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.

Quality of life of patients with cutaneous leishmaniasis: A comparative analysis of the EQ-5D-3L and CLIQ questionnaires.

PURPOSE: To evaluate the performance of the Cutaneous Leishmaniasis Impact Questionnaire (CLIQ) using the EuroQol-5 Dimension (EQ-5D-3L) as a reference standard (criterion validation); to evaluate the responsiveness of the instruments and estimate a cut-off point for the CLIQ to be able to discriminate between high and low impacts of cutaneous leishmaniasis on patients. METHODS: Between 2020 and 2022, a longitudinal validation study was conducted at a reference centre for leishmaniasis in Brazil. The EQ-5D-3L and CLIQ questionnaires were administered before, during and after treatment for cutaneous leishmaniasis. The correlation between the instruments was assessed using Spearman's correlation coefficient, responsiveness was assessed using the Wilcoxon test, and CLIQ cut-off points were proposed based on results of the EQ-5Q-3L, dichotomized between patients reporting no problems' and 'some or extreme problems'. RESULTS: There were satisfactory correlation coefficients between the two instruments before (-0.596) and during treatment (-0.551) and a low correlation between the instruments after the end of treatment (-0.389). In general, the responsiveness of the instruments was satisfactory. The CLIC scores that maximized sensitivity and specificity for recognizing impaired health status before and during treatment were 7 points and 17 points, respectively. However, at the end of treatment, based on the results for the EQ-5D-3L, the CLIC was not able to discriminate between individuals with high and low impacts of the disease. CONCLUSION: The CLIQ corresponds well with the EQ-5D-3L when applied before and during treatment but does not seem to be appropriate for follow-up evaluations after the end of treatment.

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.

A review of non-invasive samples and tools in kala-azar diagnosis and test of cure.

The recurrence of visceral leishmaniasis (VL), also called kala-azar (KA), in endemic regions of tropical countries like India, is primarily attributed to asymptomatic VL, post-kala azar dermal leishmaniasis (PKDL), and human immunodeficiency virus (HIV) co-infection. To effectively manage VL cases and elimination targets, an early and rapid diagnosis as well as accurate field surveillance is highly essential. The traditional sampling methods like bone marrow (BM), spleen, and lymph node (LN) tissue aspirations are invasive, painful, tedious, and prone to nosocomial infections, require skilled persons and hospital facilities, and are not feasible in rural areas. Therefore, there is an urgent requirement for the adoption of a patient-friendly, non-invasive, non-hospitalized sampling procedure that ensures an effective VL diagnosis. This review aims to meticulously evaluate the most recent scientific research that focuses on the precision, feasibility, and applicability of non-invasive sampling (NIS) and techniques for the diagnosis and test of cure of VL, particularly in resource-limited settings. Apart from that, the non-invasive techniques (NIT) that have shown promising results while monitoring VL treatment response and relapse are also reviewed. The limitations associated with NIT and possible improvements in this regard are discussed as well to improve the diagnosis and management of VL.

Disseminated Leishmaniasis, a Severe Form of Leishmania braziliensis Infection.

Disseminated leishmaniasis (DL) is an emergent severe disease manifesting with multiple lesions. To determine the relationship between immune response and clinical and therapeutic outcomes, we studied 101 DL and 101 cutaneous leishmaniasis (CL) cases and determined cytokines and chemokines in supernatants of mononuclear cells stimulated with leishmania antigen. Patients were treated with meglumine antimoniate (20 mg/kg) for 20 days (CL) or 30 days (DL); 19 DL patients were instead treated with amphotericin B, miltefosine, or miltefosine and meglumine antimoniate. High levels of chemokine ligand 9 were associated with more severe DL. The cure rate for meglumine antimoniate was low for both DL (44%) and CL (60%), but healing time was longer in DL (p = 0.003). The lowest cure rate (22%) was found in DL patients with >100 lesions. However, meglumine antimoniate/miltefosine treatment cured all DL patients who received it; therefore, that combination should be considered as first choice therapy.

Self-reporting of hearing loss and tinnitus in the diagnosis of ototoxicity by meglumine antimoniate in patients treated for American tegumentary Leishmaniasis.

INTRODUCTION: American Tegumentary Leishmaniasis (ATL) treatment is based on pentavalent antimonials (Sb5+), but these drugs have been associated to several adverse effects. Hearing loss and tinnitus during treatment with meglumine antimoniate (MA) have already been reported. This study aimed to describe the usefulness of self-reporting of hearing loss and tinnitus in diagnosing MA-induced ototoxicity. METHODS: A prospective longitudinal study was conducted with 102 patients with parasitological diagnosis of ATL, treated with different MA schemes. The presence of clinical auditory toxicity was defined as the emergence or worsening of self-reporting hearing loss and/or tinnitus during monitoring. Measures of sensitivity, specificity, and the positive and negative predictive value of the patient's self-reporting of hearing loss and tinnitus in relation to the result of the audiometric test (considered the gold standard) were calculated. RESULTS: The age of the evaluated patients ranged from 15 to 81 years, with a median of 41 years, and most were male (73.5%). Seventy-five patients (73.5%) had cutaneous leishmaniasis and 27 (26.5%) mucosal leishmaniasis. Eighty-six patients (84.3%) received intramuscular (IM) treatment and 16 (15.7%) were treated with intralesional MA. During treatment, 18 (17,6%) had tinnitus and 7 (6,9%) had complaint of hearing loss. 53 (52%) patients had cochlear toxicity confirmed by tone threshold audiometry and high frequency audiometry, from which 60% received a dose of 20 mg Sb5+/kg/day (p = 0.015) and 96.2% were treated with IM MA (p = 0.001). Tinnitus has greater specificity and positive predictive value than hearing loss, with a low number of false positives, but with a high false negative value. CONCLUSION: Although the large number of false negatives suggests that self-report of hearing loss or tinnitus cannot be considered a good screening test for referring the patient to an audiometry, the low number of false positives suggests the need to value the patient's complaint for referral. Otherwise, this study reinforces the importance of audiological monitoring during treatment with MA, especially in those patients with self-reporting of hearing loss or tinnitus when treated with 20 mg Sb5+/kg/day via IM.

Diagnosis of Cutaneous Leishmaniasis Using FTIR Spectroscopy and Machine Learning: An Animal Model Study.

Cutaneous leishmaniasis (CL) is a polymorphic and spectral skin disease caused by Leishmania spp. protozoan parasites. CL is difficult to diagnose because conventional methods are time-consuming, expensive, and low-sensitive. Fourier transform infrared spectroscopy (FTIR) with machine learning (ML) algorithms has been explored as an alternative to achieve fast and accurate results for many disease diagnoses. Besides the high accuracy demonstrated in numerous studies, the spectral variations between infected and noninfected groups are too subtle to be noticed. Since variability in sample set characteristics (such as sex, age, and diet) often leads to significant data variance and limits the comprehensive understanding of spectral characteristics and immune responses, we investigate a novel methodology for diagnosing CL in an animal model study. Blood serum, skin lesions, and draining popliteal lymph node samples were collected from Leishmania (Leishmania) amazonensis-infected BALB/C mice under experimental conditions. The FTIR method and ML algorithms accurately differentiated between infected (CL group) and noninfected (control group) samples. The best overall accuracy (∼72%) was obtained in an external validation test using principal component analysis and support vector machine algorithms in the 1800-700 cm-1 range for blood serum samples. The accuracy achieved in analyzing skin lesions and popliteal lymph node samples was satisfactory; however, notable disparities emerged in the validation tests compared to results obtained from blood samples. This discrepancy is likely attributed to the elevated sample variability resulting from molecular compositional differences. According to the findings, the successful functioning of prediction models is mainly related to data analysis rather than the differences in the molecular composition of the samples.

Big is not better: Comparing two alpha-Gal-bearing glycotopes in neoglycoproteins as biomarkers for Leishmania (Viannia) braziliensis infection.

The protozoan parasite Leishmania (Viannia) braziliensis is among Latin America's most widespread Leishmania species and is responsible for tegumentary leishmaniasis (TL). This disease has multiple clinical presentations, with cutaneous leishmaniasis (CL) being the most frequent. It manifests as one or a few localized skin ulcers, which can spread to other body areas. Hence, early diagnosis and treatment, typically with pentavalent antimonials, is critical. Traditional diagnostic methods, like parasite culture, microscopy, or the polymerase chain reaction (PCR) for detection of the parasite DNA, have limitations due to the uneven distribution of parasites in biopsy samples. Nonetheless, studies have revealed high levels of parasite-specific anti-α-Gal antibodies in L. (V.) braziliensis-infected patients. Previously, we demonstrated that the neoglycoprotein NGP28b, consisting of the L. (Leishmania) major type-2 glycoinositolphospholipid (GIPL)-3-derived trisaccharide Galpα1,6Galpα1,3Galfß conjugated to bovine serum albumin (BSA) via a linker, acts as a reliable serological biomarker (BMK) for L. (V.) braziliensis infection in Brazil. This indicates the presence of GIPL-3 or a similar structure in this parasite, and its terminal trisaccharide either functions as or is part of an immunodominant glycotope. Here, we explored whether extending the trisaccharide with a mannose unit would enhance its efficacy as a biomarker for the serological detection of L. (V.) braziliensis. We synthesized the tetrasaccharide Galpα1,6Galpα1,3Galfß1,3Manpα(CH2)3SH (G31SH) and conjugated it to maleimide-functionalized BSA to afford NGP31b. When we assessed the efficacy of NGP28b and NGP31b by chemiluminescent enzyme-linked immunosorbent assay on a cohort of CL patients with L. (V.) braziliensis infection from Bolivia and Argentina against a healthy control group, both NGPs exhibited similar or identical sensitivity, specificity, and accuracy. This finding implies that the mannose moiety at the reducing end is not part of the glycotope recognized by the parasite-specific anti-α-Gal antibodies in patients' sera, nor does it exert a relevant influence on the terminal trisaccharide's conformation. Moreover, the mannose does not seem to inhibit glycan-antibody interactions. Therefore, NGP31b is a viable and dependable BMK for the serodiagnosis of CL caused by L. (V.) braziliensis.

The need for culture in tegumentary leishmaniasis diagnosis in Bolivia: A comparative evaluation of four parasitological techniques using two sampling methods.

Leishmaniases are zoonotic diseases caused by protozoa of the genus Leishmania. In Bolivia, leishmaniasis occurs mainly in the cutaneous form (CL) followed by the mucosal or mucocutaneous form (ML or MCL), grouped as tegumentary leishmaniosis (TL), while cases of visceral leishmaniasis (VL) are rare. The cases of TL are routinely diagnosed by parasitological methods: Direct Parasitological Exam (DPE) and axenic culture, the latter being performed only by specialized laboratories. The aim of the present study was to optimize the parasitological diagnosis of TL in Bolivia, using two sampling methods. Samples from 117 patients with suspected TL, obtained by aspiration (n = 121) and scraping (n = 121) of the edge of the lesion were tested by: direct parasitological exam, culture in TSTB medium, and miniculture and microculture in Schneider's medium. A positive laboratory result by any of the four techniques evaluated using either of the two sampling methods was considered the gold standard. Of the 117 suspected patients included, TL was confirmed in 96 (82 %), corresponding 79 of the confirmed cases (82.3 %) to CL and 16 (16.7 %) to ML. Parasitological techniques specificity was 100 % and their analytical sensitivity was greater with scraping samples in TSTB culture (98 %). Scraping samples in TSTB and miniculture correlated well with the reference (Cohen's kappa coefficient=0.88) and showed good reliability (Cronbach's alpha coefficient ≥0.91). Microculture provided positive results earlier than the other culture methods (mean day 4.5). By day 14, 98 % of positive cultures had been detected. Scraping sampling and miniculture were associated with higher culture contamination (6 % and 17 %, respectively). Bacterial contamination predominated, regardless of the sampling and culture method, while filamentous fungi and mixed contamination were more frequently observed in cultures from scraping samples. In conclusion: (i) scraping samples proved more suitable for the diagnosis of TL as they increased analytical sensitivity, are less traumatic for the patient and are safer for laboratory personnel than aspirates; (ii) culture, mainly in TSBT medium, should be used for the diagnosis of TL due to its high sensitivity (doubling the number of cases diagnosed by DPE) and its low cost compared to other culture media.

Detection of Internal Transcribed Spacer 1 and hsp70 Genetic Markers Using Restriction Fragment Length Polymorphisms and Sequencing in Identification of Leishmania Species Causing Tegumentary Leishmaniasis in Brazil.

The identification of Leishmania species that cause tegumentary leishmaniasis (TL) is important for taxonomic and prognostic purposes. Molecular analysis using different Leishmania genomic targets is the most useful method for identifying Leishmania species. Therefore, we evaluated the performance of ribosomal RNA internal transcribed spacer 1 (ITS1) and heat shock protein (hsp70) genetic markers by polymerase chain reaction (PCR), followed by restriction fragment length polymorphism analysis (RFLP) and sequencing, for identification of Leishmania species. Samples from 84 Brazilian patients were amplified. Internal transcribed spacer 1 PCR followed by RFLP (HaeIII) [ITS1-RFLP (HaeIII)] identified 46.4% (39/84) of the samples as compatible with the Viannia subgenus. Internal transcribed spacer 1 PCR followed by sequencing (ITS1-sequencing) identified Leishmania (Viannia) braziliensis in 91.7% (77/84) of the TL samples, Leishmania (Leishmania) amazonensis in 3.6% (3/84), L. (V.) guyanensis in 2.4% (2/84), and L. (L.) infantum in 1.2% (1/84). One of the samples showed the same proportion of similarity with L. (V.) guyanensis and L. (V.) panamensis. hsp70 nested PCR followed by RFLP (HaeIII) [nested hsp70-RFLP (HaeIII)] identified 91.7% (77/84) of the samples as compatible with L. (V.) braziliensis/L. (V.) naiffi, 3.6% (3/84) with L. (L.) amazonensis, 1.2% (1/84) with L. (L.) infantum, and 3.6% (3/84) with L. (V.) guyanensis. hsp70 PCR followed by sequencing (hsp70-sequencing) identified L. (V.) braziliensis in 91.7% (77/84) of the TL samples, L. (L.) amazonensis in 3.6% (3/84), L. (V.) guyanensis in 3.6% (3/84), and L. (L.) infantum in 1.2% (1/84). Our findings clearly showed that nested hsp70-RFLP (HaeIII) is better than ITS1-RFLP (HaeIII) and that ITS1 or hsp70 PCR followed by sequencing was adequate for identifying Leishmania species. We also found that Leishmania (Viannia) braziliensis is the most common species causing TL in Brazil. Therefore, sequencing multiple target genes such as ITS1 and hsp 70 is more accurate than RFLP for identifying Leishmania species.

Case Report: An Atypical Case of Post-Kala-Azar Dermal Leishmaniasis with Ulcers and Verrucous Lesions: Clinical and Therapeutic Implications.

About 75% cases of post-kala-azar dermal leishmaniasis (PKDL) occur in India. Although the classic description of PKDL is the progression from initial hypopigmented macular lesions to papules to plaques and nodular lesions, atypical morphologies are also seen and are easily missed or misdiagnosed. We report a case of a 27-year-old man who presented to us with multiple acral ulcers and verrucous lesions for 5 years. A diagnosis of PKDL was made based on slit skin smear, histopathology, and quantitative polymerase chain reaction. The patient was given combination therapy with four doses of liposomal amphotericin B and miltefosine 50 mg twice daily for 45 days. In this report, we discuss unusual morphologies of PKDL, the pathway to the diagnosis, and the therapeutic options available along with their efficacy.

Cutaneous and Visceral Leishmaniasis Caused by the Same Zymodeme of Leishmania donovani in Kerala, India.

The tribal population in and around the Western Ghats region of India is affected by both cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL) with typical clinical symptoms. In this study, we recorded and analyzed seven CL and three VL cases from this emerging belt. All the cases were found as autochthonous transmission. Multiple genetic markers (minicircle kinetoplast DNA polymerase chain reaction and restriction fragment length polymorphism of 3'untranslated region heat shock protein (HSP) 70, a larger segment of HSP 70, and 6-phosphogluconate dehydrogenase [PGDH] gene sequences) were used to identify and characterize the parasite. It was found that both clinical manifestations are caused by zymodeme MON-37 of Leishmania donovani. We have investigated the detailed entomological and epidemiological aspects of disease transmission. An abundant population of the proven vector Phlebotomus argentipes was observed in the study villages.

A prospective longitudinal study on the elimination trend of rural cutaneous leishmaniasis in southeastern Iran: Climate change, population displacement, and agricultural transition from 1991 to 2021.

Leishmaniasis is a complex disease. Any change in weather conditions affects the humans' social and agricultural expansion and, consequently, the parasite's life cycle in terms of ecology, biodiversity, social stigma, and exclusion. This population-based prospective longitudinal investigation was conducted between 1991 and 2021 in a well-defined CL (cutaneous leishmaniasis) focus in Bam County, southeastern Iran. A robust health clinic and health surveillance system were responsible for the ongoing systematic documentation, detection, identification, and management of CL cases. The exponential smoothing method via the state space model was used in the univariate time series. The TTR, smooth, and forecast packages were used in R software. Landsat satellite images from 1991, 2001, 2011, and 2021 were employed in the physical development. During this period, the temperature increased while the rainfall and humidity decreased. The findings showed a downward trend in the standardized drought index. Also, the results showed that climate warming and ecological changes profoundly affected the area's agricultural patterns and topographical features. Furthermore, the last three decades witnessed an elimination trend for zoonotic CL (ZCL) and the predominance of anthroponotic CL (ACL). The present findings showed that the critical factors in the predominance of ACL and elimination of ZCL were rising temperature, drought, migration, unplanned urbanization, earthquake, and agrarian reform. The wall-enclosed palm tree gardens excluded the primary ZCL reservoir host. They controlled the disease while providing suitable conditions for the emergence/re-emergence of ACL in the newly established settlements and the unplanned ecozone. Therefore, robust health infrastructures, sustained financial support, and evidence-based research studies are crucial to facilitating the necessary surveillance, monitoring, and evaluation to control and eliminate the disease.

Molecular identification of Leishmania tropica in mammals occurring in human-inhabited areas of a cutaneous leishmaniasis endemic focus in North-West Pakistan.

Cutaneous Leishmaniasis is endemic in the tribal district of Khyber near the Pak-Afghan border and is caused by Leishmania tropica. In Pakistan, cutaneous leishmaniasis caused by L. tropica is considered anthroponotic and is thought to be maintained by a human-sand fly-human transmission cycle. Along with humans, other mammals may also be acting as reservoir hosts of leishmaniasis in the study area. To investigate the role of non-human mammals in the transmission of leishmaniasis, blood samples were collected from 245 animals from the CL endemic district of Khyber, Pakistan. Leishmania parasite in these samples was detected by amplifying the species-specific sequences in minicircle kinetoplast DNA, using PCR. L. tropica DNA was detected in 18 (7.35%) samples, comprising 11 cows (Bos taurus), 6 goats (Capra hircus), and 1 dog (Canus lupus familiaris). Only a single cow and dog had a leishmaniasis-like lesion, and the remaining positive samples were asymptomatic. None of the tested sheep (Ovis aries) and rat (Rattus rattus, Rattus norvegicus) was positive. The present study reports the first instance of molecular detection of L. tropica in domestic animals. Our study indicates that along with humans' cows, goats and dogs may also be playing an important role in the transmission of anthroponotic cutaneous leishmaniasis in district Khyber in particular and Pakistan in general.

Auricular leishmaniasis mimicking squamous cell carcinoma of the pinna.

Cutaneous leishmaniasis can occur on any exposed area of the body; however, the pinna is an exceptionally rare site for the disease. Caused by the parasite Leishmania, cutaneous leishmaniasis has a wide range of presentations and thus is very easy to misdiagnose or mistake for a neoplastic lesion. Here, we report the case of a middle-aged male patient presenting with a painful, ulcerated lesion on the left auricle initially suspected to be a malignancy with histopathology eventually revealing a diagnosis of auricular leishmaniasis. The patient received appropriate therapy and was found to be disease free at follow-up. These isolated lesions of the pinna often resemble neoplastic lesions and thus may escape diagnosis for months at a time, increasing patient stress as well as expenditure. In addition, prompt recognition may also help mitigate recurrence of the disease, making it worthwhile to include cutaneous leishmaniasis as part of the differential, especially in endemic areas.

A case report on para-kala-azar dermal leishmaniasis: an unresolved mystery.

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. 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.

Validation of Oxford nanopore sequencing for improved New World Leishmania species identification via analysis of 70-kDA heat shock protein.

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.