Lack of association between blood-based detection of Trypanosoma cruzi DNA and cardiac involvement in a non-endemic area.

Cases of chronic Chagas disease have been increasing in non-endemic areas due to the growth in immigration. This study examined the association between positive Trypanosoma cruzi-DNA detection in blood by PCR and presence of chagasic cardiac involvement in a cohort of immigrants in a European city. No association was found in this study between the positive T. cruzi blood PCR and cardiac involvement.

Chagas disease in European countries: the challenge of a surveillance system.

A study of aggregate data collected from the literature and official sources was undertaken to estimate expected and observed prevalence of Trypanosoma cruzi infection, annual incidence of congenital transmission and rate of underdiagnosis of Chagas disease among Latin American migrants in the nine European countries with the highest prevalence of Chagas disease. Formal and informal data sources were used to estimate the population from endemic countries resident in Europe in 2009, diagnosed cases of Chagas disease and births from mothers originating from endemic countries. By 2009, 4,290 cases had been diagnosed in Europe, compared with an estimated 68,000 to 122,000 expected cases. The expected prevalence was very high in undocumented migrants (on average 45% of total expected cases) while the observed prevalence rate was 1.3 cases per 1,000 resident migrants from endemic countries. An estimated 20 to 183 babies with congenital Chagas disease are born annually in the study countries. The annual incidence rate of congenital transmission per 1,000 pregnancies in women from endemic countries was between none and three cases. The index of under diagnosis of T. cruzi infection was between 94% and 96%. Chagas disease is a public health challenge in the studied European countries. Urgent measures need to be taken to detect new cases of congenital transmission and take care of the existing cases with a focus on migrants without legal residency permit and potential difficulty accessing care.

Genetic diversity of human zoonotic leishmaniasis in Iberian Peninsula.

Leishmaniasis caused by Leishmania (Leishmania) infantum is a zoonotic disease endemic in South Europe, from Portugal to the Middle East. The aim of the present study was to investigate the genetic diversity of L. infantum parasites in Iberian Peninsula. Twenty-four L. infantum strains isolated from immunocompetent patients with leishmaniasis from several localities of Portugal and Spain were studied. The use of kinetoplast DNA-PCR-restriction fragment length polymorphism as a molecular marker revealed intra-specific variation. No association was found between genotype and clinical form of the disease or patients age group. Two main clusters were identified with this marker: (i) zymodeme MON-1 strains and (ii) non-MON-1 strains. However, no association was found between strains variability and geographical distribution suggesting that parasite populations of different regions in the Iberian Peninsula are homogenous.

Chagas disease in Latin American migrants: a Spanish challenge.

Chagas' disease affects millions in Latin America and is the leading cause of cardiomyopathy and death due to cardiovascular disease in patients aged 30-50 years. As a consequence of immigration it has settled in several European countries, where besides imported cases, autochthonous infections arise through vertical transmission and blood/organ donation. All Latin American immigrants who attended our Unit were screened for T. cruzi infection (ELISA and IFAT ± PCR). An ECG and echocardiogram were requested for all positive patients, and oesophageal manometry, barium swallow and barium enema were requested according to patient symptoms. All patients under 50 years without severe cardiac involvement and who had not received correct treatment previously were treated with benznidazole 5 mg/kg/day for 60 days. Patients were followed-up with serology and PCR 1 month after treatment ended and every 6 months thereafter. A total of 1146 Latin Americans were screened for T. cruzi (357 positive serology results). The typical patient profile was a Bolivian female, of rural origin, in her fourth decade of life, without evidence of visceral involvement. Treatment tolerance was poor, with 29.7% discontinuing treatment due to adverse reactions. Among those with adverse reactions (52%), the most frequent were cutaneous hypersensitivity (68.7%), gastrointestinal upset (20%) and nervous system disturbances (16.2%). T. cruzi infection is no longer limited to Latin America. Poor treatment tolerance can limit current treatment options. More epidemiological data are necessary to estimate the magnitude of a problem of great relevance for public health and health resource planning.

Nodular lymphangitic subcutaneous dissemination after intralesional antimonial treatment for localized cutaneous leishmaniasis.

Human leishmaniases are protozoan diseases with diverse clinical features. They are transmitted by the bites of the Phlebotomus sand fly, and the reservoirs are usually wild and domestic animals, particularly dogs. In Spain, Leishmania infantum is the causative agent of both cutaneous and visceral leishmaniasis. Cutaneous leishmaniasis is a parasitic disease occurring throughout the Americas and in the Old World, particularly the Middle East and North Africa. It is spread by the female sandfly. We report a 33-year-old man who presented with a solitary plaque on the right elbow, which was found to contain Leishmania bodies. He was treated with intralesional meglumine antimoniate, but 2 weeks later, several subcutaneous nodules were noted on the inner right arm. PCR identified the organism as L. infantum. The patient was treated with itraconazole (200 mg/day) for 6 weeks was prescribed, which resulted in marked clinical improvement in the elbow plaque. However, because of the persistence and lack of response of the other lesions, systemic treatment with meglumine antimoniate (20 mg/kg/day) intravenously for 7 days and intramuscularly for 13 days was administered. A progressive improvement in both cutaneous and subcutaneous lesions was achieved, and the lesions resolved completely after 2 months.

Immunization with H1, HASPB1 and MML Leishmania proteins in a vaccine trial against experimental canine leishmaniasis.

The protective capabilities of three Leishmania recombinant proteins - histone 1 (H1) and hydrophilic acylated surface protein B1 (HASPB1) immunized singly, or together as a protein cocktail vaccine with Montanide, and the polyprotein MML immunized with MPL-SE adjuvant - were assessed in beagle dogs. Clinical examination of the dogs was carried out periodically under blinded conditions and the condition of the dogs defined as asymptomatic or symptomatic. At the end of the trial, we were able to confirm that following infection with L. infantum promastigotes, five out of eight dogs immunized with H1 Montanide, and four out of eight dogs immunized with either the combination of HASPB1 with Montanide or the combination of H1+HASPB1 with Montanidetrade mark, remained free of clinical signs, compared with two out of seven dogs immunized with the polyprotein MML and adjuvant MPL-SE, and two out of eight dogs in the control group. The results demonstrate that HASPB1 and H1 antigens in combination with Montanide were able to induce partial protection against canine leishmaniasis, even under extreme experimental challenge conditions.

Genomic organization and expression of the HSP70 locus in New and Old World Leishmania species.

Heat shock is believed to be a developmental inductor of differentiation in Leishmania. Furthermore, heat shock genes are extensively studied as gene models to decipher mechanisms of gene regulation in kinetoplastids. Here, we describe the organization and expression of the HSP70 loci in representative Leishmania species (L. infantum, L. major, L. tropica, L. mexicana, L. amazonensis and L. braziliensis). With the exception of L. braziliensis, the organization of the HSP70 loci was found to be well conserved among the other Leishmania species. Two types of genes, HSP70-I and HSP70-II, were found to be present in these Leishmania species except for L. braziliensis that lacks HSP70-II gene. Polymorphisms in the HSP70 locus allow the differentiation of the Old and New World species within the subgenus Leishmania. A notable discrepancy between our data and those of the L. major genome database in relation to the gene copy number composing the L. major HSP70 locus was revealed. The temperature-dependent accumulation of the HSP70-I mRNAs is also conserved among the different Leishmania species with the exception of L. braziliensis. In spite of these differences, analysis of the HSP70 synthesis indicated that the HSP70 mRNAs are also preferentially translated during heat shock in L. braziliensis.

The biological diagnosis of leishmaniasis in HIV-infected patients.

This review emphasises the particular difficulties encountered in confirming a suspected case of cutaneous or visceral leishmaniasis when that case is co-infected with HIV. HIV infection appears to have a more profound impact on the development of visceral leishmaniasis than on the evolution of the purely cutaneous disease. The various techniques available for immunological, parasitological and molecular diagnosis are presented and evaluated. The value of serodiagnosis for the detection of antileishmanial antibodies is in part dependent on the antigens used. Western blots may have a use not only in diagnosis but also in predicting the cases of HIV infection that are at most risk of developing symptomatic leishmaniasis. The presence of leishmanial parasites may still only be demonstrated incontrovertibly by the microscopical examination of smears or the culture of blood or biopsy samples. The use of cultures not only permits diagnosis but also detailed study of the parasites. The potential use of PCR in diagnosis is explored and related to other possible tests. A recommended, standardized procedure for the diagnosis of leishmaniasis in HIV-infected patients is presented.

A nested polymerase chain reaction (Ln-PCR) for diagnosing and monitoring Leishmania infantum infection in patients co-infected with human immunodeficiency virus.

We investigated a Leishmania-specific nested polymerase chain reaction (Ln-PCR) for the diagnosis and treatment monitoring of L. infantum infections in patients co-infected with human immunodeficiency virus (HIV). Peripheral blood and bone marrow samples from 89 HIV patients in Spain suspected of having leishmaniasis were examined by different diagnostic techniques (Ln-PCR, microscopy, NNN culture and indirect fluorescent antibody test). The sensitivity of Ln-PCR compared with microscopy and culture of bone marrow was 95.45% using blood and 100% when using bone marrow. 38 of these patients with confirmed leishmaniasis were entered in a chemotherapy trial (reported elsewhere), and samples from them were collected before treatment, one month after treatment ended and during follow-up (1-20 months), and examined similarly. Ln-PCR was shown to be a good method for testing efficacy of treatment and for predicting relapses after treatment (relapses were predicted on average 5 months earlier than when using classical diagnostic techniques). We suggest that Ln-PCR (especially using peripheral blood) should be the technique of choice for diagnosis, monitoring the success of treatment, and predicting relapses in patients with HIV and suspected or confirmed L. infantum infection.

Molecular tracking of infections by Leishmania infantum.

Leishmania infantum is a major opportunistic parasite in patients with acquired immune deficiency syndrome and is very variable in these subjects. Isoenzyme characterization is not able to explain this variability, since half of the stocks isolated from patients co-infected with human immunodeficiency virus and Leishmania belong to zymodeme MON-1. Amplification of L. infantum minicircles by the polymerase chain reaction (PCR) and digestion of the amplified product to reveal restriction fragment length polymorphisms (RFLP) has proved very useful in distinguishing between relapses and reinfections in co-infected, treated patients. We have confirmed the existence of a leishmaniasis outbreak among intravenous drug users in north-east Spain, previously detected by isoenzymatic analysis. We have documented persistence of the same strain of Leishmania in 2 treated co-infected patients throughout several years, regardless of the theoretical rapid evolution ascribed to kinetoplast deoxyribonucleic acid minicircle sequences. We suggest using this PCR-RFLP technique to detect reinfections in treated co-infected subjects.

HIV--Leishmania infantum co-infection: humoral and cellular immune responses to the parasite after chemotherapy.

Specific serum antibodies, peripheral blood T-cell subsets, cellular response in vitro to soluble Leishmania antigens, phenotype of stimulated cells, and serum levels of tumour necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta 1 were studied in Spain in 17 patients co-infected with HIV and Leishmania infantum who had been previously treated with pentavalent antimony. Both humoral and cellular responses to Leishmania sp. appeared diminished, 8 out of 17 patients were positive by indirect immunofluorescence, and immunoblotting detected heterogeneous antibody-binding pattern in 11 out of 13 subjects. A blastogenesis test was positive in 4 cases; 2 of them presented proliferation of CD4+ cells while CD8+ cells proliferated in the other 2 patients. Serum levels of TNF-alpha were similar to those observed in patients infected with HIV only, while serum levels of TGF-beta 1 were significantly lower in the co-infected patients. The inability of antibody response to control the parasite and the absence of specific T-cell immunity to Leishmania sp. would explain the high frequency of relapses reported in these patients. The decreased levels of TGF-beta 1 could have an important role in the interaction between the 2 pathogens.

Myocardial and skeletal muscle aging and changes in oxidative stress in relationship to rigorous exercise training.

Cardiac and skeletal muscle are very different functional tissues, and we would expect a variation in the ROS generation, in ageing and rigorous exercise-related in both tissues. We determined TBARS, total SOD, Cu, ZnSOD and MnSOD activities, and the patterns of SOD isoenzymes in skeletal muscle and heart of male Wistar rats, young and old, in rest and after rigorous exercise. There were no differences in the levels of lipoperoxidation in aged rest animals in both tissues, but the level was increased after exhaustion. The level of SOD activities was bigger in the heart than in skeletal muscle. Total SOD and Cu, ZnSOD activities were higher in old rest animals in the skeletal muscle than in young rest rats. This change did not occur in the heart. After rigorous exercise, the level of SOD activities was increased in young rats in both tissues. However, in old exhausted rats, the activities were only elevated in the heart. Different Cu, ZnSOD isoenzyme patterns showed in relation to tissues. In the skeletal muscle in old animals, the Cu, ZnSOD isoenzyme pattern was modified. The rigorous exercise did not change this pattern. The pattern of MnSOD isoenzyme was not varied in either tissue, age nor and exercise.

Dynamics of serum cytokines in patients with visceral leishmaniasis and HIV-1 co-infection.

Serum cytokine levels and peripheral T cell subpopulations of HIV-1-infected patients before, during and after active visceral leishmaniasis (VL) were analysed and compared with appropriate controls. At VL diagnosis, co-infected patients showed higher serum levels of interferon-gamma (IFN-gamma) than matched HIV-1 controls without VL, and lower serum concentrations of IL-10 than non-immunocompromised VL controls. High levels of tumour necrosis factor-alpha (TNF-alpha) and IFN-gamma were present in the sera of HIV-1-infected patients with active VL. TNF-alpha remained elevated after VL recovery. A steady decline in the CD4+ cell count, an increase of serum HIV viraemia and a progressive seroconversion for the HIV-1 p24 antigen was observed during the course of VL disease. Thus, an aberrant activation of the TNF system with possible negative immunological and virological consequences is present in HIV-1-infected patients with VL. A more extensive prospective validation of these findings in a bigger cohort of patients will nevertheless be necessary. The results support the hypothesis that different opportunistic infection agents may trigger the production of proinflammatory cytokines during immunodeficiency, and in this way accelerate the course of HIV-1 disease.

The role of serology in the diagnosis and prognosis of visceral leishmaniasis in patients coinfected with human immunodeficiency virus type-1.

To define the possible role of serology in the diagnosis and prognosis of visceral leishmaniasis (VL) in patients with human immunodeficiency virus type-1 (HIV-1) infection, the dynamics of humoral immune responses was investigated in 20 coinfected patients. Sequential sera obtained before, during, and after VL diagnosis were analyzed by an indirect immunofluorescent antibody test (IFAT), a recombinant ELISA (using the rK39 protein), and immunoblotting. During the active course of the disease, positive results were found by IFAT or ELISA in 22% of the cases and by immunoblotting in 78% of the cases. A great variability in the response was observed during the follow-up with a trend to more positive results near the time of VL diagnosis. Forty-six percent of the patients were positive by IFAT or ELISA on at least one time point before VL and 37.5% were positive during the period following treatment. These results confirm the limited usefulness of the IFAT and ELISA in the diagnosis of VL in coinfected patients and demonstrate their low ability to predict the development or the outcome of disease. In these patients, immunoblotting could be a useful tool for studying the natural course of leishmaniasis, although it has limited value for diagnosis or treatment control.

Leishmania and human immunodeficiency virus coinfection: the first 10 years.

Over 850 Leishmania-human immunodeficiency virus (HIV) coinfection cases have been recorded, the majority in Europe, where 7 to 17% of HIV-positive individuals with fever have amastigotes, suggesting that Leishmania-infected individuals without symptoms will express symptoms of leishmaniasis if they become immunosuppressed. However, there are indirect reasons and statistical data demonstrating that intravenous drug addiction plays a specific role in Leishmania infantum transmission: an anthroponotic cycle complementary to the zoonotic one has been suggested. Due to anergy in patients with coinfection, L. infantum dermotropic zymodemes are isolated from patient viscera and a higher L. infantum phenotypic variability is seen. Moreover, insect trypanosomatids that are currently considered nonpathogenic have been isolated from coinfected patients. HIV infection and Leishmania infection each induce important analogous immunological changes whose effects are multiplied if they occur concomitantly, such as a Th1-to-Th2 response switch; however, the consequences of the viral infection predominate. In fact, a large proportion of coinfected patients have no detectable anti-Leishmania antibodies. The microorganisms share target cells, and it has been demonstrated in vitro how L. infantum induces the expression of latent HIV-1. Bone marrow culture is the most useful diagnostic technique, but it is invasive. Blood smears and culture are good alternatives. PCR, xenodiagnosis, and circulating-antigen detection are available only in specialized laboratories. The relationship with low levels of CD4+ cells conditions the clinical presentation and evolution of disease. Most patients have visceral leishmaniasis, but asymptomatic, cutaneous, mucocutaneous, diffuse cutaneous, and post-kala-azar dermal leishmaniasis can be produced by L. infantum. The digestive and respiratory tracts are frequently parasitized. The course of coinfection is marked by a high relapse rate. There is a lack of randomized prospective treatment trials; therefore, coinfected patients are treated by conventional regimens. Prophylactic therapy is suggested to be helpful in preventing relapses.

Variability of Leishmania (Leishmania) infantum among stocks from immunocompromised, immunocompetent patients and dogs in Spain.

Leishmania (Leishmania) infantum is the causative agent of both the cutaneous and visceral forms of leishmaniasis in southwest Europe; the dog is the main reservoir. In order to identify the L. (L.) infantum zymodemes present in Spain, a total number of 85 Leishmania stocks isolated from dogs (31), HIV-positive patients (46) with visceral or cutaneous leishmaniasis, a patient with visceral leishmaniasis complicating renal transplantation (1) and immunocompetent patients (7) with visceral or cutaneous leishmaniasis, have been characterized by isoenzyme typing. All canine stocks were MON-1, which is the most widespread zymodeme in the Mediterranean area. In immunocompetent patients three zymodemes were found: MON-1 (2), MON-24 (2) and MON-34 (3). Nine different zymodemes were obtained in stocks from HIV co-infected patients, indicating a higher variability of L. (L.) infantum amongst them: MON-1 (in 21 stocks), MON-24 (7), MON-28 (1), MON-29 (3), MON-33 (7), MON-34 (1) and MON-183 (4). Two new zymodemes, MON-198 (1) and MON-199 (1), were described among HIV patients from Spain. The stock from the renal transplanted patient was MON-1. The exclusive presence of certain zymodemes in immunocompromised patients and their absence in typical cases of cutaneous and visceral leishmaniasis and in infected dogs suggests two possibilities: (i) an anthroponotic pattern of leishmaniasis where intravenous drug user-infected patients act as potential reservoir for these new zymodemes. In the latter, syringes could act as the vehicles for infected monocytes; (ii) the cellular immune system could select virulent from non-virulent zymodemes in immunocompetent visceral leishmaniasis patients.

Epidemiology of canine leishmaniasis in the Madrid region, Spain.

A cross-sectional serological survey was carried out in the Madrid Autonomous Region (Comunidad de Madrid) in order to study and describe canine leishmaniasis epidemiology. The presence of leishmaniasis-specific antibodies was ascertained by immunofluorescence testing, 591 dogs were screened, revealing a prevalence of 5.25% (95% confidence interval 7.4-3.6), with no difference being encountered between rural and periurban areas. Age-specific prevalence exhibits a peak at 2-3 years and another at 7-8 years. Incidence or force of infection by occupation is as follows: pet dogs 0.059 (95% confidence interval 0.009-0.108) and working dogs 0.035 (95% confidence interval 0.012-0.057), there being a ratio between infection rates of 1.7, viz., indicating a 70% greater risk of infection among pet than among working dogs. The basic case reproduction number R0 is 1.06, suggesting that very intense control measures would not be needed for a drop in prevalence and incidence of infection to be achieved.