Sequence analysis of the 3-untranslated region of HSP70 (type I) genes in the genus Leishmania: Its usefulness as a molecular marker for species identification

Background: The Leishmaniases are a group of clinically diverse diseases caused by parasites of the genus Leishmania. To distinguish between species is crucial for correct diagnosis and prognosis as well as for treatment decisions. Recently, sequencing of the HSP70 coding region has been applied in phylogenetic studies and for identifying of Leishmania species with excellent results. Methods: In the present study, we analyzed the 3-untranslated region (UTR) of Leishmania HSP70-type I gene from 24 strains representing eleven Leishmania species in the belief that this non-coding region would have a better discriminatory capacity for species typing than coding regions. Results: It was observed that there was a remarkable degree of sequence conservation in this region, evenbetween species of the subgenus Leishmania and Viannia. In addition, the presence of many microsatellites was a common feature of the 3-UTR of HSP70-I genes in the Leishmania genus. Finally, we constructed dendrograms based on global sequence alignments of the analyzed Leishmania species and strains, the results indicated that this particular region of HSP70 genes might be useful for species (or species complex) typing, improving for particular species the discrimination capacity of phylogenetic trees based on HSP70 coding sequences. Given the large size variation of the analyzed region between the Leishmania and Viannia subgenera, direct visualization of the PCR amplification product would allow discrimination between subgenera, and a HaeIII-PCR-RFLP analysis might be used for differentiating some species within each subgenera. Conclusions: Sequence and hylogenetic analyses indicated that this region, which is readily amplified using a single pair of primers from both Old and New World Leishmania species, might be useful as a molecular marker for species discrimination. © 2012 Requena et al.; licensee BioMed Central Ltd.Ministerio de Ciencia y Tecnología (BFU2009-08986); Fondo de Investigaciones Sanitarias (ISCIII-RETIC RD06/0021/0008-FEDER and ISCIII-RETIC RD06/0021/0009-FEDER); Agencia Española de Cooperación Internacional para el Desarrollo (AECID, A/024740/09); Fundación Ramón ArecesPeer Reviewe

Risk factors for visceral leishmaniasis in a new epidemic site in Amhara Region, Ethiopia

We conducted a case-control study to evaluate risk factors for visceral leishmaniasis during an epidemic in a previously unaffected district of Ethiopia. We also collected blood and bone marrow specimens from dogs in the outbreak villages. In multivariable analyses of 171 matched case-control pairs, dog ownership, sleeping under an acacia tree during the day, and habitually sleeping outside at night were associated with significantly increased risk. Specimens from 7 (3.8%) dogs were positive by immunofluorescent antibody test (IFAT) and both enzyme-linked immunosorbent assays (ELISAs), whereas Leishmania DNA was detected in 5 (2.8%) bone marrow aspirates (from 3 seropositive and 2 seronegative dogs). Insecticide-treated nets may only protect a portion of those at risk. Further research on the vectors, the role of the dog in the transmission cycle, and the effect of candidate interventions are needed to design the best strategy for control

Papular dermatitis due to Leishmania infantum infection in seventeen dogs: diagnostic features, extent of the infection and treatment outcome

BACKGROUND: This study describes immunological responses, diagnostic features, follow up and treatment outcomes from seventeen dogs with papular dermatitis due to Leishmania infection diagnosed by cytology or real time-PCR. METHODS: Specific Leishmania humoral and cellular immune responses were evaluated by means of an immunofluorescence antibody test in all cases and a delayed-type hypersensitivity (DTH) reaction to leishmanin in eight cases. The extent of infection was studied in several tissues including blood, lymph node, conjunctival and oral swabs, by means of PCR, at the time of diagnosis and during follow-up. Culture was performed on nine dogs from cutaneous lesions and lymph node aspirates and molecular typing was carried out on isolates based on ITS-1, ITS-2 and Haspb gene sequencing analysis. RESULTS: Cytological and molecular results from fine needle aspirates of papules were diagnostic in 8 out of 13 (61.5%) cases and in 14 out of 15 dogs (93.3%), respectively. In all dogs, specific anti-Leishmania antibody levels were low or absent. Blood and lymph node PCRs and lymph node culture were negative in all dogs. Three out of the nine dogs (33%) were positive by culture from cutaneous lesions. The three isolates were identified as ITS type A, however, polymorphism was observed in the Haspb gene (PCR products of 626 bp, 962 bp and 371 bp). DTH response was positive in all tested dogs at the time of diagnosis. The majority of dogs were successfully treated with only N-methylglucamine antimoniate, after which cutaneous lesions disappeared or were reduced to depigmented, flattened scars. All dogs remained seronegative and the majority of dogs were negative by PCR in several tissues during follow-up. CONCLUSIONS: This study points out that papular dermatitis due to L. infantum is probably an underestimated benign cutaneous problem, associated with a parasite specific cell mediated immunity and a poor humoral immune response. Papular dermatitis is seen in young dogs, and appears to be a mild disease with restricted parasite dissemination and a good prognosis. PCR can be used as a non-invasive method to routinely evaluate papules if Leishmania infection is suspected in cases in which parasites are not visualized by cytology.The authors thank Dr. Carmen Cañavate (Instituto de Salud Carlos III, Madrid, Spain) for kindly providing L. infantum promastigotes for leishmanin skin test; Laura Perillo for her collaboration in cytopathology; Antonino Lombardo (Studio Veterinario Lombardo, Mascalucia, CT, Italy) for his collaboration in collecting the clinical cases. The authors are grateful to Francesca Soutter (Royal Veterinary College) for the English revision of the manuscript. The authors are also grateful to technicians of the CreNaL laboratory, IZS, Sicily for their technical help. The authors also thank the reviewers for the constructive critical revision of the manuscript. Laia Solano-Gallego holds a Ramón y Cajal senior researcher contract awarded by the Spanish Ministerio de Economia y Competitividad and the European Social Fund. Publication of the CVBD9 thematic series has been sponsored by Bayer HealthCare - Animal Health division.S

DNA sequence analysis suggests that cytb-nd1 PCR-RFLP may not be applicable to sandfly species identification throughout the Mediterranean region

Molecular methods are increasingly used for both species identification of sandflies and assessment of their population structure. In general, they are based on DNA sequence analysis of targets previously amplified by PCR. However, this approach requires access to DNA sequence facilities, and in some circumstances, it is time-consuming. Though DNA sequencing provides the most reliable information, other downstream PCR applications are explored to assist in species identification. Thus, it has been recently proposed that the amplification of a DNA region encompassing partially both the cytochrome-B (cytb) and the NADH dehydrogenase 1 (nd1) genes followed by RFLP analysis with the restriction enzyme Ase I allows the rapid identification of the most prevalent species of phlebotomine sandflies in the Mediterranean region. In order to confirm the suitability of this method, we collected, processed, and molecularly analyzed a total of 155 sandflies belonging to four species including Phlebotomus ariasi, P. papatasi, P. perniciosus, and Sergentomyia minuta from different regions in Spain. This data set was completed with DNA sequences available at the GenBank for species prevalent in the Mediterranean basin and the Middle East. Additionally, DNA sequences from 13 different phlebotomine species (P. ariasi, P. balcanicus, P. caucasicus, P. chabaudi, P. chadlii, P. longicuspis, P. neglectus, P. papatasi, P. perfiliewi, P. perniciosus, P. riouxi, P. sergenti, and S. minuta), from 19 countries, were added to the data set. Overall, our molecular data revealed that this PCR-RFLP method does not provide a unique and specific profile for each phlebotomine species tested. Intraspecific variability and similar RFLP patterns were frequently observed among the species tested. Our data suggest that this method may not be applicable throughout the Mediterranean region as previously proposed. Other molecular approaches like DNA barcoding or phylogenetic analyses would allow a more precise molecular species identification.This work was supported in part by Instituto de Salud Carlos III (MPY-1248/12) grant and (PI14CIII/00016) funded by AESI. Ivonne Pamela Llanes-Acevedo was granted a student fellowship by COLCIENCIAS/COLFUTURO.S

Molecular Diagnosis of Leishmaniasis in Spain: Development and Validation of Ready-To-Use Gel-Form Nested and Real-Time PCRs To Detect Leishmania spp

Leishmaniasis is an endemic parasitic disease in at least 98 countries. In Spain, it is considered a zoonosis caused by Leishmania infantum, with an annual incidence of 0.62 cases/100,000 inhabitants. The predominant clinical manifestations are the cutaneous (CL) and visceral forms (VL), and the diagnosis is performed by parasitological, serological, and molecular tests. At the WHO Collaborating Center for Leishmaniasis (WHOCCLeish), routine diagnostic tests are based on a nested PCR (Ln-PCR), culture, and serological tests. To simplify our PCR protocol, we aimed to develop and validate a ready-to-use nested gel-form PCR (LeishGelPCR) and a duplex real-time PCR (qPCR) that allowed simultaneous detection of Leishmania and mammalian DNA as an internal control (Leish-qPCR). Clinical validation was performed in 200 samples from the WHOCCLeish collection; 92 and 85 out of 94 and 87 samples were positive by LeishGelPCR and Leish-qPCR, respectively, showing a sensitivity of 98% in both approaches. The specificity was 100% for LeishGelPCR and 98% for Leish-qPCR. The limits of detection of both protocols were similar (0.5 and 0.2 parasites/reaction). Parasite loads in VL and CL forms were similar, although high loads were observed when invasive samples were tested. In conclusion, LeishGelPCR and Leish-qPCR showed excellent performance in the diagnosis of leishmaniasis. These new forms of 18S rRNA gene PCR are equivalent to Ln-PCR and can be introduced in the algorithm for CL and VL diagnosis. IMPORTANCE Although the gold standard for diagnosis of leishmaniasis is the microscopic observation of amastigotes, molecular techniques are becoming a cost-efficient alternative. Currently, PCR is a routine resource that is used in many reference microbiology laboratories. In this article, we have described two ways to improve the reproducibility and usability of the molecular detection of Leishmania spp. These new approaches could be introduced even in middle- and low-resource laboratories; one is a ready-to-use gel-form system of a nested PCR and the other is a real-time PCR. We show why molecular diagnosis is the best methodology to confirm a clinical suspicion of leishmaniasis with higher sensitivity than traditional methods, thus facilitating early diagnosis and timely treatment of human leishmaniasis.This research was supported by Subprograma Retos de Colaboración, Ministerio de Economía y Competitividad (RTC-2016-5245-1) and Agreement CNM-Mundo Sano Foundation-Spain (MVP 1379).S

Differentiation and Gene Flow among European Populations of Leishmania infantum MON-1

Visceral leishmaniasis is caused by protozoan parasites of the genus Leishmania. This disease is a public health problem in countries bordering the Mediterranean, in China, and South America. Until now, isoenzyme analysis, a method with several advantages but also some limitations, is the gold standard for typing the causative agent L. infantum. We have developed a new method based on hypervariable DNA markers, the microsatellites. Its higher discriminatory power, genotype-based analysis, the possibility to use biological material instead of parasite cultures, and the fast analysis are the major improvements. We could demonstrate for the first time that there exist different geographically determined populations within the predominant zymodeme of L. infantum, which has important epidemiological implications. We also tested for relationships between genotype and clinical picture and/or host background. Leishmania is considered to reproduce mainly clonally; however, we found some indication for recombination in our study. Our work constitutes a solid basis for further population and epidemiological studies of L. infantum by completing the existing microsatellite database by analysing strains from other endemic foci

Implications of zoonotic and vector-borne parasites to free-roaming cats in central Spain

Cats are definitive hosts and reservoirs for several parasites, some of which are responsible for serious zoonotic diseases. We conducted a case-control study of data from a trap-neuter-return (TNR) programme (years 2014-2017) designed to examine the prevalence of zoonotic parasites in free-roaming cats living in urban areas of central Spain. In the animal population tested (n = 263), we detected a 29.2% prevalence of endoparasites, including high rates of cestodes (12.9%) and Toxocara cati (11.7%). While faecal samples showed no Toxoplasma gondii oocysts, the seroprevalence of T. gondii infection was 24.2%. Antibodies to Leishmania infantum were detected in 4.8% of the animals, though all skin and blood samples analyzed were PCR negative for this parasite. Ectoparasites (ticks and fleas) were found in 4.6% of the cat population, and 10.6% of the cats were detected with Otodectes cynotis. Finally, 6.3% and 7.9% cats tested positive for feline leukaemia virus and feline immunodeficiency virus, respectively. Our study provides useful information for animal-welfare and public-health, as the parasites detected can affect native wild animals through predation, competition and disease transmission. Our detection of zoonotic parasites such as L. infantum, T. gondii, T. cati, Giardia duodenalis and several ectoparasites prompts an urgent need for health control measures in stray cats.S

Changes in the microbiological diagnosis and epidemiology of cutaneous leishmaniasis in real-time PCR era: A six-year experience in a referral center in Barcelona

Leishmania; Reacción en cadena de la polimerasa; EspañaLeishmania; Polymerase chain reaction; SpainLeishmania; Reacció en cadena de la polimerasa; EspanyaBackground Leishmaniasis is a neglected disease caused by different species of the protozoa Leishmania spp. Cutaneous lesions are the most common clinical manifestation. This disease is prevalent in tropical and subtropical areas, including the Mediterranean basin. In Spain, Leishmania (L.) infantum is the only endemic species, but imported cases are often diagnosed. Different classical parasitological methods can be performed for cutaneous leishmaniasis (CL) diagnosis; but currently molecular techniques serve as a relevant tool for the detection and characterization of Leishmania parasites. We aimed to evaluate clinical and epidemiological characteristics of CL diagnosed patients by real-time PCR in a tertiary hospital over a six-year period. Methodology/Principal findings Clinical, epidemiological and microbiological data were retrospectively collected and analyzed. In our study, CL was confirmed in 59 (31.4%) out of 188 patients by real-time PCR, showing an increase over recent years: 11 cases of CL between 2014 and 2016 and 48 between 2017 and 2019. Real-time PCR was performed on skin swabs and/or biopsies samples, with a positivity of 38.5% and 26.5%, respectively. Results were 100% concordant when biopsy and skin swab were performed simultaneously. L. (L.) infantum was the most frequent species detected (50%), followed by L. (L.) major (45%) and Viannia subgenus (5%), which were detected only in imported cases. L. (L.) major was almost entirely detected in travelers/migrants from Morocco. Multiple and atypical skin lesions were more common in imported cases than in autochthonous cases (44.4% vs. 21.8%). Conclusions/Significance An increase in both autochthonous and imported CL cases has been observed in past years in our hospital. Molecular techniques assist in improving CL diagnosis and characterization of the Leishmania species, mainly in imported cases.The author(s) received no specific funding for this work