Visceral Leishmaniosis caused by Leishmania (L.) mexicana in a Mexican patient with human immunodeficiency virus infection

A 36 year old male was admitted in December 1997 to hospital with afternoon fever, malaise and hepatosplenomegaly. He also had a dry cough, dyspnoea and anaemia. Pneumonia caused by Pneumocystis carinii and human immunodeficiency virus (HIV) infection were documented. The HIV infection was confirmed in 1997 with 290,000 virus copies. The patient had been in the Mexican State of Chiapas which is known to be endemic for visceral leishmaniosis (VL) and localized cutaneous leishmaniosis (LCL). The visceral symptoms were diagnosed as VL and the causal agent was identified as Leishmania (L.) mexicana. Identification of Leishmania was carried out by the analysis of amplified DNA with specific primers belonging to the Leishmania subgenus and by dot blot positive hybridisation of these polymerase chain reaction derived products with kDNA from the L. (L.) mexicana MC strain used as probe. This is the first case in Mexico of VL caused by a species of Leishmania that typically produces a cutaneous disease form.

Optimisation de la cryo microscopie électronique pour les études des minicercles d'ADN

RESUME : Bien que les propriétés physiques de la structure de l'ADN aient été intensivement étudiées pendant plus de 50 ans il y a encore beaucoup de questions importantes qui attendent des réponses. Par exemple, qu'arrive-t-il à la structure de la double hélice d'ADN nue (sans protéines liées) lorsqu'elle est fortement courbée, de la même manière que dans les nucléosomes? Cet ADN nu est-il facilement plié (il reste dans le régime élastique) ou réduit-il la contrainte de flexion en formant des sites hyperflexibles «kinks» (il sort du régime élastique en cassant l'empilement des paires de bases à certains endroits) ? La microscopie électronique peut fournir une réponse à cette question par visualisation directe des minicercles d'ADN de la longueur d'un tour de nucléosome (environ 90 paires de bases). Pour que la réponse soit scientifiquement valide, on doit observer les molécules d'ADN lorsqu'elles sont en suspension dans la solution d'intérêt et sans que des colorations, produits chimiques ou fixatifs n'aient été ajoutés, étant donné que ceux-ci peuvent changer les propriétés de l'ADN. La technique de la cryo-microscopie électronique (cryo-EM) développée par le groupe de Jacques Dubochet au début des années 80, permet la visualisation directe des molécules d'ADN suspendues dans des couche minces vitrifiées de solutions aqueuses. Toutefois, le faible contraste qui caractérise la cryo-EM combinée avec la très petite taille des minicercles d'ADN rendent nécessaire l'optimisation de plusieurs étapes, aussi bien dans la préparation des échantillons que dans le processus d'acquisition d'images afin d'obtenir deux clichés stéréo qui permettent la reconstruction 3-D des minicercles d'ADN. Dans la première partie de ma thèse, je décris l'optimisation de certains paramètres pour la cryoEM et des processus d'acquisition d'image utilisant comme objets de test des plasmides et d'autres molécules d'ADN. Dans la deuxième partie, je .décris comment j'ai construit les minicercles d'ADN de 94 bp et comment j'ai introduit des modifications structurelles comme des coupures ou des lacunes. Dans la troisième partie, je décris l'analyse des reconstructions des rninicercles d'ADN. Cette analyse, appuyée par des tests biochimiques, indique fortement que des molécules d'ADN sont capables de former de petites molécules circulaires de 94 bp sans dépasser les limites d'élasticité, indiquant que les minicercles adoptent une forme circulaire régulière où la flexion est redistribuée le long la molécule. ABSTRACT : Although physical properties of DNA structure have been intensively studied for over 50 years there are still many important questions that need to be answered. For example, what happens to protein-free double-stranded DNA when it is strongly bent, as in DNA forming nucleosomes? Is such protein-free DNA smoothly bent (i.e. it remains within elastic limits of DNA rigidity) or does it release its bending stress by forming sharp kinks (i.e. it exits the elastic regime and breaks the stacking between neighbouring base-pairs in localized regions)? Electron microscopy can provide an answer to this question by directly visualizing DNA minicircles that have the size of nucleosome gyres (ca 90 bp). For the answer to be scientifically valid, one needs to observe DNA molecules while they are still suspended in the solution of interest and no staining chemicals or fixatives have been added since these can change the properties of the DNA. CryoEM techniques developed by Jacques Dubochet's group beginning in the 1980's permit direct visualization of DNA molecules suspended in cryo-vitrified layers of aqueous solutions. However, a relatively weak contrast of cryo-EM preparations combined with the very small size of the DNA minicircles made it necessary to optimize many of the steps and parameters of the cryo-EM specimen preparation and image acquisition processes in order to obtain stereo-pairs of images that permit the 3-D reconstruction of the observed DNA minicircles. In the first part of my thesis I describe the optimization of the cryo-EM preparation and the image acquisition processes using plasmid size DNA molecules as a test object. In the second part, I describe how I formed the 94 by DNA minicircles and how I introduced structural modifications like nicks or gaps. In the third part, I describe the cryo-EM analysis of the constructed DNA minicircles. That analysis, supported by biochemical tests, strongly indicates that DNA minicircles as small as 94 by remain within the elastic limits of DNA structure, i.e. the minicircles adopt a regular circular shape where bending is redistributed along the molecules.

Cotton Rats (Sigmodon hispidus) and Black Rats (Rattus rattus) as Possible Reservoirs of Leishmania spp. in Lara State, Venezuela

A total of 519 wild animals belonging to eleven species were collected during a two year study in a cutaneous leishmaniasis endemic area in Venezuela (La Matica, Lara State). The animals were captured in home-made Tomahawk-like traps baited with maize, bananas or other available local fruits, and parasites were isolated from 27 specimens. Two different species were found naturally infected with flagellates, i.e., cotton rats (Sigmodon hispidus) and black rats (Rattus rattus). Characterization of the parasites using PCR, kDNA restriction pattern and hybridization with species-specific probes revealed the presence of Leishmania (L.) mexicana in three of the black rats and Leishmania (V.) braziliensis in two others. The latter species was also identified in the single positive specimen of S. hispidus. The results suggested both species of animals as possible reservoirs of Leishmania sp.

Frequency of Infection of Lutzomyia Phlebotomines with Leishmania braziliensis in a Brazilian Endemic Area as Assessed by Pinpoint Capture and Polymerase Chain Reaction

Leishmania infected of Lutzomyia spp. are rare in endemic areas. We tested the hypothesis that there is clustering of infected vectors by combining pinpoint capture with sensitive L. braziliensis kDNA minicircle specific PCR/dot blot in an endemic area in the State of Bahia. Thirty out of 335 samples (10 to 20 sand flies/sample; total of 4,027 female sand flies) were positive by PCR analysis and dot blot leading to a underestimated overall rate of 0.4% positive phlebotomines. However, 83.3% of the positive samples were contributed by a single sector out of four sectors of the whole studied area. This resulted in a rate of 1.5% Leishmania positive phlebotomines for this sector, far above rates of other sectors. Incidence of American cutaneous leishmaniasis cases for this sector was about twice that for other sectors. Our results show that there is a non-homogeneous distribution of Leishmania-infected vectors. Such a clustering may have implications in control strategies against leishmaniasis, and reinforces the necessity of understanding the ecological and geographical factors involved in leishmanial transmission.

Schizodeme analysis of Trypanosoma cruzi Colombian strain clones isolated from the acute phase of murine infection

Colombian strain of Trypanosoma cruzi, biodeme Type III (T. cruzi I), has been cloned by micromanipulation at two phases of the acute infection: early (10 days ) and advanced (30 days). Twelve clones were obtained therefrom. Characterization by their biological and biochemical behavior showed an identity among the several clones and their parental strain, albeit with different degrees of virulence. Molecular characterization of the kinetoplast DNA (kDNA) after amplification by polymerase chain reaction revealed identical profiles of the bands from the kDNA minicircle by the analysis of restriction fragment lenght polymorphism for the isolated clones, their parental strain, and to the clones isolated at two different phases of the infection. Results suggest the predominance of a "principal clone", in the composition of the Colombian strain, responsible for the biological and biochemical behavior. However, no relationship was detected between the molecular profile of kDNA and the degree of virulence presented by the several clones.

Hitchhiking Trypanosoma cruzi minicircle DNA affects gene expression in human host cells via LINE-1 retrotransposon

The horizontal transfer of Trypanosoma cruzi mitochondrial minicircle DNA to the genomes of naturally infected humans may play an important role in the pathogenesis of Chagas disease. Minicircle integrations within LINE-1 elements create the potential for foreign DNA mobility within the host genome via the machinery associated with this retrotransposon. Here we document integration of minicircle DNA fragments in clonal human macrophage cell lines and their mobilization over time. The movement of an integration event in a clonal transfected cell line was tracked at three months and three years post-infection. The minicircle sequence integrated into a LINE-1 retrotransposon; one such foreign fragment subsequently relocated to another genomic location in association with associated LINE-1 elements. The p15 locus was altered at three years as a direct effect of minicircle/LINE-1 acquisition, resulting in elimination of p15 mRNA. Here we show for the first time a molecular pathology stemming from mobilization of a kDNA/LINE-1 mutation. These genomic changes and detected transcript variations are consistent with our hypothesis that minicircle integration is a causal component of parasite-independent, autoimmune-driven lesions seen in the heart and other target tissues associated with Chagas disease.

Polymerase chain reaction with two molecular targets in mucosal leishmaniasis' diagnosis: a validation study

We validated the polymerase chain reaction (PCR) with a composite reference standard in 61 patients clinically suspected of having mucosal leishmaniasis, 36 of which were cases and 25 were non-cases according to this reference standard. Patient classification and test application were carried out independently by two blind observers. One pair of primers was used to amplify a fragment of 120 bp in the conserved region of kDNA and another pair was used to amplify the internal transcript spacers (ITS) rDNA. PCR showed 68.6% (95% CI 59.2-72.6) sensitivity and 92% (95% CI 78.9-97.7) specificity; positive likelihood ratio: 8.6 (95% CI 2.8-31.3) and negative likelihood ratio: 0.3 (95% CI 0.3-0.5), when kDNA molecular target was amplified. The test performed better on sensitivity using this target compared to the ITS rDNA molecular target which showed 40% (95% CI 31.5-42.3) sensitivity and 96% (95% CI 84.1-99.3) specificity; positive likelihood ratio: 10 (95% CI 2.0-58.8) and negative likelihood ratio: 0.6 (95% CI 0.6-0.8). The inter-observer agreement was excellent for both tests. Based upon results obtained and due to low performance of conventional methods for diagnosing mucosal leishmaniasis, we consider PCR with kDNA as molecular target is a useful diagnostic test and the ITS rDNA molecular target is useful when the aim is to identify species.

Isolation and molecular identification of Leishmania chagasi from a bat (Carollia perspicillata) in northeastern Venezuela

This report describes the isolation of a Leishmania chagasi strain from a bat (Carollia perspicillata), and its identification using biological methods and molecular characterization. The parasites were isolated in an artificial culture medium from a blood sample extracted from a bat heart. The isolate was then inoculated into the footpads of Balb/c mice, which subsequently developed a typical nodular leishmanial lesion; the parasites were confirmed as Leishmania by smear and histopathology. Molecular characterization of the parasites was performed by polymerase chain reaction with species-specific primers, kDNA restriction pattern following Hae III endonuclease digestion and dot blot hybridization using a kDNA probe. This report demonstrates that bats can be hosts for L. chagasi species and suggests the need for studies to determine whether they may be involved in foci of visceral leishmaniasis.

Trypanosoma cruzi: blood parasitism kinetics and their correlation with heart parasitism intensity during long-term infection of Beagle dogs

The goals of the present study were to evaluate the kinetics of blood parasitism by examination of fresh blood, blood culture (BC) and PCR assays and their correlation with heart parasitism during two years of infection in Beagle dogs inoculated with the Be-78, Y and ABC Trypanosoma cruzi strains. Our results showed that the parasite or its kDNA is easily detected during the acute phase in all infected animals. On the other hand, a reduced number of positive tests were verified during the chronic phase of the infection. The frequency of positive tests was correlated with T. cruzi strain. The percentage of positive BC and blood PCR performed in samples from animals inoculated with Be-78 and ABC strains were similar and significantly larger in relation to animals infected with the Y strain.Comparison of the positivity of PCR tests performed using blood and heart tissue samples obtained two years after infection showed two different patterns associated with the inoculated T. cruzi strain: (1) high PCR positivity for both blood and tissue was observed in animals infected with Be-78 or ABC strains; (2) lower and higher PCR positivity for the blood and tissue, respectively, was detected in animals infected with Y strains. These data suggest that the sensitivity of BC and blood PCR was T. cruzi strain dependent and, in contrast, the heart tissue PCR revealed higher sensitivity regardless of the parasite stock.

Efficacy of benznidazol treatment for asymptomatic chagasic patients from state of Rio Grande do Sul evaluated during a three years follow-up

The efficacy of benznidazol on the treatment of chagasic patients from the state of Rio Grande do Sul was evaluated during a three-year follow-up. A cohort of 80 asymptomatic chronic chagasic patients or blood bank donors (49 male and 31 female) was studied. Their ages varied from 17-42 years, with a mean and a median of 30 and 35 years, respectively. The 80 patients presented positive serology, hemoculture and polymerase chain reaction (PCR). They were treated with 5 mg/Kg benznidazol twice a day for 60 days. Serological, parasitological and PCR methods were used to evaluate response. Serology was performed using commercial ELISA and indirect immunofluorescence (IFI) tests, parasitemia was monitored by hemoculture in LIT medium and PCR with primers S35/S36 was used to amplify a Trypanosoma cruzi 330 bp kDNA repetitive sequence. PCR positivity of 240 seropositive individuals was compared using DNA preparations from whole blood/guanidine EDTA (GE), buffy-coat/GE and frozen buffy-coat. Fifty non-chagasic individuals were used as negative controls. PCR positivity was 86.7% for the frozen buffy-coat, 71.7% for the GE/buffy-coat and 69.2% for the GE/whole blood. The hemocultures became negative just after treatment and remained negative during the three years of follow-up. In the third year after treatment, 9/80 (11.3%) patients presented negative PCR and, from those, four also presented negative serological tests. Furthermore, a reduction in three serological titers was observed in 27/80 (33.8%) of the patients treated. Taken together, the results show that four of the 80 (5.0%) chronic chagasic patients from the state of Rio Grande do Sul were cured after treatment with benznidazol.

Genus-specific kinetoplast-DNA PCR and parasite culture for the diagnosis of localised cutaneous leishmaniasis: applications for clinical trials under field conditions in Brazil

The positivities of two methods for the diagnosis of localised cutaneous leishmaniasis (CL) were estimated in 280 patients enrolled in a clinical trial. The trial was conducted in an endemic area of Leishmania (Viannia) braziliensis and trial participants were patients with skin ulcers and positive leishmanin skin tests. Patients underwent aspirative skin punctures of the ulcerated lesions and lymph nodes for in vitro cultures, which were processed under field conditions at the local health centre. Skin lesion biopsies were tested at a reference laboratory using kinetoplastid DNA (kDNA)-PCR to detect DNA. The median time required to obtain a positive culture from the skin samples was seven days and the contamination rate of the samples was 1.8%. The positivities of the cultures from skin lesions, kDNA-PCR and the combination of the two methods were 78.2% (95% CI: 73-82.6%), 89.3% (95% CI: 85.1-92.4%) and 97.1% (95% CI: 94.5-98.5%). We conclude that parasite culture is a feasible method for the detection of Leishmania in field conditions and that the combination of culture and PCR has a potential role for the diagnosis of CL in candidates for clinical trials.

A century of research: what have we learned about the interaction of Trypanosoma cruzi with host cells?

Since the discovery of Trypanosoma cruzi and the brilliant description of the then-referred to "new tripanosomiasis" by Carlos Chagas 100 years ago, a great deal of scientific effort and curiosity has been devoted to understanding how this parasite invades and colonises mammalian host cells. This is a key step in the survival of the parasite within the vertebrate host, and although much has been learned over this century, differences in strains or isolates used by different laboratories may have led to conclusions that are not as universal as originally interpreted. Molecular genotyping of the CL-Brener clone confirmed a genetic heterogeneity in the parasite that had been detected previously by other techniques, including zymodeme or schizodeme (kDNA) analysis. T. cruzi can be grouped into at least two major phylogenetic lineages: T. cruzi I, mostly associated with the sylvatic cycle and T. cruzi II, linked to human disease; however, a third lineage, T. cruziIII, has also been proposed. Hybrid isolates, such as the CL-Brener clone, which was chosen for sequencing the genome of the parasite (Elias et al. 2005, El Sayed et al. 2005a), have also been identified. The parasite must be able to invade cells in the mammalian host, and many studies have implicated the flagellated trypomastigotes as the main actor in this process. Several surface components of parasites and some of the host cell receptors with which they interact have been described. Herein, we have attempted to identify milestones in the history of understanding T. cruzi- host cell interactions. Different infective forms of T. cruzi have displayed unexpected requirements for the parasite to attach to the host cell, enter it, and translocate between the parasitophorous vacuole to its final cytoplasmic destination. It is noteworthy that some of the mechanisms originally proposed to be broad in function turned out not to be universal, and multiple interactions involving different repertoires of molecules seem to act in concert to give rise to a rather complex interplay of signalling cascades involving both parasite and cellular components.

The biological in vitro effect and selectivity of aromatic dicationic compounds on Trypanosoma cruzi

Trypanosoma cruzi is a parasite that causes Chagas disease, which affects millions of individuals in endemic areas of Latin America. One hundred years after the discovery of Chagas disease, it is still considered a neglected illness because the available drugs are unsatisfactory. Aromatic compounds represent an important class of DNA minor groove-binding ligands that exhibit potent antimicrobial activity. This study focused on the in vitro activity of 10 aromatic dicationic compounds against bloodstream trypomastigotes and intracellular forms of T. cruzi. Our data demonstrated that these compounds display trypanocidal effects against both forms of the parasite and that seven out of the 10 compounds presented higher anti-parasitic activity against intracellular parasites compared with the bloodstream forms. Additional assays to determine the potential toxicity to mammalian cells showed that the majority of the dicationic compounds did not considerably decrease cellular viability. Fluorescent microscopy analysis demonstrated that although all compounds were localised to a greater extent within the kinetoplast than the nucleus, no correlation could be found between compound activity and kDNA accumulation. The present results stimulate further investigations of this class of compounds for the rational design of new chemotherapeutic agents for Chagas disease.

A comparison of molecular markers to detect Lutzomyia longipalpis naturally infected with Leishmania (Leishmania) infantum

The aim of the present study was to detect natural infection by Leishmania (Leishmania) infantum in Lutzomyia longipalpis captured in Barcarena, state of Pará, Brazil, through the use of three primer sets. With this approach, it is unnecessary to previously dissect the sandfly specimens. DNA of 280 Lu. longipalpis female specimens were extracted from the whole insects. PCR primers for kinetoplast minicircle DNA (kDNA), the mini-exon gene and the small subunit ribosomal RNA (SSU-rRNA) gene of Leishmania were used, generating fragments of 400 bp, 780 bp and 603 bp, respectively. Infection by the parasite was found with the kDNA primer in 8.6% of the cases, with the mini-exon gene primer in 7.1% of the cases and with the SSU-rRNA gene primer in 5.3% of the cases. These data show the importance of polymerase chain reaction as a tool for investigating the molecular epidemiology of visceral leishmaniasis by estimating the risk of disease transmission in endemic areas, with the kDNA primer representing the most reliable marker for the parasite.

Evolution of Trypanosoma cruzi: clarifying hybridisations, mitochondrial introgressions and phylogenetic relationships between major lineages

Several different models of Trypanosoma cruzi evolution have been proposed. These models suggest that scarce events of genetic exchange occurred during the evolutionary history of this parasite. In addition, the debate has focused on the existence of one or two hybridisation events during the evolution of T. cruzi lineages. Here, we reviewed the literature and analysed available sequence data to clarify the phylogenetic relationships among these different lineages. We observed that TcI, TcIII and TcIV form a monophyletic group and that TcIII and TcIV are not, as previously suggested, TcI-TcII hybrids. Particularly, TcI and TcIII are sister groups that diverged around the same time that a widely distributed TcIV split into two clades (TcIVS and TcIVN). In addition, we collected evidence that TcIII received TcIVSkDNA by introgression on several occasions. Different demographic hypotheses (surfing and asymmetrical introgression) may explain the origin and expansion of the TcIII group. Considering these hypotheses, genetic exchange should have been relatively frequent between TcIII and TcIVS in the geographic area in which their distributions overlapped. In addition, our results support the hypothesis that two independent hybridisation events gave rise to TcV and TcVI. Consequently, TcIVS kDNA was first transferred to TcIII and later to TcV and TcVI in TcII/TcIII hybridisation events.