Sofia Cortes

PCR as a rapid and sensitive tool in the diagnosis of human and canine leishmaniasis using Leishmania donovani s.l.-specific kinetoplastid primers

This study was performed in order to test the efficacy of a new polymerase chain reaction (PCR) assay for the diagnosis of both human and canine leishmaniasis caused by Leishmania infantum. The new primers were developed on the basis of a complete DNA sequence of the L. infantum kinetoplast minicircle. Specificity and sensitivity were evaluated by testing bone marrow spots on filter paper and skin biopsy samples, and the PCR results were compared to data from in vitro cultures. Leishmania strains from different foci, as well as other trypanosomatids and opportunistic pathogenic micro-organisms, were also included in this study. The results show that the primers are highly specific, detecting only L. donovani s.l. DNA, and sensitive for the detection of parasite DNA in biological samples from three different geographical regions of Portugal (north, centre and south) and from Brazil.

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

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.

QUANTIFICATION OF LEISHMANIA INFANTUM PARASITES IN TISSUE BIOPSIES BY REAL-TIME POLYMERASE CHAIN REACTION AND POLYMERASE CHAIN REACTION–ENZYME-LINKED IMMUNOSORBENT ASSAY

Most of the experimental studies of Leishmania spp. infection require the determination of the parasite load in different tissues. Quantification of parasites by microscopy is not very sensitive and is time consuming, whereas culture microtitrations remain laborious and can be jeopardized by microbial contamination. The aim of this study was to quantify Leishmania infantum parasites by real-time polymerase chain reaction (PCR) using specific DNA TaqMan® probes and to compare the efficacy of detection of this technique with a PCR–enzyme-linked immunosorbent assay (ELISA). For this purpose, spleen and liver samples from L. infantum–infected mice were collected during a 3-mo longitudinal study and analyzed by both methods. PCR– ELISA failed to quantify Leishmania spp. DNA in samples with very low or very high numbers of parasites. Real-time PCR was more sensitive than PCR–ELISA, detecting down to a single parasite, and enabled the parasite quantification over a wide, 5-log range. In summary, this study developed a method for absolute quantification of L. infantum parasites in infected organs using real-time TaqMan® PCR.

Cell mediated immunity and specific IgG1 and IgG2 antibody response in natural and experimental canine leishmaniosis.

In the present study, we have followed up Leishmania infantum infection in dogs: (1) naturally infected; (2) experimentally infected with amastigotes; and (3) experimentally infected with culture promastigotes. The main objective was to evaluate the differences of the humoral and cellular immune responses of each group. Sera from 12 beagle dogs were analysed for total anti-leishmanial antibodies and IgG1 and IgG2 subclasses by enzyme-linked immunosorbent assay (ELISA). Lymphoproliferation to L. infantum antigen was also performed. All naturally infected animals were symptomatic with a marked humoral response. Dogs inoculated with amastigotes were asymptomotic and presented lower antibody titres than naturally infected. Dogs inoculated with culture promastigotes were asymptomotic with no significant humoral response. Strong proliferative responses to Leishmania antigen was observed in dogs inoculated with promastigotes. In our experimental model, IgG1 antibody levels presented a similar pattern in all infected animals, and IgG2 reactivity was high in naturally infected dogs.

Risk factors for canine leishmaniasis in an endemic Mediterranean region.

Human visceral leishmaniasis is an emergent/re-emergent parasitic zoonotic disease in Europe caused by Leishmania infantum, with domestic dog as its main reservoir host. This study presents the results of a canine epidemiological survey in a mediterranean region where human and canine leishmaniasis (CanL) are endemic - Portugal. The main goal was to identify risk factors, which can be relevant for Leishmania infection control. The national survey was carried out in January 2009 with a screening of 3974 dogs from all 18 districts of mainland Portugal. Direct Agglutination Test was used for the detection of anti-Leishmania antibodies in canine blood. An overall CanL true prevalence of 6.31% was observed. Apparent prevalence at district level ranged from 0.88% to 16.16%, with the highest prevalence in the interior regions. Identified risk factors for positivity were: dogs of 2 years and older (adjusted odds ratio OR=5.39); spending exclusively/most of the time outdoors (OR=2.51); origin from the interior of Portugal in comparison to littoral/coast districts (OR=2.51); not having long fur (OR=2.03); and being pure exotic (OR=1.67). The results confirm the leishmaniasis endemicity in Portugal and the dynamic character of prevalence as new foci emerged and old foci lost their importance. The dogs age, fur size, district and living outdoors as opposed to indoors were more important than dog breeds and insecticide treatment in the transmission of Leishmania infection. The future of CanL prevention and control rely on an integrated approach involving veterinarians, dog owners and health authorities in order to reduce the canine infection risk and consequently, the human zoonotic visceral leishmaniasis.

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.

Infectivity of promastigotes and amastigotes of Leishmania infantum in a canine model for leishmaniosis

Seven dogs experimentally infected with amastigotes or culture promastigotes of Leishmania infantum MON-1 were observed for a period of up to 38 months. The course of infection was monitored by clinical and parasitological examinations, haematological and serum protein analysis, and by anti-leishmania antibody levels. Two of the three amastigote-inoculated dogs developed a symptomatic infection with haematological and protein alterations, and a strong humoral immune response. The third dog was asymptomatic with no haematological or protein alterations and developed a steady humoral response. Four promastigote-inoculated dogs remained asymptomatic throughout the observation period, with only transient antibody responses to leishmanial antigen, and no haematological or protein alterations. The detection of the parasite in biological material obtained at necropsy showed that dogs with no clinical signs or other manifestations of disease may be infected. This indicates that asymptomatic carriers may be present in the canine population, but not identifiable by the usual serological tests, and suggests that epidemiological surveys based on serology may underestimate the prevalence of canine leishmaniosis and the parasite transmission risk.

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.

Detection of Leishmania in immunocompromised patients using peripheral blood spots on filter paper and the polymerase chain reaction

Abstract The aim of the present study was to investigate whether the polymerase chain reaction could be used to detect Leishmania infantum in peripheral blood spots of immunocompromised patients. Although visceral leishmaniasis in immunocompromised individuals is routinely diagnosed by direct microscopy or by culture of biopsy material, both methods have disadvantages. In order to evaluate an alternative method of diagnosis, blood spots were collected on filter paper from 24 immunocompromised individuals with visceral leishmaniasis diagnosed by bone marrow microscopy or culture. The samples were tested using the polymerase chain reaction. Leishmania DNA was detected in 15 of 20 patients who had not yet begun treatment for Leishmania infection and in two of four patients undergoing treatment. Using microscopy or culture, parasites were detected in 5 of 19 and 8 of 19 fresh blood samples, respectively. The results suggest that the polymerase chain reaction can be used with blood spots on filter paper as an initial screening method for immunocompromised patients suspected to have Leishmania infection.

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.