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Typing of Leishmania isolates from vectors and leporids of the Madrid (Spain) outbreak.

ABSTRACT: In 2009, a large outbreak of leishmaniasis, associated with environmental changes, was declared near Madrid (Spain), in which Phlebotomus perniciosus was the vector, whereas the main reservoirs were hares and rabbits. Analysis of isolates from humans, vectors and leporids from the focus identified the Leishmania infantum ITS-Lombardi genotype. However, multilocus enzyme electrophoresis (MLEE), the reference technique for Leishmania typing, and sequencing of the hsp70 gene, a commonly used marker, were not performed. In the present study, 19 isolates from P. perniciosus (n = 11), hares (n = 5) and rabbits (n = 3) from the outbreak area, all characterized as ITS-Lombardi in previous studies, were analysed by MLEE and hsp70 sequencing. The hsp70 results confirmed that all the analysed strains are L. infantum. However, by MLEE, 4 different zymodemes of L. infantum were identified based on variable mobilities of the NP1 enzyme: MON-34 (NP1¹⁰⁰, n = 11), MON-80 (NP1¹³⁰, n = 6), MON-24 (NP1¹⁴⁰, n = 1) and MON-331 (NP1¹⁵⁰, n = 1). The relative frequency of these zymodemes does not correspond to their usual occurrence in Spain. Moreover, MON-34 and MON-80 were found in P. perniciosus, hares and rabbits for the first time. These findings continue to provide insights into the outbreak and call for further studies with a higher number of strains.

SUBMITTER: Fernandez-Arevalo A

PROVIDER: S-EPMC10941034 | biostudies-literature | 2024 Feb

REPOSITORIES: biostudies-literature

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Typing of Leishmania isolates from vectors and leporids of the Madrid (Spain) outbreak.

Fernández-Arévalo Anna A González Estela E Ballart Cristina C Martín-Martín Inés I Tebar Silvia S Muñoz Carme C Jiménez Maribel M Molina Ricardo R Gállego Montserrat M

Parasitology 20231218 2

In 2009, a large outbreak of leishmaniasis, associated with environmental changes, was declared near Madrid (Spain), in which Phlebotomus perniciosus was the vector, whereas the main reservoirs were hares and rabbits. Analysis of isolates from humans, vectors and leporids from the focus identified the Leishmania infantum ITS-Lombardi genotype. However, multilocus enzyme electrophoresis (MLEE), the reference technique for Leishmania typing, and sequencing of the hsp70 ...[more]

PMID: 38105582

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Project description:BackgroundLeishmania infantum is the main etiological agent of both visceral and cutaneous clinical forms of leishmaniasis in the Mediterranean area. Leishmania/HIV coinfection in this area is characterized by a chronic course and frequent recurrences of clinical episodes. The present study using Multilocus Microsatellite Typing (MLMT) analysis, a highly discriminative tool, aimed to genetically characterize L. infantum isolates taken from monitored Leishmania/HIV coinfected patients presenting successive clinical episodes.MethodsIn this study, by the analysis of 20 microsatellite loci, we studied the MLMT profiles of 25 L. infantum isolates from 8 Leishmania/HIV coinfected patients who had experienced several clinical episodes. Two to seven isolates per patient were taken before and after treatment, during clinical and non-clinical episodes, with time intervals of 6 days to 29 months. Genetic diversity, clustering and phenetic analyses were performed.ResultsMLMT enabled us to study the genetic characteristics of the 25 L. infantum isolates, differentiating 18 genotypes, corresponding to a genotypic diversity of 0.72. Fifteen genotypes were unique in the total sample set and only 3 were repeated, 2 of which were detected in different patients. Both clustering and phylogenetic analyses provided insights into the genetic links between the isolates; in five patients isolates showed clear genetic links: either the genotype was exactly the same or only slightly different. In contrast, the isolates of the other three patients were dispersed in different clusters and some could be the result of mixing between populations.ConclusionsOur data indicated a great MLMT variability between isolates from coinfected patients and no predominant genotype was observed. Despite this, almost all clinical episodes could be interpreted as a relapse rather than a reinfection. The results showed that diverse factors like an intrapatient evolution over time or culture bias could influence the parasite population detected in the patient, making it difficult to differentiate between relapse and reinfection.

Project description:Leishmaniasis is a vector-borne disease of worldwide distribution, currently present in 98 countries. Since late 2010, an unusual increase of human visceral and cutaneous leishmaniasis cases has been observed in the south-western Madrid region, totaling more than 600 cases until 2015. Some hosts, such as human, domestic dog and cat, rabbit (Oryctolagus cuniculus), and hare (Lepus granatensis), were found infected by the parasite of this disease in the area. Hares were described as the most important reservoir due to their higher prevalence, capacity to infect the vector, and presence of the same strains as in humans. Various measures were adopted to prevent and control the disease, and since 2013 there was a slight decline in the human sickness. We used a mathematical model to evaluate the efficacy of each measure in reducing the number of infected hosts. We identified in the present model that culling both hares and rabbits, without immediate reposition of the animals, was the best measure adopted, decreasing the proportion of all infected hosts. Particularly, culling hares was more efficacious than culling rabbits to reduce the proportion of infected individuals of all hosts. Likewise, lowering vector contact with hares highly influenced the reduction of the proportion of infected hosts. The reduction of the vector density per host in the park decreased the leishmaniasis incidence of hosts in the park and the urban areas. On the other hand, the reduction of the vector density per host of the urban area (humans, dogs and cats) decreased only their affected population, albeit at a higher proportion. The use of insecticide-impregnated collar and vaccination in dogs affected only the infected dogs' population. The parameters related to the vector contact with dog, cat or human do not present a high impact on the other hosts infected by Leishmania. In conclusion, the efficacy of each control strategy was determined, in order to direct future actions in this and in other similar outbreaks. The present mathematical model was able to reproduce the leishmaniasis dynamics in the Madrid outbreak, providing theoretical support based on successful experiences, such as the reduction of human cases in Southwest Madrid, Spain.

Project description:BACKGROUND:Some wild animals have been recognized as potential reservoirs of Leishmania infantum infection (e.g. carnivores, lagomorphs, rodents, etc.). Leishmania infantum was also identified infecting humans and lagomorphs (i.e. hares and rabbits) over the period of 2009-2016, with the latter acting as the main reservoirs involved in the human leishmaniosis outbreak in Madrid. RESULTS:Two cases of clinical leishmaniosis are reported in orangutans (Pongo pygmaeus pygmaeus) housed at two different centres in Madrid. The first is the case of a 36-year-old male orangutan with severe weight loss and apathy. A complete blood count and biochemical profile revealed anaemia, neutropenia, hypoalbuminaemia and elevated transaminases. Hepato-splenomegaly was also observed. Four months later, due to worsening of clinical signs (mainly bilateral epistaxis), blood and bone marrow samples were collected. Amastigotes of L. infantum were detected in macrophages from a bone marrow aspirate and by specific polymerase chain reaction. The second case was a 34-year-old female orangutan with severe weight loss and apathy and no other apparent clinical signs. A complete blood count and biochemical profile revealed anaemia, pancytopenia and hypoalbuminaemia. Splenomegaly and pericardial effusion were also observed. As leishmaniosis was included in the differential diagnosis, both blood and bone marrow samples were collected. Leishmania infantum infection was confirmed by microscopy, molecular diagnosis and serology (immunofluorescence antibody test). Both animals were treated daily with oral miltefosine for 28 days; allopurinol was also given uninterruptedly in Case 2 for at least 6 months. During follow-up, though good clinical recovery was clear, a lack of parasitological cure was confirmed molecularly in both blood and bone marrow samples from the two orangutans. In both habitats, the presence of the sand fly vector identified as Phlebotomus perniciosus was confirmed. CONCLUSIONS:To our knowledge, this is the first report of L. infantum infection in great apes and in the endangered species P. p. pygmaeus. We are presently looking for L. infantum in other non-human primates living in the same peri-urban areas. If detected, we will examine the impacts of this serious disease on these critically endangered species.

Project description:BackgroundThe flagellate protozoan Giardia duodenalis is an enteric parasite causing human giardiasis, a major gastrointestinal disease of global distribution affecting both developing and industrialised countries. In Spain, sporadic cases of giardiasis have been regularly identified, particularly in pediatric and immigrant populations. However, there is limited information on the genetic variability of circulating G. duodenalis isolates in the country.MethodsIn this longitudinal molecular epidemiological study we report the diversity and frequency of the G. duodenalis assemblages and sub-assemblages identified in 199 stool samples collected from 184 individual with symptoms compatible with giardiasis presenting to two major public hospitals in Madrid for the period December 2013-January 2015. G. duodenalis cysts were initially detected by conventional microscopy and/or immunochomatography on stool samples. Confirmation of the infection was performed by direct immunofluorescence and real-time PCR methods. G. duodenalis assemblages and sub-assemblages were determined by multi-locus genotyping of the glutamate dehydrogenase (GDH) and β-giardin (BG) genes of the parasite. Sociodemographic and clinical features of patients infected with G. duodenalis were also analysed.Principal findingsOf 188 confirmed positive samples from 178 giardiasis cases a total of 124 G. duodenalis isolates were successfully typed at the GDH and/or the BG loci, revealing the presence of sub-assemblages BIV (62.1%), AII (15.3%), BIII (4.0%), AI (0.8%), and AIII (0.8%). Additionally, 6.5% of the isolates were only characterised at the assemblage level, being all of them assigned to assemblage B. Discordant genotype results AII/AIII or BIII/BIV were also observed in 10.5% of DNA isolates. A large number of multi-locus genotypes were identified in G. duodenalis assemblage B, but not assemblage A, isolates at both the GDH and BG loci, confirming the high degree of genetic variability observed in other molecular surveys. BIV was the most prevalent genetic variant of G. duodenalis found in individuals with symptomatic giardiasis in the population under study.ConclusionsHuman giardiasis is an ongoing public health problem in Spain affecting primarily young children under four years of age but also individuals of all age groups. Our typing and sub-typing results demonstrate that assemblage B is the most prevalent G. duodenalis assemblage circulating in patients with clinical giardiasis in Central Spain. Our analyses also revealed a large genetic variability in assemblage B (but not assemblage A) isolates of the parasite, corroborating the information obtained in similar studies in other geographical regions. We believe that molecular data presented here provide epidemiological evidence at the population level in support of the existence of genetic exchange within assemblages of G. duodenalis.

Project description:Leishmaniasis is a debilitating infectious disease that has a variety of clinical forms. In China, visceral leishmaniasis (VL) is the most common symptom, and L. donovani and/or L. infantum are the likely pathogens. In this study, multilocus sequence typing (MLST) of five enzyme-coding genes (fh, g6pdh, icd, mpi, pgd) and two conserved genes (hsp70, lack) was used to investigate the phylogenetic relationships of Chinese Leishmania strains. Concatenated alignment of the nucleotide sequences of the seven genes was analyzed and phylogenetic trees were constructed using neighbor-joining and maximum parsimony models. A set of additional sequences from 25 strains (24 strains belong to the L. donovani complex and one strain belongs to L. gerbilli) were retrieved from GenBank to infer the molecular evolutionary history of Leishmania from China and other endemic areas worldwide. Phylogenetic analyses consolidated Chinese Leishmania into four groups: (i) one clade A population comprised 13 isolates from different foci in China, which were pathogenic to humans and canines. This population was subdivided into two subclades, clade A1 and clade A2, which comprised sister organisms to the remaining members of the worldwide L. donovani complex; (ii) a population in clade B consisted of one reference strain of L. turanica and five Chinese strains from Xinjiang; (iii) clade C (SELF-7 and EJNI-154) formed a population that was closely related to clade B, and both isolates were identified as L. gerbilli; and (iv) the final group, clade D, included Sauroleishmania (LIZRD and KXG-E) and was distinct from the other strains. We hypothesize that the phylogeny of Chinese Leishmania is associated with the geographical origins rather than with the clinical forms (VL or CL) of leishmaniasis. To conclude, this study provides further molecular information on Chinese Leishmania isolates and the Chinese isolates appear to have a more complex evolutionary history than previously thought.

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Typing of Leishmania isolates from vectors and leporids of the Madrid (Spain) outbreak.

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Typing of <i>Leishmania</i> isolates from vectors and leporids of the Madrid (Spain) outbreak.

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