Publications by authors named "Carmen Chicharro"

28 Publications

  • Page 1 of 1

Detection of cutaneous leishmaniasis in three communities of Oti Region, Ghana.

PLoS Negl Trop Dis 2021 05 24;15(5):e0009416. Epub 2021 May 24.

School of Public Health, University of Ghana, Accra, Ghana.

Background: Cutaneous leishmaniasis (CL) is the most common type of leishmaniasis, a neglected tropical disease caused by parasites of the genus Leishmania. In Ghana, some studies in the Volta region have detected Leishmania parasites among persons with skin ulcers.

Methodology/principal Findings: Using a cross-sectional study design, the prevalence of CL in three communities of the Oti Region of Ghana was investigated. Demographic and epidemiological data were obtained by a structured interviewer administered questionnaire. A total of 426 (12.4%) out of 3,440 participants screened had at least one skin ulcer. Of 595 skin ulcers sampled and tested by PCR for Leishmania infection, 150 (25.2%) ulcers from 136 individuals tested positive, accounting for an overall CL prevalence of 31.9% among persons with skin ulcers. Individual community CL prevalence of 23.2%, 29.8%, and 36.8% was observed in Ashiabre, Keri, and Sibi Hilltop respectively among persons with skin ulcers.

Conclusions/significance: Confirmation of CL in the study area suggests an active cycle of transmission of Leishmania infection. The observation of skin ulcers which tested negative to Leishmania infection suggests a need to test for additional causes of skin ulcers such as Treponema pallidum pertenue and Mycobacterium ulcerans in the study area.
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http://dx.doi.org/10.1371/journal.pntd.0009416DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8177633PMC
May 2021

Loop-Mediated Isothermal Amplification Allows Rapid, Simple and Accurate Molecular Diagnosis of Human Cutaneous and Visceral Leishmaniasis Caused by When Compared to PCR.

Microorganisms 2021 Mar 16;9(3). Epub 2021 Mar 16.

WHO Collaborating Centre for Leishmaniasis, National Center for Microbiology, Instituto de Salud Carlos III, 28220 Majadahonda, Spain.

Loop-mediated isothermal amplification allows the rapid, sensitive and specific amplification of DNA without complex and expensive equipment. We compared the diagnostic performance of Loopamp™ Detection Kit (Eiken Chemical Co., Ltd., Tokyo, Japan) with conventional and real-time polymerase chain reaction (PCR) for human cutaneous and visceral leishmaniasis caused by . A total of 230 DNA samples from cutaneous (CL) and visceral (VL) leishmaniasis cases and controls from Spain, characterized by nested PCR (LnPCR) were tested by: (i) the Loopamp™ Detection Kit (Loopamp), run on Genie III real-time fluorimeter (OptiGene, UK); and (ii) real-time quantitative PCR (qPCR). The Loopamp test returned 98.8% (95% confidence interval-CI: 96.0-100.00) sensitivity and specificity of 97.7% (95% CI: 92.2-100) on VL samples, and 100% (95% CI: 99.1-100) sensitivity and 100.0% (95% CI: 98.8-100.0) specificity on CL samples. The Loopamp time-to-positivity () obtained by real-time fluorimetry showed excellent concordance (C = 97.91%) and strong correlation (r = 0.799) with qPCR's cycle threshold (). The performance of Loopamp is comparable to that of LnPCR and qPCR in the diagnosis of cutaneous and visceral leishmaniasis due to . The excellent correlation between the and should be further investigated to determine the accuracy of Loopamp to quantify parasite load in tissues.
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http://dx.doi.org/10.3390/microorganisms9030610DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7999953PMC
March 2021

Occurrence and Genetic Diversity of Protist Parasites in Captive Non-Human Primates, Zookeepers, and Free-Living Sympatric Rats in the Córdoba Zoo Conservation Centre, Southern Spain.

Animals (Basel) 2021 Mar 5;11(3). Epub 2021 Mar 5.

Parasitology Reference and Research Laboratory, Spanish National Centre for Microbiology, 28220 Madrid, Spain.

Little information is currently available on the epidemiology of parasitic and commensal protist species in captive non-human primates (NHP) and their zoonotic potential. This study investigates the occurrence, molecular diversity, and potential transmission dynamics of parasitic and commensal protist species in a zoological garden in southern Spain. The prevalence and genotypes of the main enteric protist species were investigated in faecal samples from NHP ( = 51), zookeepers ( = 19) and free-living rats ( = 64) by molecular (PCR and sequencing) methods between 2018 and 2019. The presence of spp. was also investigated in tissues from sympatric rats using PCR. sp. (45.1%), (27.5%), (21.6%), (3.9%), and (2.0%) (but not spp.) were detected in NHP. (10.5%) and sp. (10.5%) were identified in zookeepers, while spp. (45.3%), (14.1%), and sp. (6.25%) (but not spp.) were detected in rats. ST1, ST3, and ST8 and sub-assemblage AII were identified in NHP, and ST1 in zookeepers. isolates failed to be genotyped in human samples. In rats, four (, , and rat genotypes IV and V), one (assemblage G), and three (ST4) genetic variants were detected. Our results indicate high exposure of NHP to zoonotic protist species. Zoonotic transmission of ST1 was highly suspected between captive NHP and zookeepers.
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http://dx.doi.org/10.3390/ani11030700DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8035673PMC
March 2021

Cutaneous leishmaniasis: A pathological study of 360 cases with special emphasis on the contribution of immunohistochemistry and polymerase chain reaction to diagnosis.

J Cutan Pathol 2020 Nov 8;47(11):1018-1025. Epub 2020 Sep 8.

Department of Pathology, Hospital Universitario de Fuenlabrada, Madrid, Spain.

Background: Traditional methods for the diagnosis of leishmaniasis yield poor sensitivity, which limits its effectiveness in lesions with a low parasite burden.

Methods: Retrospective pathologic study of 360 cases of cutaneous leishmaniasis and analysis of the different diagnostic methods used.

Results: In 93% of the lesions, histopathology showed a dense and diffuse inflammatory infiltrate, consisting of lymphocytes, histiocytes and plasma cells, which occupied the superficial and mid dermis and variably extended to deep dermis and superficial subcutis (standard pattern). The remaining cases exhibited atypical features, such as perivascular, interstitial or perifollicular inflammatory patterns, folliculitis or panniculitis. Granulomas were identified in 84% of biopsies, most of them as small, poorly formed, non-necrotizing histiocytic aggregates. Amastigotes were visualized by routine histopathologic exam in 36% of biopsies. Immunohistochemistry stained 17 of 26 lesions (65%) negative by conventional stains. PCR provided the correct diagnosis in 218 cases (58% of the series) negative for Leishmania by other techniques.

Conclusions: Biopsies negative for Leishmania by traditional diagnostic methods that show the histopathologic standard pattern, those with atypical features from patients with clinical suspicion of cutaneous leishmaniasis in endemic areas, should be studied by immunohistochemistry and/or PCR for Leishmania in order to reach the definitive diagnosis.
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http://dx.doi.org/10.1111/cup.13785DOI Listing
November 2020

Latest trends in Leishmania infantum infection in dogs in Spain, Part I: mapped seroprevalence and sand fly distributions.

Parasit Vectors 2020 Apr 21;13(1):204. Epub 2020 Apr 21.

Grupo de Investigación Epicontrol-Carnívoros, Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, Madrid, Spain.

Background: This report describes L. infantum infection seroprevalence in dogs in Spain through data obtained from peer-reviewed literature and a cross-sectional serological survey assessing epidemiological and habitat variables as risk factors for infection. The study also provides preliminary sand fly species distribution data and indicates factors affecting their distribution and density.

Methods: Three different studies were conducted in Spain: (i) a peer-reviewed literature seroprevalence survey (1985-2019); (ii) a cross-sectional serological survey (2011-2016); and (iii) a preliminary entomological survey (2013-2014). In the cross-sectional serological survey, 1739 dogs from 74 different locations including 25 Spanish provinces were tested for L. infantum by indirect immunofluorescence antibody test (IFAT) (antibody titre ≥ 1:100). Seroprevalence of L. infantum infection was analysed by province and bioclimatic zone. Statistics were used to analyse relationships between several dog- and environment-related variables and L. infantum seroprevalence. In parallel, during 2013-2014, sand flies were collected across the Iberian Peninsula and the Balearic Islands using CDC light traps to examine relationships between habitat-related factors and sand fly species densities (number of sand flies per trap per hour).

Results: The literature review revealed that the provinces showing the highest seroprevalence were Balearic Islands (57.1%), Ourense (35.6%), Málaga (34.6%) and Cáceres (34.2%), and those showing the lowest seroprevalence were Vizcaya (0%), Cantabria (2.0%) and Álava (3.3%). In our survey, anti-Leishmania IgG antibodies were detected in 176 of the 1739 dogs rendering a seroprevalence of 10.12%. Percentage seroprevalence distributions significantly varied among bioclimatic belts. Seropositivity for L. infantum was related to size (large breed dogs versus small) and were significantly higher in younger dogs (≤ 1 years-old). In the entomological survey, 676 sand flies of five species were captured: 562 (83.13%) Phlebotomus perniciosus; 64 (9.47%) Sergentomyia minuta; 38 (5.62%) P. ariasi: 6 (0.89%) P. sergenti; and 6 (0.89%) P. papatasi. Phlebotomus perniciosus showed a greater density in the thermo-Mediterranean than in the meso-Mediterranean zone. Densities of S. minuta and P. ariasi were significantly higher in rural habitats.

Conclusions: This updated seroprevalence map of L. infantum infection in dogs in Spain defines non-endemic, hypoendemic, endemic and hyperendemic areas, and confirms P. perniciosus as the most abundant sand fly vector in Spain.
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http://dx.doi.org/10.1186/s13071-020-04081-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7171843PMC
April 2020

Molecular typing reveals the co-existence of two transmission cycles of American cutaneous leishmaniasis in the Andean Region of Venezuela with Lutzomyia migonei as the vector.

Mem Inst Oswaldo Cruz 2018 Dec 6;113(12):e180323. Epub 2018 Dec 6.

Universidad de Carabobo, Facultad de Ciencias de la Salud, Centro Nacional de Referencia de Flebotomos y otros Vectores, Instituto de Investigaciones Biomédicas Dr Francisco J Triana-Alonso, Maracay, Venezuela.

BACKGROUND The transmission routes for American cutaneous leishmaniasis (ACL) are in flux, so studies examining its transmission in humans, mammalian hosts, and sand fly vectors are urgently needed. OBJECTIVES The aim of this work was understand the epidemiological cycles of Leishmania spp., which causes ACL in the Andean Region of Venezuela, by identifying the Leishmania and the sand fly species involved in human and dog infections. METHODS Thirty-one biopsies from patients in Mérida and Táchira states with suspected ACL were studied by both parasitological tests (cultures and hamster inoculation) and a molecular test [Internal transcribed spacer 1 (ITS1) nested polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)]. We also conducted a survey to detect Leishmania infection in dogs (Immunifluorescence antibody test and ITS1 nested PCR-RFLP) and sand flies (ITS1 nested PCR-RFLP) from El Carrizal, a highly endemic focus of ACL in Venezuela. FINDINGS Three different Leishmania species were identified in the clinical samples from humans (Leishmania braziliensis, L. guyanensis, and L. mexicana) and dogs (L. guyanensis and L. mexicana). The predominant sand fly species found were those from the Verrucarum group (infected with L. mexicana) and Lutzomyia migonei (infected with L. guyanensis and L. mexicana). MAIN CONCLUSIONS We show that Lu. migonei may be the putative vector in two ACL epidemiological cycles, involving L. guyanensis and L. mexicana. We also report for the first time the presence of L. guyanensis in domestic animals.
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http://dx.doi.org/10.1590/0074-02760180323DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6282108PMC
December 2018

Cellular Markers of Active Disease and Cure in Different Forms of -Induced Disease.

Front Cell Infect Microbiol 2018 13;8:381. Epub 2018 Nov 13.

WHO Collaborating Centre for Leishmaniasis, National Centre for Microbiology, Instituto de Salud Carlos III, Madrid, Spain.

Increased numbers of peripheral blood mononucleocytes (PBMC) and increased IFN-γ secretion following challenge of blood samples with soluble antigen (SLA), have been proposed as biomarkers of specific cell-mediated immunity, indicating that treatment of visceral leishmaniasis (VL) has been successful. However, infection may manifest as cutaneous leishmaniasis (CL), and less commonly as localized leishmanial lymphadenopathy (LLL) or mucosal leishmaniasis (ML). The present work examines the value of these biomarkers as indicators of cured leishmaniasis presenting in these different forms. Blood samples were collected before and after treatment from patients living in Fuenlabrada (Madrid, Spain), an endemic area recently the center of a leishmaniasis outbreak. All samples were subjected to -specific PCR, serological tests (IFAT and rK39-ICT), and the SLA-cell proliferation assay (SLA-CPA), recording PBMC proliferation and the associated changes in IFN-γ production. Differences in the results recorded for the active and cured conditions were only significant for VL. PCR returned positive results in 67% of patients with active VL and in 3% of those with cured leishmaniasis. Similarly, rK39-ICT returned a positive result in 77% of active VL samples . 52% in cured VL samples, and IFAT in 90% . 56%; in the SLA-CPA, PBMC proliferation was seen in 16% . 90%, and an associated increase in IFN-γ production of 14 and 84%, respectively. The present findings reinforce the idea that PBMC proliferation and increased IFN-γ production in SLA-stimulated PBMC provide biomarkers of clinical cure in VL. Other tests are urgently needed to distinguish between the cured and active forms of the other types of clinical leishmaniasis caused by .
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http://dx.doi.org/10.3389/fcimb.2018.00381DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6243388PMC
September 2019

Genome Dynamics during Environmental Adaptation Reveal Strain-Specific Differences in Gene Copy Number Variation, Karyotype Instability, and Telomeric Amplification.

mBio 2018 11 6;9(6). Epub 2018 Nov 6.

Unité de Parasitologiemoléculaire et Signalisation, Institut Pasteur, Paris, France

Protozoan parasites of the genus adapt to environmental change through chromosome and gene copy number variations. Only little is known about external or intrinsic factors that govern genomic adaptation. Here, by conducting longitudinal genome analyses of 10 new clinical isolates, we uncovered important differences in gene copy number among genetically highly related strains and revealed gain and loss of gene copies as potential drivers of long-term environmental adaptation in the field. In contrast, chromosome rather than gene amplification was associated with short-term environmental adaptation to culture. Karyotypic solutions were highly reproducible but unique for a given strain, suggesting that chromosome amplification is under positive selection and dependent on species- and strain-specific intrinsic factors. We revealed a progressive increase in read depth towards the chromosome ends for various isolates, which may represent a nonclassical mechanism of telomere maintenance that can preserve integrity of chromosome ends during selection for fast growth. Together our data draw a complex picture of genomic adaptation in the field and in culture, which is driven by a combination of intrinsic genetic factors that generate strain-specific phenotypic variations, which are under environmental selection and allow for fitness gain. Protozoan parasites of the genus cause severe human and veterinary diseases worldwide, termed leishmaniases. A hallmark of biology is its capacity to adapt to a variety of unpredictable fluctuations inside its human host, notably pharmacological interventions, thus, causing drug resistance. Here we investigated mechanisms of environmental adaptation using a comparative genomics approach by sequencing 10 new clinical isolates of the , , and complexes that were sampled across eight distinct geographical regions. Our data provide new evidence that parasites adapt to environmental change in the field and in culture through a combination of chromosome and gene amplification that likely causes phenotypic variation and drives parasite fitness gains in response to environmental constraints. This novel form of gene expression regulation through genomic change compensates for the absence of classical transcriptional control in these early-branching eukaryotes and opens new venues for biomarker discovery.
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http://dx.doi.org/10.1128/mBio.01399-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6222132PMC
November 2018

Evaluation of fluorimetry and direct visualization to interpret results of a loop-mediated isothermal amplification kit to detect Leishmania DNA.

Parasit Vectors 2018 04 17;11(1):250. Epub 2018 Apr 17.

WHO Collaborating Centre for Leishmaniasis, National Centre for Microbiology, Instituto de Salud Carlos III, Madrid, Spain.

Background: Nucleic acid amplification tests (NAATs) have proven to be advantageous in the diagnosis of leishmaniases, allowing sensitive diagnosis of: (i) cutaneous leishmaniasis in long duration lesions and (ii) visceral leishmaniasis using a less-invasive sample like peripheral blood, in opposition to tissue aspiration required for parasite demonstration by microscopy. Despite their benefits, the implementation of NAATs for leishmaniasis diagnosis at the point-of-care has not been achieved yet, mostly due to the complexity and logistical issues associated with PCR-based methods.

Methods: In this work, we have evaluated the performance of a ready-to-use loop-mediated isothermal amplification (LAMP) kit using two real time fluorimeters to amplify leishmanial DNA obtained by silica column-based and Boil & Spin protocols.

Results: The different approaches used to run and interpret the LAMP reactions showed a performance equivalent to PCR and real-time PCR, using spiked and clinical samples. The time to positivity obtained with real-time fluorimetry showed an excellent correlation with both Ct values and parasite load from real-time quantitative PCR.

Conclusions: The results obtained open the possibility of using a highly stable, ready-to-use LAMP kit for the accurate diagnosis of leishmaniasis at the point-of-care. Furthermore, the feasibility of relating time to positivity, determined with a portable real-time fluorimeter, with the parasite burden could have a wider application in the management of leishmaniasis, such as in treatment efficacy monitoring or as a pharmacodynamics tool in clinical trials.
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http://dx.doi.org/10.1186/s13071-018-2836-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5905109PMC
April 2018

Validation of rK39 immunochromatographic test and direct agglutination test for the diagnosis of Mediterranean visceral leishmaniasis in Spain.

PLoS Negl Trop Dis 2018 03 1;12(3):e0006277. Epub 2018 Mar 1.

WHO Collaborating Centre for Leishmaniasis, National Centre for Microbiology, Instituto de Salud Carlos III, Majadahonda, Spain.

Background: Visceral leishmaniasis (VL), the most severe form of leishmaniasis, is endemic in Europe with Mediterranean countries reporting endemic status alongside a worrying northward spread. Serological diagnosis, including immunochromatographic test based on the recombinant antigen rK39 (rK39-ICT) and a direct agglutination test (DAT) based on the whole parasite antigen, have been validated in regions with high VL burden, such as eastern Africa and the Indian subcontinent. To date, no studies using a large set of patients have performed an assessment of both methods within Europe.

Methodology/principal Findings: We selected a range of clinical serum samples from patients with confirmed VL (including HIV co-infection), Chagas disease, malaria, other parasitic infections and negative samples (n = 743; years 2009-2015) to test the performance of rK39-ICT rapid test (Kalazar Detect Rapid Test; InBios International, Inc., USA) and DAT (ITM-DAT/VLG; Institute of Tropical Medicine Antwerp, Belgium). An in-house immunofluorescence antibody test (IFAT), was included for comparison. Estimated sensitivities for rK39-ICT and DAT in HIV-negative VL patients were 83.1% [75.1-91.2] and 84.2% [76.3-92.1], respectively. Sensitivity was reduced to 67.3% [52.7-82.0] for rK39 and increased to 91.3% [82.1-100.0] for DAT in HIV/VL co-infected patients. The in-house IFAT was more sensitive in HIV-negative VL patients, 84.2% [76.3-92.1] than in HIV/VL patients, 79.4% [73.3-96.2]. DAT gave 32 false positives in sera from HIV-negative VL suspects, compared to 0 and 2 for rK39 and IFAT, respectively, but correctly detected more HIV/VL patients (42/46) than rK39 (31/46) and IFAT (39/46).

Conclusions/significance: Though rK39-ICT and DAT exhibited acceptable sensitivity and specificity a combination with other tests is required for highly sensitive diagnosis of VL cases in Spain. Important variation in the performance of the tests were seen in patients co-infected with HIV or with other parasitic infections. This study can help inform the choice of serological test to be used when screening or diagnosing VL in a European Mediterranean setting.
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http://dx.doi.org/10.1371/journal.pntd.0006277DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5849364PMC
March 2018

Molecular detection of Leishmania infantum and Leishmania tropica in rodent species from endemic cutaneous leishmaniasis areas in Morocco.

Parasit Vectors 2017 Oct 2;10(1):454. Epub 2017 Oct 2.

Ecology and the Environment Laboratory L2E, (URAC 32, CNRST ERACNERS 06), Faculty of Sciences Semlalia, Cadi Ayyad University, Marrakesh, Morocco.

Background: Leishmaniasis remains a major public health problem in African nations, including Morocco, where little is known about the vertebrate reservoirs involved in the causal parasites' transmission cycles. The present study investigates the role of rodent species as potential reservoirs of Leishmania spp. in central Morocco, where both L. tropica and L. infantum have been reported.

Methods: Rodents were caught from 22 sites in central Morocco, by using Sherman metal traps, and identified morphologically. For each specimen, genomic DNA was extracted from different tissues using the Speed Tools DNA extraction Kit. Then, samples were PCR-analyzed, targeting the SSU rRNA gene to detect Leishmania spp. DNA, followed by amplification of the internal transcribed spacer 1 (ITS1) and its sequencing to identify the species.

Results: A total of 197 rodents belonging to ten species were captured and identified: Rattus rattus (40.61%), Mus musculus (25.38%), Apodemus sylvaticus (8.63%), Mus spretus (7.11%), Meriones shawi (5.58%), Rattus norvegicus (4.57%), Meriones libycus (3.05%), Mastomys erythroleucus (2.03%), Gerbillus campestris (2.03%) and Lemniscomys barbarus (1.01%). Molecular analysis revealed the presence of Leishmania species in 18 specimens: six R. rattus (out of 80 captured; 7.5%), 11 M. musculus (out of 50 captured; 22%), and one R. norvegicus (out of 9 captured; 11.11%).

Conclusions: To the best of our knowledge, L. infantum and L. tropica were identified in rodent species for the first time in Morocco. These findings suggest that rodent species may be involved in L. infantum and L. tropica transmission cycles in this country but that further studies are needed to confirm their role as reservoirs of Leishmania species in Morocco.
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http://dx.doi.org/10.1186/s13071-017-2398-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5625640PMC
October 2017

Comparison of Leishmania typing results obtained from 16 European clinical laboratories in 2014.

Euro Surveill 2016 Dec;21(49)

Hospital for Tropical Diseases, London, United Kingdom.

Leishmaniasis is endemic in southern Europe, and in other European countries cases are diagnosed in travellers who have visited affected areas both within the continent and beyond. Prompt and accurate diagnosis poses a challenge in clinical practice in Europe. Different methods exist for identification of the infecting Leishmania species. Sixteen clinical laboratories in 10 European countries, plus Israel and Turkey, conducted a study to assess their genotyping performance. DNA from 21 promastigote cultures of 13 species was analysed blindly by the routinely used typing method. Five different molecular targets were used, which were analysed with PCR-based methods. Different levels of identification were achieved, and either the Leishmania subgenus, species complex, or actual species were reported. The overall error rate of strains placed in the wrong complex or species was 8.5%. Various reasons for incorrect typing were identified. The study shows there is considerable room for improvement and standardisation of Leishmania typing. The use of well validated standard operating procedures is recommended, covering testing, interpretation, and reporting guidelines. Application of the internal transcribed spacer 1 of the rDNA array should be restricted to Old World samples, while the heat-shock protein 70 gene and the mini-exon can be applied globally.
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http://dx.doi.org/10.2807/1560-7917.ES.2016.21.49.30418DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5291127PMC
December 2016

LEISHMANIA INFANTUM INFECTION IN BENNETT'S WALLABIES (MACROPUS RUFOGRISEUS RUFOGRISEUS) IN A SPANISH WILDLIFE PARK.

J Zoo Wildl Med 2016 Jun;47(2):586-93

Although dogs are the main reservoir for human Leishmania infantum infection, the disease has also been reported in other domestic and wild mammals. In 2011, a fatal case of naturally acquired leishmaniosis was described for the first time in a Bennett's wallaby (Macropus rufogriseus rufogriseus) kept in a wildlife park in Madrid (Spain). This study was designed to assess the infection status of twelve Bennett's wallabies in the same park one year after this incident. Phlebotomus perniciosus, the main vector of L. infantum in Spain, was screened for using sticky and Centers for Disease Control miniature light traps. L. infantum infection was confirmed by molecular diagnosis in four animals, but only one wallaby returned a positive serology result. The presence of the sand fly vector was also confirmed in this habitat. These results suggest that the first case of L. infantum in a wallaby in this park was not an isolated incident and stress the need for further work to determine the role of this parasite in the morbidity and mortality of these macropods. Madrid was recently the scene of an outbreak of human cutaneous and visceral leishmaniosis. Epidemiological studies have so far revealed the widespread presence of L. infantum infection in animals other than the dog. Our ongoing work suggests a risk of L. infantum infection not only among captive animals in Madrid, but also among threatened species or even species that are already extinct in the wild.
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http://dx.doi.org/10.1638/2014-0216.1DOI Listing
June 2016

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

Parasitol Res 2016 Mar 12;115(3):1287-95. Epub 2016 Jan 12.

Neglected Tropical Diseases Programme, Foundation for Innovative New Diagnostics-FIND, Chemin des Mines 9, Campus Biotech, 1202, Geneva, Switzerland.

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.
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http://dx.doi.org/10.1007/s00436-015-4865-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4759228PMC
March 2016

Hemophagocytic lymphohistiocytosis in children with visceral leishmaniasis.

Pediatr Infect Dis J 2015 Jun;34(6):667-9

From the *Pediatric Infectious Diseases and Immunodeficiencies Unit, 12 de Octubre University Hospital, Madrid, Spain; †Pediatric Hematology/Oncology Unit, 12 de Octubre University Hospital, Madrid, Spain; ‡WHO Collaborating Centre for Leishmaniasis, Servicio de Parasitología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain; §Department of Pediatrics, Fuenlabrada University Hospital, Fuenlabrada, Spain; ¶Pediatric Infectious Diseases Unit, Hospital Infantil Universitario Niño Jesús, Madrid, Spain; ‖Department of Pediatrics, Severo Ochoa University Hospital, Leganés, Spain; **Department of Pediatrics, Getafe University Hospital, Getafe, Spain; ††Department of Pediatrics, Móstoles University Hospital, Móstoles, Spain; and ‡‡Department of Pediatrics, Ramón y Cajal University Hospital, Madrid, Spain.

Acquired hemophagocytic lymphohistiocitosis (HLH) syndrome can be a complication of visceral leishmaniasis (VL). A multicenter prospective study was conducted to determine the frequency of HLH syndrome in children with VL. Twenty-four children with VL were identified, and 10 (41%) developed HLH syndrome. VL should be ruled out in all children with HLH criteria living in or coming from endemic areas.
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http://dx.doi.org/10.1097/INF.0000000000000685DOI Listing
June 2015

A randomised, double-blind, controlled efficacy trial of the LiESP/QA-21 vaccine in naïve dogs exposed to two leishmania infantum transmission seasons.

PLoS Negl Trop Dis 2014 Oct 9;8(10):e3213. Epub 2014 Oct 9.

Unit of Vector-borne Diseases and International Health, MIPI Department, Istituto Superiore di Sanità, Rome, Italy.

Canine leishmaniasis is an important zoonosis caused by uncontrolled infection with Leishmania infantum, where an inappropriate immune response is not only responsible for permitting this intracellular parasite to multiply, but is also responsible for several of the pathological processes seen in this disease. Effective canine vaccines are therefore a highly desirable prevention tool. In this randomised, double-blinded, controlled trial, the efficacy of the LiESP/QA-21 vaccine (CaniLeish, Virbac, France) was assessed by exposing 90 naïve dogs to natural L. infantum infection during 2 consecutive transmission seasons, in two highly endemic areas of the Mediterranean basin. Regular PCR, culture, serological and clinical examinations were performed, and the infection/disease status of the dogs was classified at each examination. The vaccine was well-tolerated, and provided a significant reduction in the risk of progressing to uncontrolled active infection (p = 0.025) or symptomatic disease (p = 0.046), with an efficacy of 68.4% and a protection rate of 92.7%. The probability of becoming PCR positive was similar between groups, but the probability of returning to a PCR negative condition was higher in the vaccinated group (p = 0.04). In conclusion, we confirmed the interest of using this vaccine as part of a comprehensive control program for canine leishmaniasis, and validated the use of a protocol based on regular in-depth assessments over time to assess the efficacy of a canine leishmaniasis vaccine.
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http://dx.doi.org/10.1371/journal.pntd.0003213DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4191955PMC
October 2014

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

Parasit Vectors 2014 Mar 24;7:120. Epub 2014 Mar 24.

Departament de Medicina i Cirurgia Animal, Facultat de Veterinaria, Universitat Autònoma de Barcelona, Cerdanyola 08193 Barcelona, Spain.

Unlabelled: :

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.
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http://dx.doi.org/10.1186/1756-3305-7-120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3987822PMC
March 2014

Factors associated with Leishmania asymptomatic infection: results from a cross-sectional survey in highland northern Ethiopia.

PLoS Negl Trop Dis 2012 27;6(9):e1813. Epub 2012 Sep 27.

Centro Nacional de Medicina Tropical, Instituto de Salud Carlos III, Madrid, Spain.

Background: In northern Ethiopia the prevalence of visceral leishmaniasis is steadily rising posing an increasing public health concern. In order to develop effective control strategies on the transmission of the disease it is important to generate knowledge on the epidemiological determinants of the infection.

Methodology/principal Findings: We conducted a cross-sectional survey on children 4-15 years of age using a multi staged stratified cluster sampling on high incidence sub-districts of Amhara regional state, Ethiopia. The survey included a socio-demographic, health and dietary questionnaire, and anthropometric measurements. We performed rK39-ICT and DAT serological tests in order to detect anti-Leishmania antibodies and carried out Leishmanin Skin Test (LST) using L.major antigen. Logistic regression models were used. Of the 565 children surveyed 56 children were positive to infection (9.9%). The individual variables that showed a positive association with infection were increasing age, being male and sleeping outside [adjusted odds ratios (95% CI): 1.15 (1.03, 1.29), 2.56 (1.19, 5.48) and 2.21 (1.03, 4.71) respectively] and in relation to the household: past history of VL in the family, living in a straw roofed house and if the family owned sheep [adjusted OR (95% CI): 2.92 (1.25, 6.81), 2.71 (1.21, 6.07) and 4.16 (1.41, 12.31) respectively].

Conclusions/significance: A behavioural pattern like sleeping outside is determinant in the transmission of the infection in this area. Protective measures should be implemented against this identified risk activity. Results also suggest a geographical clustering and a household focalization of the infection. The behaviour of the vector in the area needs to be clarified in order to establish the role of domestic animals and house materials in the transmission of the infection.
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http://dx.doi.org/10.1371/journal.pntd.0001813DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3459849PMC
January 2013

Low prevalence of Leishmania infection in post-epidemic areas of Libo Kemkem, Ethiopia.

Am J Trop Med Hyg 2012 Jun;86(6):955-8

Centro Nacional de Epidemiología, Instituto de Salud Carlos III, CIBER en Epidemiología y Salud Pública (CIBERESP), Madrid, Spain.

In Libo Kemkem (a district of Amhara region, Ethiopia), no cases of kala-azar had ever been reported until 2005 when an outbreak occurred. Over one-third of those cases were children under 15 years of age. The aim of the present study was to determine the prevalence of Leishmania infection in children aged 4-15 years. A cross-sectional survey was conducted in 2009. Children participating in the survey were selected using a three-stage cluster sampling method. A total of 386 children were included in the study. The overall prevalence of Leishmania infection (direct agglutination test- and/or rK39 immunochromatographic test- and/or leishmanin skin test-positive subjects) in this population was 1.02% (95% confidence interval = 0-4.54), and prevalence was higher in boys and children older than 12 years. Only one case of active disease was encountered. The results suggest that the conditions responsible for the outbreak no longer reign. However, active surveillance remains necessary.
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http://dx.doi.org/10.4269/ajtmh.2012.11-0436DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3366538PMC
June 2012

Usefulness of the rK39-immunochromatographic test, direct agglutination test, and leishmanin skin test for detecting asymptomatic Leishmania infection in children in a new visceral leishmaniasis focus in Amhara State, Ethiopia.

Am J Trop Med Hyg 2012 May;86(5):792-8

Armauer Hansen Research Institute, Addis Ababa, Ethiopia.

In areas where visceral leishmaniasis is anthroponotic, asymptomatically infected patients may play a role in transmission. Additionally, the number of asymptomatic patients in a disease-endemic area will also provide information on transmission dynamics. Libo Kemkem and Fogera districts (Amhara State, Ethiopia) are now considered newly established areas to which visceral leishmaniasis is endemic. In selected villages in these districts, we conducted a study to assess the usefulness of different approaches to estimate the asymptomatic infection rate. Of 605 participants, the rK39 immunochromatographic test was able to detect asymptomatic infection in 1.5% (9 of 605), direct agglutination test in 5.3% (32 of 605), and leishmanin skin test in 5.6% (33 of 589); the combined use of serologic methods and leishmanin skin test enabled detecting asymptomatic infection in 10.1% (61 of 605). We conclude that the best option to detect asymptomatic infection in this new visceral leishmaniasis-endemic focus is the combined use of the direct agglutination test and the leishmanin skin test.
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http://dx.doi.org/10.4269/ajtmh.2012.11-0196DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3335682PMC
May 2012

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.

Parasit Vectors 2012 Apr 28;5:87. Epub 2012 Apr 28.

Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autonoma de Madrid, 28049 Madrid, Spain.

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, even between 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 phylogenetic 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.
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http://dx.doi.org/10.1186/1756-3305-5-87DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3425316PMC
April 2012

Evaluation of two rK39 dipstick tests, direct agglutination test, and indirect fluorescent antibody test for diagnosis of visceral leishmaniasis in a new epidemic site in highland Ethiopia.

Am J Trop Med Hyg 2011 Jan;84(1):102-6

World Health Organization Collaborating Center for Leishmaniasis, National Center of Microbiology, Instituto de Salud Carlos III, Madrid, Spain.

We assessed the performance characteristics of two rK39 immunochromatographic tests, a direct agglutination test (DAT), and an indirect immunofluorescent antibody test (IFAT) in the site of a new epidemic of visceral leishmaniasis (VL) in northwestern Ethiopia. The study population was composed of 179 patients with suspected VL and 67 controls. The sensitivities of Kalazar Detect(®), DiaMed-IT Leish(®), DAT, and IFAT in 35 polymerase chain reaction-confirmed VL cases were 94.3%, 91.4%, 91.4%, and 100%, respectively, and the specificities were 98.5%, 94%, 98.5%, and 98.5%, respectively. In a Bayesian latent class analysis of all 246 specimens, the estimated sensitivities were 90.5%, 89%, 88.8%, and 96% for Kalazar Detect(®), DiaMed-IT Leish(®), DAT, and IFAT, respectively; DAT showed the highest estimated specificity (97.4%). Both rK39 immunochromatographic tests perform as well as DAT, and are suitable for VL diagnosis in first-level health centers in this area of Ethiopia.
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http://dx.doi.org/10.4269/ajtmh.2011.10-0229DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3005501PMC
January 2011

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

Am J Trop Med Hyg 2009 Jul;81(1):34-9

Malaria & Other Vector Borne Diseases, Prevention and Control Program, Ministry of Health, Addis Ababa, 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.
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July 2009

Differentiation and gene flow among European populations of Leishmania infantum MON-1.

PLoS Negl Trop Dis 2008 Jul 9;2(7):e261. Epub 2008 Jul 9.

Institut für Mikrobiologie und Hygiene, Charité Universitätsmedizin Berlin, Berlin, Germany.

Background: Leishmania infantum is the causative agent of visceral and cutaneous leishmaniasis in the Mediterranean region, South America, and China. MON-1 L. infantum is the predominating zymodeme in all endemic regions, both in humans and dogs, the reservoir host. In order to answer important epidemiological questions it is essential to discriminate strains of MON-1.

Methodology/principal Findings: We have used a set of 14 microsatellite markers to analyse 141 strains of L. infantum mainly from Spain, Portugal, and Greece of which 107 strains were typed by MLEE as MON-1. The highly variable microsatellites have the potential to discriminate MON-1 strains from other L. infantum zymodemes and even within MON-1 strains. Model- and distance-based analysis detected a considerable amount of structure within European L. infantum. Two major monophyletic groups-MON-1 and non-MON-1-could be distinguished, with non-MON-1 being more polymorphic. Strains of MON-98, 77, and 108 were always part of the MON-1 group. Among MON-1, three geographically determined and genetically differentiated populations could be identified: (1) Greece; (2) Spain islands-Majorca/Ibiza; (3) mainland Portugal/Spain. All four populations showed a predominantly clonal structure; however, there are indications of occasional recombination events and gene flow even between MON-1 and non-MON-1. Sand fly vectors seem to play an important role in sustaining genetic diversity. No correlation was observed between Leishmania genotypes, host specificity, and clinical manifestation. In the case of relapse/re-infection, only re-infections by a strain with a different MLMT profile can be unequivocally identified, since not all strains have individual MLMT profiles.

Conclusion: In the present study for the first time several key epidemiological questions could be addressed for the MON-1 zymodeme, because of the high discriminatory power of microsatellite markers, thus creating a basis for further epidemiological investigations.
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http://dx.doi.org/10.1371/journal.pntd.0000261DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2438616PMC
July 2008

Immunogenicity of HSP-70, KMP-11 and PFR-2 leishmanial antigens in the experimental model of canine visceral leishmaniasis.

Vaccine 2008 Mar 13;26(15):1902-11. Epub 2008 Feb 13.

WHO Collaborating Centre for Leishmaniasis, Centro Nacional de Microbiología, Inst. de Salud Carlos III, Majadahonda, Spain.

Zoonotic visceral leishmaniasis (ZVL) is a parasitic disease caused by Leishmania infantum/L. chagasi that is emerging as an important medical and veterinary problem. Dogs are the domestic reservoir for this parasite and, therefore, the main target for controlling the transmission to humans. In the present work, we have evaluated the immunogenicity of the Leishmania infantum heat shock protein (HSP)-70, paraflagellar rod protein (PFR)-2 and kinetoplastida membrane protein (KMP)-11 recombinant proteins in dogs experimentally infected with the parasite. We have shown that peripheral blood mononuclear cells (PBMC) from experimentally infected dogs proliferated in response to these recombinant antigens and against the soluble leishmanial antigen (SLA). We have also quantified the mRNA expression level of the cytokines induced in PBMC upon stimulation with the HSP-70, PFR-2 and KMP-11 proteins. These recombinant proteins induced an up-regulation of IFN-gamma. HSP-70 and PFR-2 also produced an increase of the TNF-alpha transcripts abundance. No measurable induction of IL-10 was observed and low levels of IL-4 mRNA were produced in response to the three mentioned recombinant antigens. Serum levels of specific antibodies against HSP-70, PFR-2 and KMP-11 recombinant proteins were also determined in these animals. Our study showed that HSP-70, KMP-11 and PFR-2 proteins are recognized by infected canines. Furthermore, these antigens produce a Th1-type immune response, suggesting that they may be involved in protection. The identification as vaccine candidates of Leishmania antigens that elicit appropriate immune responses in the canine model is a key step in the rational approach to generate a vaccine for canine visceral leishmaniasis.
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http://dx.doi.org/10.1016/j.vaccine.2008.01.042DOI Listing
March 2008

Comparison of new diagnostic tools for management of pediatric Mediterranean visceral leishmaniasis.

J Clin Microbiol 2006 Jul;44(7):2343-7

WHO Collaborating Centre for Leishmaniasis, Servicio de Parasitología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Ctra. Pozuelo-Majadahonda km 2, 28220 Majadahonda, Madrid, Spain.

New techniques are available for diagnosing leishmaniasis, but their efficacy in the identification of pediatric visceral leishmaniasis (VL) has not been compared with that of traditional methods. Blood, bone marrow, and urine samples were taken from 25 children with VL during their first clinical episode, 22 days after the start of treatment with liposomal amphotericin B (3 mg/kg/day on 6 days over a 10-day period), and when a relapse was suspected during follow-up. The results obtained suggest that antibody detection techniques, the antigen detection in urine (KAtex kit), and Leishmania nested PCR (LnPCR) analysis of the blood could be used for diagnosis of the first clinical episode. After treatment, clinical improvement was associated with negativization of Novy-MacNeal-Nicolle culture and microscopy of bone marrow aspirate, KAtex test, and LnPCR blood analysis results. Interestingly, LnPCR analysis of the bone marrow aspirate showed that sterile cure was not achieved in eight patients, two of which suffered a relapse within 10 to 20 weeks. All of the new noninvasive techniques tested showed high diagnostic sensitivity. However, LnPCR analysis of the bone marrow was the most sensitive; this test was able to detect the persistence of parasites and predict potential relapses.
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http://dx.doi.org/10.1128/JCM.02297-05DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1489479PMC
July 2006

Comparison of molecular markers for strain typing of Leishmania infantum.

Infect Genet Evol 2006 Nov 11;6(6):440-6. Epub 2006 Apr 11.

Laboratory of Molecular Parasitology, Instituut voor Tropische Geneeskunde, Nationalestraat 155, B-2000 Antwerpen, Belgium.

The epidemiology of Leishmania infantum, the etiological agent of visceral leishmaniasis, is changing rapidly; hence powerful typing tools are required in order to monitor the parasite populations spreading and to adapt adequate control measures. We compared here the resolving power of four molecular methods at the zymodeme level: PCR-RFLP analysis of kDNA minicircles (kDNAPCR-RFLP) and antigen genes (cysteine proteinase b and major surface protease, cpb- and gp63PCR-RFLP), multilocus microsatellite typing (MLMT) and random amplification of polymorphic DNA (RAPD) were applied to samples of 25 L. infantum MON-1 strains obtained from different hosts (HIV+ patients, HIV- patients and dogs) coming from three Spanish foci: Madrid, Mallorca and Ibiza. While RAPD was not sufficiently resolving, the other three methods allowed genotyping within the zymodeme. KDNAPCR-RFLP and MLMT were the most discriminatory and appeared the most adequate for strain fingerprinting. In an eco-geographical context, cpbPCR-RFLP, MLMT and kDNAPCR-RFLP were all informative: they showed here a similar picture, with the existence of cluster(s) of isolates from the islands and other one(s) of mixed composition (Madrid and the islands). None of the markers revealed an association with the host type or the clinical form. In general, there was a significant correlation between each pair of distances calculated from the cpb, microsatellite and kDNA data, respectively, but visual inspection of the trees revealed a better congruence between cpb and microsatellite trees. The methods used here are complementary and each adapted to answer specific epidemiological questions. Their choice should be the result of a compromise between the required resolving power, the genetic features of the respective markers and the technical aspects.
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http://dx.doi.org/10.1016/j.meegid.2006.02.003DOI Listing
November 2006

Relapses versus reinfections in patients coinfected with Leishmania infantum and human immunodeficiency virus type 1.

J Infect Dis 2002 May 22;185(10):1533-7. Epub 2002 Apr 22.

World Health Organization Collaborating Centre for Leishmaniasis, Servicio de Parasitología, Centro Nacional de Microbiología, Madrid, Spain.

In the Mediterranean basin, Leishmania infantum is a major opportunistic parasite in people with acquired immunodeficiency syndrome (AIDS), and up to 9% of the patients with AIDS suffer from newly acquired or reactivated visceral leishmaniasis. Distinguishing between reinfections and relapses in these patients is important because some apparent treatment failures occur in patients with new rather than reactivated infections. Isoenzyme characterization is limited for use in determining relapsed versus newly acquired leishmaniasis in human immunodeficiency virus (HIV)-infected patients because of the variability of L. infantum and the predominance of the MON-1 zymodeme in people coinfected with HIV. A seminested polymerase chain reaction (PCR) was used to amplify L. infantum minicircle kinetoplast DNA, and, after digestion, the restriction fragment-length polymorphism (RFLP) profiles showed that 3 (7.5%) of 40 patients coinfected with L. infantum and HIV had a new infection, whereas isoenzyme characterization indicated that all 40 patients had infection relapses. These results suggest the utility of this PCR-RFLP analysis in detecting leishmaniasis reinfection in HIV-positive patients.
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http://dx.doi.org/10.1086/340219DOI Listing
May 2002
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