapy with pentavalent antimonials is not indicated. All observed lesions healed and no amastigotes were found in the smears after clinical healing.

Histopathological findings and diagnosis of cutaneous leishmaniasis, confirmed by PCR, in an endemic region of Brazil.

INTRODUCTION: Diagnosis of cutaneous leishmaniasis (CL) is difficult, and the correct use of histopathological criteria can be useful in clinical practice. The present study evaluates the association between histopathological findings and the results of polymerase chain reaction (PCR) in clinically suspected cases of CL. METHODOLOGY: Skin samples were received in a laboratory from an endemic region of Brazil for over nine years. Associations were analyzed by means of the Chi square test with a 5% level of significance. RESULTS: Of the 222 examined samples, 190 (85.6%) tested positive by PCR. All 25 cases identified by microscopic examination also tested positive by PCR. Except for the more intense inflammatory infiltrate, all other evaluated histological variables (ulceration, epidermal hyperplasia, hyperkeratosis, presence of granuloma, neutrophils, histiocytes, lymphocytes, plasmocytes, and necrosis) were not significantly associated with PCR positivity. CONCLUSIONS: The intensity of the inflammatory infiltrate is a good indicator of the occurrence of CL. Histopathological aspects are useful to increase the predictive values of CL diagnoses, but PCR is still necessary to confirm or exclude the disease.

Clinical polymorphism of zoonotic cutaneous leishmaniasis: combination of the clinical and the parasitological diagnosis.

Zoonotic cutaneous leishmaniasis (ZCL) is a neglected tropical disease caused by Leishmania (L.) major. This zoonosis is characterized by a broad-spectrum clinical polymorphism and may be underestimated and poorly treated since it is a simulator of various dermatoses. The aim of our study was to analyze the clinical polymorphism of patients with ZCL. A total of 142 patients with confirmed CL based on the microscopic examination of skin lesion biopsies were included in this study. Molecular typing of Leishmania species revealed that all patients were infected with L. major. In total, 14 clinical forms were observed. Six were typical and eight were atypical. The typical ZCL forms are grouped as follows: papular (26.76%), ulcero-crusted (26.05%), ulcerated (13.38%), impetiginous (9.86%), nodular (9.15%), and papulo-nodular (5.63%) lesions. In atypical ZCL forms, we described erythematous (2.81%), erysipeloid (1.4%), sporotrichoid, (1.4%), keratotic (0.7%) lupoid (0.7%), lichenoid (0.7%), psoriasiform (0.7%), and zosteriform (0.7%) lesions. Here, the lichenoid and the keratotic forms caused by L. major were reported for the first time in Tunisia. These findings will help physicians to be aware of the unusual lesions of ZCL that could be confused with other dermatological diseases. For this reason, it will be necessary to improve the diagnosis of CL especially in endemic areas. Such large clinical polymorphism caused by L. major may be the result of a complex association between the vector microbiota, the parasite, and the host immune state, and further studies should be carried out in order to reveal the mechanisms involved in clinical polymorphism of ZCL.

[Complex cutaneous leishmaniasis with bone involvement]. / Komplexe kutane Leishmaniose mit ossärer Beteiligung.

We report an immunocompromised patient with a complex cutaneous leishmaniasis (CL) who suffered from singular bone involvement of the little left toe due to Leishmania (L.) infantum infection. The diagnosis was confirmed by positive polymerase chain reaction (PCR) testing in skin and bone tissue. The patient was successfully treated with miltefosine. In immunocompromised patients with CL, extracutaneous manifestations should always be ruled out. This is the first case report describing osseous involvement in CL due to Leishmania infantum.

ELISA with recombinant antigen Lb6H validated for the diagnosis of American tegumentary leishmaniasis.

American tegumentary leishmaniasis (ATL) diagnosis is an open question, and the search for a solution is urgent. The available tests that detect the etiological agent of the infection are specific for ATL diagnosis. However, they present disadvantages, such as low sensitivity and the need for invasive procedures to obtain the samples. Immunological methods (leishmanin skin test and search for anti-Leishmania antibodies) are good alternatives to the etiological diagnosis of ATL. Presently, we face problems with disease confirmation due to the discontinuity in the production of leishmanin skin test antigen, particularly in resource-poor settings. Aiming to diagnose ATL, we validated rLb6H-ELISA for IgG antibodies using 1,091 samples from leishmaniasis patients and healthy controls, divided into four panels, living in 19 Brazilian endemic and non-endemic states. The rLb6H-ELISA showed a sensitivity of 98.6% and a specificity of 100.0%, with the reference panel comprising 70 ATL patient samples and 70 healthy controls. The reproducibility evaluation showed a coefficient of variation of positive samples ≤ 8.20% for repeatability, ≤ 17,97% for reproducibility, and ≤ 8.12% for homogeneity. The plates sensitized with rLb6H were stable at 4°C and -20°C for 180 days and 37°C for seven days, indicating 12 months of validity. In samples of ATL patients from five research and healthcare centers in endemic and non-endemic areas, rLb6H-ELISA showed a sensitivity of 84.0%; no significant statistical difference was observed among the five centers (chi-square test, p = 0.13). In samples of healthy controls from four areas with different endemicity, a specificity of 92.4% was obtained; lower specificity was obtained in a visceral leishmaniasis high endemicity locality (chi-square test, p<0.001). Cross-reactivity was assessed in 166 other disease samples with a positivity of 13.9%. Based on the good diagnostic performance and the reproducibility and stability of the antigen, we suggest using ELISA-rLb6H to diagnose ATL.

Evaluation of blood based quantitative PCR as a molecular diagnostic tool for post kala-azar dermal leishmaniasis (PKDL).

BACKGROUND: Post kala-azar dermal leishmaniasis (PKDL) is a consequential dermal manifestation of visceral leishmaniasis (VL), serving as a parasite reservoir. The traditional diagnostic approach, which requires an invasive skin biopsy is associated with inherent risks and necessitates skilled healthcare practitioners in sterile settings. There is a critical need for a rapid, less invasive method for Leishmania detection. The main objective of this study was to evaluate and compare the diagnostic efficacy of PCR and qPCR in detecting PKDL, utilizing both skin and blood samples and to assess the utility of blood samples for molecular diagnosis. METHODS AND RESULTS: 73 individuals exhibiting clinical symptoms of PKDL and who had tested positive for rK39 rapid diagnostic test (RDT) were enrolled in this study. For the diagnosis of PKDL, both PCR and real-time quantitative PCR (qPCR), employing SYBR Green and TaqMan assays, were performed on blood and skin matched samples. qPCR results using both TaqMan and SYBR Green assay, indicated higher parasite loads in the skin compared to blood, as evident by the Ct values. Importantly, when blood samples were used for PKDL diagnosis by qPCR, an encouraging sensitivity of 69.35% (TaqMan assay) and 79.36% (SYBR Green) were obtained, compared to 8.2% with conventional PCR. CONCLUSION: The findings of the study suggest the potential utility of blood for molecular diagnosis by qPCR, offering a less invasive alternative to skin biopsies in field setting for the early detection of parasitaemia in PKDL patients and effective management and control of the disease.

Clinical and parasitological features of Leishmania infection among gold miners in the Oiapoque basin, an international Brazil-French Guiana border.

Gold miners working illegally in mines live in poor health conditions related to their strenuous work and precarious housing. Therefore, they are at higher risk for infectious diseases. American tegumentary leishmaniasis (ATL) appears to be of great concern to the population living in the Guiana Shield region. Our aim was to describe their demographic characteristics, the clinical features of cutaneous leishmaniasis (CL), and the frequency of Leishmania infection in people working in illegal gold mines in French Guiana. A cross-sectional study was carried out from October to December 2019 in Oiapoque city, Amapá, Brazil. Indeed, many gold miners working in French Guiana are originally from Brazil, and from Oiapoque in particular. A total of 105 participants from 31 different mining sites in French Guiana were recruited. Suspected Leishmania infection was confirmed by the following: detection of kDNA in blood or the lesion site; detection of specific antibodies; or detection of IFN-γ release after blood incubation with leishmanial antigens (IGRA-Leish). Nine active CL cases, 38 healed ATL (hATL) and 58 cases with no history of ATL (noATL), were identified. Only half of the treated hATL (50.0%; n = 14) reported having been assisted by a health care unit and the others treated themselves. PCR-kDNA for Leishmania was positive in the blood of 100% of CL cases. Curiously, blood PCR-kDNA was positive in 13% of hATL patients and in 15.5% of noATL patients. The IGRA-Leish was positive in 60.5% of hATL and in 37.9% of noATL. In addition to scars suggestive of CL, 71% of hATL had laboratory evidence of Leishmania infection. Restriction fragment polymorphism (RFLP) of the hsp70 gene identified a sympatric circulation of L. (V.) guyanensis (n = 4), L. (V.) braziliensis (n = 1), L. (L.) amazonensis (n = 2), L. (V.) shawi (n = 1) and L. (V.) naiffi/shawi (n = 1). Taking the laboratory techniques and the clinical evaluations together, 76% (n = 80) of the 105 participants had evidence of Leishmania infection. These results suggests that illegal gold miners working in French Guiana are at high risk for infection with different species of Leishmania, but their illegal condition and remoteness make it difficult for them to access health services.

A Case of Cutaneous Leishmaniasis with Mucosal Involvement in the Northern United States.

BACKGROUND: Cutaneous leishmaniasis (CL) is a vector-borne parasitic infection endemic to many sub-tropical regions worldwide. In the Americas, Leishmania braziliensis is responsible for most reported CL cases. Variable symptom presentation and susceptibility to secondary infection make diagnosing CL a difficult proposition for physicians who may not encounter cases frequently. CASE REPORT: We present the case of a 50-year-old man with multiple progressive lesions, diagnosed initially as a bacterial infection, who presented to a North American emergency department after several unsuccessful trials of antibiotic therapy. Eventually, polymerase chain reaction testing of a wound biopsy sample confirmed the presence of L. braziliensis. After a complicated course, the patient's infection resolved after tailored antiparasitic therapy. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: This case highlights the need to include travel history in the evaluation of atypical dermatologic infections.

Imported leishmaniasis in Denmark.

Leishmaniasis is transmitted by sandflies and involves cutaneous, mucocutaneous, or visceral disease. Sporadic, imported cases in Denmark emphasize the need for greater awareness. The incidence is stable with at least ten verified cases per year. Diagnostic methods include PCR- and antibody tests with a high positivity rate for PCR (17%) and a low positivity rate for antibody (1.4%). The latter should be used only when visceral disease is suspected. Immunosuppressed patients are at particular risk. Treatment strategies are chosen according to the severity of the condition, as argued in this review.

Sampling is decisive to determination of Leishmania (Viannia) species.

BACKGROUND: Accuracy of molecular tools for the identification of parasites that cause human cutaneous leishmaniasis (CL) could largely depend on the sampling method. Non-invasive or less-invasive sampling methods such as filter paper imprints and cotton swabs are preferred over punch biopsies and lancet scrapings for detection methods of Leishmania based on polymerase chain reaction (PCR) because they are painless, simple, and inexpensive, and of benefit to military and civilian patients to ensure timely treatment. However, different types of samples can generate false negatives and there is a clear need to demonstrate which sample is more proper for molecular assays. METHODOLOGY: Here, we compared the sensitivity of molecular identification of different Leishmania (Viannia) species from Peru, using three types of sampling: punch biopsy, filter paper imprint and lancet scraping. Different composite reference standards and latent class models allowed to evaluate the accuracy of the molecular tools. Additionally, a quantitative PCR assessed variations in the results and parasite load in each type of sample. PRINCIPAL FINDINGS: Different composite reference standards and latent class models determined higher sensitivity when lancet scrapings were used for sampling in the identification and determination of Leishmania (Viannia) species through PCR-based assays. This was consistent for genus identification through kinetoplastid DNA-PCR and for the determination of species using FRET probes-based Nested Real-Time PCR. Lack of species identification in some samples correlated with the low intensity of the PCR electrophoretic band, which reflects the low parasite load in samples. CONCLUSIONS: The type of clinical sample can directly influence the detection and identification of Leishmania (Viannia) species. Here, we demonstrated that lancet scraping samples consistently allowed the identification of more leishmaniasis cases compared to filter paper imprints or biopsies. This procedure is inexpensive, painless, and easy to implement at the point of care and avoids the need for anesthesia, surgery, and hospitalization and therefore could be used in resource limited settings for both military and civilian populations.

Comparison of Intralesional Sodium Stibogluconate versus Intralesional Meglumine Antimoniate for the Treatment of Leishmania major Cutaneous Leishmaniasis.

Israel is endemic for Old-World cutaneous leishmaniasis. The most common species is Leishmania major. However, the available treatment options are limited. This study's objective was to compare the authors' experience with different antimony intralesional treatments of Leishmania major cutaneous leishmaniasis. A retrospective evaluation was undertaken for cases of Leishmania major cutaneous leishmaniasis treated by pentavalent antimony in a university-affiliated medical centre in Israel. The previous treatment of intralesional sodium stibogluconate (Pentostam®) was compared with the current treatment of meglumine antimoniate (Glucantime®). One hundred cases of cutaneous leishmaniasis were treated during the study period, of whom 33 were treated with intralesional sodium stibogluconate and 67 were treated with intralesional meglumine antimoniate. The patients were 78 males and 22 females, mean age 24 (range 10-67) and there was a total of 354 skin lesions. Within 3 months from treatment, 91% (30/33) of the intralesional sodium stibogluconate group and 88% (59/67) of the intralesional meglumine antimoniate group had complete healing of the cutaneous lesions after an average of 3 treatment cycles (non-statistically significant). In conclusion, the 2 different medications have the same efficacy and safety for treating cutaneous leishmaniasis. Pentavalent antimoniate intralesional infiltration treatment is safe, effective, and well tolerated with minimal side effects for Old-World cutaneous leishmaniasis.

Clinical and laboratory characterization of cutaneous leishmaniasis in Chinese migrant workers returned from Iraq.

BACKGROUND: Imported cutaneous leishmaniasis (CL) is a growing problem with increasing global travel to endemic areas. Returned travelers with CL are easy to be misdiagnosed and mistreated due to the lack of awareness for the disease to the physicians in non-endemic region that may lead to unfavorable outcome. Our study intends to summarize the characteristics of Leishmania infection imported from Iraq, so as to help Chinese physicians diagnose and treat the disease. All CL patients were treated with intralesional injection of antimony. METHODS: The definitive diagnosis of CL is based on the parasite identification by microscopic examination directly on lesion smear or parasite culture, PCR amplification of Leishmania-specific internal transcribed spacer 1 (ITS-1). The phylogenetic analysis, the immunopathological examination and the cytokine detection were proceeded after the diagnosis. RESULTS: We have identified 25 CL cases in migrant Chinese workers returned from Iraq for the first time with L. major as the major species of infected Leishmania parasite. Clinical features of the Iraq-imported CL include the history of skin exposure to sandflies bite and the lesions mostly on the exposed limbs. More ulcerative wet lesion was observed than nodular dry lesion. PCR is not only used to detect Leishmania parasite with high sensitivity, but also to identify the species of infected parasite through sequencing the amplified Leishmania-specific ITS-1 gene. The phylogenetic analysis based on the amplified ITS-1 sequences revealed that the infected Leishmania was closed related to the species and strains endemic in Iraq. The immunopathological examination revealed the T-cell filtrated cellular immune response with less B cells and NK cells involved. The cytokine profile measured in the skin lesion also confirmed the Th1 cellular response with higher expression levels of IFN-γ, IL-6 and IL-8. The skin lesions in CL patients were healed after being treated locally with antimony. CONCLUSIONS: The clinical and parasitological features of these Chinese CL cases imported from Iraq provide useful information for the diagnosis and treatment of CL that is not commonly seen in Chinese local population.

Portable smartphone-based molecular test for rapid detection of Leishmania spp.

PURPOSE: Leishmaniasis, caused by the parasite of the genus Leishmania, is a neglected tropical disease which is endemic in more than 60 countries. In South-East Asia, Brazil, and East Africa, it mainly occurs as kala-azar (visceral leishmaniasis, VL), and subsequently as post kala-azar dermal leishmaniasis (PKDL) in a smaller portion of cases. As stated per WHO roadmap, accessibility to accurate diagnostic methods is an essential step to achieve elimination. This study aimed to test the accuracy of a portable minoo device, a small battery-driven, multi-use fluorimeter operating with isothermal technology for molecular diagnosis of VL and PKDL. METHODS: Fluorescence data measured by the device within 20 min are reported back to the mobile application (or app) via Bluetooth and onward via the internet to a backend. This allows anonymous analysis and storage of the test data. The test result is immediately returned to the app displaying it to the user. RESULTS: The limit of detection was 11.2 genome copies (95% CI) as determined by screening a tenfold dilution range of whole Leishmania donovani genomes using isothermal recombinase polymerase amplification (RPA). Pathogens considered for differential diagnosis were tested and no cross-reactivity was observed. For its diagnostic performance, DNA extracted from 170 VL and PKDL cases, comprising peripheral blood samples (VL, n = 96) and skin biopsies (PKDL, n = 74) from India (n = 108) and Bangladesh (n = 62), was screened. Clinical sensitivity and specificity were 88% and 91%, respectively. CONCLUSION: Minoo devices can offer a convenient, cheaper alternative to other molecular diagnostics. Its easy handling makes it ideal for use in low-resource settings to identify parasite burden.

Simple and promising paper-based electrochemical platform for serological detection of American tegumentary leishmaniasis.

BACKGROUND: American tegumentary leishmaniasis (ATL) is an endemic neglected tropical disease (NTD), its conventional treatment is toxic, slow, and invasive. Rapid diagnosis is crucial for the clinical management of suspected patients, so the development and use of low-cost, miniaturised and portable devices could be the key. OBJECTIVES: This work aimed to develop a simple paper-based electrochemical platform for the serological detection of ATL. METHODS: Platform was fabricated in Whatman N°1 paper, contains a hydrophobic zone generated by wax printing, two pencil graphite electrodes, and uses specific crude extracts (CA) antigens for ATL immuno-determination. The platform performance was analysed by measuring the relative impedance change for different antigen-antibody combinations. Then, 10 serum human samples previously diagnosed by the gold standard (five positive ATL cases and five non-ATL cases) were evaluated. FINDINGS: The platform presented a linear response for the charge transfer resistance (ΔRct) and the interface reactance (ΔXc). Also, optimal working conditions were established (1/60 serum dilution and 180 µg/mL CA concentration). Then, the platform permits to distinguish between ATL and non-ATL (p < 0.05) human serum samples. MAIN CONCLUSIONS: Our platform could allow the diagnosis, management, and monitoring of leishmaniasis while being an extremely simple and environmentally friendly technology.