Maria Odete Afonso

Leishmaniasis in Portugal: enzyme polymorphism of Leishmania infantum based on the identification of 213 strains

This study reports isoenzyme polymorphism of Leishmania strains isolated in different regions of Portugal between 1982 and 2005. A total of 213 strains were obtained from cases of visceral and cutaneous leishmaniasis isolated from immunocompetent patients (adults and children) and immunocompromised adults, as well as from dogs and sandflies. Four zymodemes were identified: MON‐1, MON‐24, MON‐29 and MON‐80. Zymodeme MON‐1 was identified in 96.7% of the strains, predominating in both immunocompetent and immunocompromised human patients, and it was the only zymodeme isolated from dogs. Isoenzyme diversity in HIV‐infected patients was higher than in the immunocompetent group, in which all the strains from visceral leishmaniasis were MON‐1. The domestic dog was confirmed as the reservoir host of zoonotic leishmaniasis in Portugal and Phlebotomus perniciosus and Phlebotomus ariasi as vectors. The overall low enzyme polymorphism observed in the Portuguese foci contrasts with the neighbouring foci in Spain.

Predicting the distribution of canine leishmaniasis in western Europe based on environmental variables.

The domestic dog is the reservoir host of Leishmania infantum, the causative agent of zoonotic visceral leishmaniasis endemic in Mediterranean Europe. Targeted control requires predictive risk maps of canine leishmaniasis (CanL), which are now explored. We databased 2187 published and unpublished surveys of CanL in southern Europe. A total of 947 western surveys met inclusion criteria for analysis, including serological identification of infection (504, 369 dogs tested 1971-2006). Seroprevalence was 23 2% overall (median 10%). Logistic regression models within a GIS framework identified the main environmental predictors of CanL seroprevalence in Portugal, Spain, France and Italy, or in France alone. A 10-fold cross-validation approach determined model capacity to predict point-values of seroprevalence and the correct seroprevalence class (<5%, 5-20%, >20%). Both the four-country and France-only models performed reasonably well for predicting correctly the <5% and >20% seroprevalence classes (AUC >0 70). However, the France-only model performed much better for France than the four-country model. The four-country model adequately predicted regions of CanL emergence in northern Italy (<5% seroprevalence). Both models poorly predicted intermediate point seroprevalences (5-20%) within regional foci, because surveys were biased towards known rural foci and Mediterranean bioclimates. Our recommendations for standardizing surveys would permit higher-resolution risk mapping.

Seasonal Dynamics of Phlebotomine Sand Fly Species Proven Vectors of Mediterranean Leishmaniasis Caused by Leishmania infantum.

Background The recent geographical expansion of phlebotomine vectors of Leishmania infantum in the Mediterranean subregion has been attributed to ongoing climate changes. At these latitudes, the activity of sand flies is typically seasonal; because seasonal phenomena are also sensitive to general variations in climate, current phenological data sets can provide a baseline for continuing investigations on sand fly population dynamics that may impact on future scenarios of leishmaniasis transmission. With this aim, in 2011–2013 a consortium of partners from eight Mediterranean countries carried out entomological investigations in sites where L. infantum transmission was recently reported. Methods/Principal Findings A common protocol for sand fly collection included monthly captures by CDC light traps, complemented by sticky traps in most of the sites. Collections were replicated for more than one season in order to reduce the effects of local weather events. In each site, the trapping effort was left unchanged throughout the survey to legitimate inter-seasonal comparisons. Data from 99,000 collected specimens were analyzed, resulting in the description of seasonal dynamics of 56,000 sand flies belonging to L. infantum vector species throughout a wide geographical area, namely P. perniciosus (Portugal, Spain and Italy), P. ariasi (France), P. neglectus (Greece), P. tobbi (Cyprus and Turkey), P. balcanicus and P. kandelakii (Georgia). Time of sand fly appearance/disappearance in collections differed between sites, and seasonal densities showed variations in each site. Significant correlations were found between latitude/mean annual temperature of sites and i) the first month of sand fly appearance, that ranged from early April to the first half of June; ii) the type of density trend, varying from a single peak in July/August to multiple peaks increasing in magnitude from May through September. A 3-modal trend, recorded for P. tobbi in Cyprus, represents a novel finding for a L. infantum vector. Adults ended the activity starting from mid September through November, without significant correlation with latitude/mean annual temperature of sites. The period of potential exposure to L.infantum in the Mediterranean subregion, as inferred by adult densities calculated from 3 years, 37 sites and 6 competent vector species, was associated to a regular bell-shaped density curve having a wide peak center encompassing the July-September period, and falling between early May to late October for more than 99% of values. Apparently no risk for leishmaniasis transmission took place from December through March in the years considered. We found a common pattern of nocturnal females activity, whose density peaked between 11 pm and 2 am. Conclusions Despite annual variations, multiple collections performed over consecutive years provided homogeneous patterns of the potential behavior of leishmaniasis vectors in selected sites, which we propose may represent sentinel areas for future monitoring. In the investigated years, higher potential risk for L. infantum transmission in the Mediterranean was identified in the June-October period (97% relative vector density), however such risk was not equally distributed throughout the region, since density waves of adults occurred earlier and were more frequent in southern territories.

The first detection of Leishmania major in naturally infected Sergentomyia minuta in Portugal

Phlebotomine sandflies of the genus Sergentomyia are widely distributed throughout the Old World. It has been suggested that Sergentomyia spp are involved in the transmission of Leishmania in India and Africa, whereas Phlebotomus spp are thought to be the sole vectors of Leishmania in the Old World. In this study, Leishmania major DNA was detected in one Sergentomyia minuta specimen that was collected in the southern region of Portugal. This study challenges the dogma that Leishmania is exclusively transmitted by species of the genus Phlebotomus in the Old World.

Stray Dogs and Leishmaniasis in Urban Areas, Portugal

To the Editor: In southern Europe, zoonotic visceral leishmaniasis caused by Leishmania infantum used to be considered a rural disease, but it is becoming more prevalent in urban areas. Outbreaks in urban/periurban settings are associated with the urbanization of natural zoonotic foci (1). The presence of a high number of stray dogs in urban/periurban settlements may contribute to the spread and increase of new infections. A canine survey was performed twice a month from December 1, 2002, through December 31, 2003. A total of 374 dogs from urban areas of Lisbon were screened for leishmaniasis. Owners voluntarily brought 277 domestic dogs; 97 stray dogs were from public shelters. Indirect fluorescent assay was used for detection of anti-Leishmania antibodies using a cut-off of 1/64, and popliteal lymph node aspirates for Novy, Nicolle, and MacNeal cultures were tested (2). A high overall prevalence (19.2%) of canine leishmaniasis was found, despite use of conventional tests only. The infection rate would probably have been higher had more sensitive techniques, such as molecular tools, been used. During the 1980s, Abranches et al. (2) performed a similar seroepidemiologic survey and found a prevalence rate of 5.5%. Our results show an increase of canine leishmaniasis cases in Lisbon. In our study, the prevelance of infection in domestic dogs was 18.4% (51/277), and the prevelance in stray dogs was 21.6% (21/97), with no statistical difference (p = 0.48, significance level 95%). These results support the importance of the role of stray dogs in parasite transmission in Lisbon but differ from the 7.8% seroprevalence found in Madrid, where 1,803 stray dogs were studied over a 10-year period (3). However, the sample size and duration of both studies are different. In other urban areas of large European cities and Brazil, the existence of a high canine seroprevalence has shown an urbanization of the parasitosis (4,5). This is associated with an increase in 1-family homes with gardens in the peripheries of cities. Dogs are commonly kept in these gardens, which can provide good habitats for sandflies. On the other hand, the development of suburban areas can also lead to an increase of solid waste and deficient sanitary conditions, thus attracting infected stray dogs. The difference in percentage of domestic dogs (39.21%) and stray dogs (28.57%) that appeared healthy, although infected, was not statistically significant (p = 0.39). The percentage of apparently healthy dogs was lower than expected, as different studies have shown that more than half of the seropositive dogs are asymptomatic (3,6). Moreover, stray dogs are more likely to experience deficient health and nutritional conditions, and we thus expected larger differences between the 2 groups of animals. Of note, asymptomatic infected dogs can be a source of infection to the vectors, although symptomatic dogs are more effective reservoirs (6). Along with the canine survey, from June through September a total of 488 sandflies were collected from 99 biotopes selected from the studied areas where canine or human cases have been diagnosed. The vectors were morphologically identified by standard entomologic keys (7) as follows: 392 (80.33%) Phlebotomus perniciosus, 93 (19.06%) Ph. ariasi, and 3 (0.61%) Ph. sergenti. Phlebotomine density ranged from 0.08 to 7.70 specimens/CDC trap/night. Ph. ariasi was found infected, reflecting an overall infection rate of 1.22% (1/82). In Portugal, Ph. ariasi and Ph. perniciosus are the proven vectors of L. infantum (8). Although phlebotomine infection was proven in Lisbon, it was low when compared with the canine infection rate, highlighting the need for a more extensive vectorial study in these areas. From 2002 through 2006, 20 new cases of kala-azar in immunocompetent patients (16 children and 4 adults) were diagnosed in our laboratory. In spite of the number of new cases being higher in immunocompromised persons, namely, HIV-infected patients, generally only the cases of immunocompetent persons reflect natural zoonotic transmission. Immunocompromised patients can also experience the reactivation of an old latent infection or be infected by zoonotic transmission or by anthroponotic transmission without a vector. Despite some studies that have shown a direct relationship between the prevalence of leishmaniasis in canine and human populations, canine leishmaniasis is much more prevalent and more widely distributed than visceral leishmaniasis, and it does not strongly correlate with the prevalence in humans (6). Moreover, Ph. ariasi and Ph. perniciosus are known to be preferentially zoophilic. In domestic dogs, if the owner takes preventive measures, the infection risk may be reduced. Stray dogs, however, are an easier target for infection and sandfly biting due to precarious physical conditions and outdoor living habits that make canine leishmaniasis control much more difficult. In conclusion, sanitary conditions and animal health must be improved to prevent the transmission risk of leishmaniasis by this group of animals. The absence of surveillance or preventive measures and equilibrium rupture in the ecologic system could contribute to the emergence of human leishmaniasis in urban areas.

Entomological and ecological studies in a new potential zoonotic leishmaniasis focus in Torres Novas municipality, Central Region, Portugal.

In Portugal human and canine leishmaniasis are caused by Leishmania infantum, and Phlebotomus perniciosus and P. ariasi are the proven vectors. Three main foci were identified in eightys decade: Trás-os-Montes and Alto Douro region, Lisbon region and Algarve region, but according to OnLeish observatory data, canine leishmaniasis cases have been reported from several other regions, for which sand fly species and their infection rates are unknown. This study is the first phlebotomine survey in Torres Novas municipality, Santarém District, Portugal. The main objectives were to identify the phlebotomine species, their bioecological aspects, Leishmania infection rate and the risk factors for the presence of phlebotomine species in the municipality. From June to November, 2010, 275 biotopes were surveyed with CDC light-traps. Captures covered the 17 parishes of the municipality and included domestic, peridomestic and sylvatic biotopes. Specimens were identified morphologically and females were used for molecular detection of Leishmania and bloodmeal identification. Simple and multiple logistic regression analysis were used to identify risk factors for phlebotomine presence. Nonparametric tests were used to compare densities of independent groups. A total of 1262 sand flies were captured and identified, and four species detected: P. perniciosus (73.69%), P. ariasi (8.16%), P. sergenti (6.58%) and Sergentomyia minuta (11.57%). In 71.4% localities at least one L. infantum proven vector species was present. Risk factors were identified as: high average temperatures and low relative humidities, sheltered locations and absence of strong wind, presence of pine trees as dominant vegetation, peridomestic biotopes, particularly sheep pens or proximity of sheep, poultry and house martin nests. L. infantum infection rate was 4% for P. ariasi and 0.48% for the total of Larroussius females. P. perniciosus females exhibited an opportunistic behavior, feeding in a wide variety of vertebrate hosts. The high abundance and distribution of proven vector species, together with a canine leishmaniasis seroprevalence of 7.93% in the District, and the capture of a gravid infective sand fly female, suggests that Torres Novas municipality is a potential zoonotic leishmaniasis focus in the country.

Molecular detection of Leishmania DNA and identification of blood meals in wild caught phlebotomine sand flies (Diptera: Psychodidae) from southern Portugal

BackgroundZoonotic visceral leishmaniasis caused by Leishmania infantum which is transmitted by phlebotomine sand flies (Diptera, Psychodidae) is endemic in the Mediterranean basin. The main objectives of this study were to (i) detect Leishmania DNA and (ii) identify blood meal sources in wild caught female sand flies in the zoonotic leishmaniasis region of Algarve, Portugal/Southwestern Europe.MethodsPhlebotomine sand flies were collected using CDC miniature light traps and sticky papers. Sand flies were identified morphologically and tested for Leishmania sp. by PCR using ITS-1 as the target sequence. The source of blood meal of the engorged females was determined using the cyt-b sequence.ResultsOut of the 4,971 (2,584 males and 2,387 females) collected sand flies, Leishmania DNA was detected by PCR in three females (0.13%), specifically in two specimens identified on the basis of morphological features as Sergentomyia minuta and one as Phlebotomus perniciosus. Haematic preferences, as defined by the analysis of cyt-b DNA amplified from the blood-meals detected in the engorged female specimens, showed that P. perniciosus fed on a wide range of domestic animals while human and lizard DNA was detected in engorged S. minuta.ConclusionsThe anthropophilic behavior of S. minuta together with the detection of Leishmania DNA highlights the need to determine the role played by this species in the transmission of Leishmania parasites to humans. In addition, on-going surveillance on Leishmania vectors is crucial as the increased migration and travelling flow elevate the risk of introduction and spread of infections by Leishmania species which are non-endemic.

Leishmania infection and host-blood feeding preferences of phlebotomine sandflies and canine leishmaniasis in an endemic European area, the Algarve Region in Portugal

The Algarve Region (AR) in southern Portugal, which is an international tourist destination, has been considered an endemic region of zoonotic leishmaniasis caused by Leishmania infantum since the 1980s. In the present study, phlebotomine and canine surveys were conducted to identify sandfly blood meal sources and to update the occurrence of Leishmania infection in vectors and dogs. Four sandfly species were captured: Phlebotomus perniciosus, Phlebotomus ariasi, Phlebotomus sergenti and Sergentomyia minuta. In one P. perniciosus female, L. infantum DNA was detected. Blood meal tests showed that this species had no host preferences and was an opportunistic feeder. An overall canine leishmaniasis (CanL) seroprevalence of 16.06% was found; the seroprevalence was 3.88% in dogs housed in kennels and 40.63% in dogs that attended veterinary clinics. The simultaneous occurrence of dogs and P. perniciosus infected with L. infantum in the AR indicates that the region continues to be an endemic area for CanL. Our results reinforce the need for the systematic spatial distribution of phlebotomine populations and their Leishmania infection rates and the need to simultaneously perform pathogen monitoring in both invertebrate and vertebrate hosts to investigate the transmission, distribution and spreading of Leishmania infection.

First molecular detection of Leishmania tarentolae-like DNA in Sergentomyia minuta in Spain

Phlebotomine sand flies (Diptera, Psychodidae) are vectors of multiple Leishmania species, among which Leishmania infantum stands out as a being frequently pathogenic to humans and dogs in Mediterranean countries. In this study, Sergentomyia minuta sand flies were collected using CDC miniature light traps in different 431 biotopes from Southwest Spain. A total of 114 females were tested for the presence of Leishmania DNA by targeting ITS-1 and cyt-B sequences by PCR. Leishmania DNA was detected in one S. minuta. Characterization of the obtained DNA sequences by phylogenetic analyses revealed close relatedness with Leishmania tarentolae Wenyon, 1921 as well as with both human and canine pathogenic strains of Asian origin (China), previously described as Leishmania sp. To our knowledge, this is the first report of phlebotomine sand flies naturally infected with L. tarentolae-like in Spain. The possible infection of sand flies with novel Leishmania species should be taken into consideration in epidemiological studies of vector species in areas where leishmaniosis is endemic.

Exploring the utility of phylogenetic analysis of cytochrome oxidase gene subunit I as a complementary tool to classical taxonomical identification of phlebotomine sand fly species (Diptera, Psychodidae) from southern Europe.

Phlebotomine sand flies (Diptera, Psychodidae) are known to be vectors of several pathogens such as Leishmania and Phlebovirus genera. The identification of phlebotomine sand fly species is currently based on morphological characters, and requires considerable taxonomic expertise and skilfulness, but may be complemented by DNA-based analyses for (i) accurate species identification and (ii) for estimating sand fly diversity. The aim of this study was to evaluate the utility of mitochondrial cytochrome oxidase gene subunit I (cox1) sequence analysis as a complementary tool to classical taxonomical for the identification of the most prevalent phlebotomine sand fly species from southern Europe (i.e. Phlebotomus ariasi, P. perniciosus, P. sergenti and Sergentomyia minuta). Phylogenetic analyses of cox1 sequences allowed conclusive assignment of most of the sand flies into individual species, and revealed the genetic heterogeneity that characterizes some of the identified genetic clusters. Nevertheless, it showed some limitations, as it failed to (i) allocate correctly all of all species of a given subgenus to a single lineage, or (ii) conclusively identify sequences amplified from individuals classified morphologically as P. ariasi. A more extensive analysis of cox1 sequences together with morphometric characterization of specimens from different geographic areas/regions might be useful for the correct assessment of the phylogenetic relationship within the P. ariasi/P. chadlii cluster and/or help to ascertain the usefulness of cox1 for molecular taxonomy of sand flies.