Repetitive elements cause assembly fragmentation in complex eukaryotic genomes, limiting the study of their variability. The genome of Trypanosoma cruzi, the parasite that causes Chagas disease, has a high repetitive content, including multigene families. Although many T. cruzi multigene families encode surface proteins that play pivotal roles in host-parasite interactions, their variability is currently underestimated, as their high repetitive content results in collapsed gene variants. To estimate sequence variability and copy number variation of multigene families, we developed a read-based approach that is independent of gene-specific read mapping and de novo assembly. This methodology was used to estimate the copy number and variability of MASP, TcMUC, and Trans-Sialidase (TS), the three largest T. cruzi multigene families, in 36 strains, including members of all six parasite discrete typing units (DTUs). We found that these three families present a specific pattern of variability and copy number among the distinct parasite DTUs. Inter-DTU hybrid strains presented a higher variability of these families, suggesting that maintaining a larger content of their members could be advantageous. In addition, in a chronic murine model and chronic Chagasic human patients, the immune response was focused on TS antigens, suggesting that targeting TS conserved sequences could be a potential avenue to improve diagnosis and vaccine design against Chagas disease. Finally, the proposed approach can be applied to study multicopy genes in any organism, opening new avenues to access sequence variability in complex genomes. IMPORTANCE Sequences that have several copies in a genome, such as multicopy-gene families, mobile elements, and microsatellites, are among the most challenging genomic segments to study. They are frequently underestimated in genome assemblies, hampering the correct assessment of these important players in genome evolution and adaptation. Here, we developed a new methodology to estimate variability and copy numbers of repetitive genomic regions and employed it to characterize the T. cruzi multigene families MASP, TcMUC, and transsialidase (TS), which are important virulence factors in this parasite. We showed that multigene families vary in sequence and content among the parasite\'s lineages, whereas hybrid strains have a higher sequence variability that could be advantageous to the parasite\'s survivability. By identifying conserved sequences within multigene families, we showed that the mammalian host immune response toward these multigene families is usually focused on the TS multigene family. These TS conserved and immunogenic peptides can be explored in future works as diagnostic targets or vaccine candidates for Chagas disease. Finally, this methodology can be easily applied to any organism of interest, which will aid in our understanding of complex genomic regions.

BACKGROUND: Plasmodium malariae is the most neglected of the six human malaria species and it is still unknown which is the mechanism underlying the long latency of this Plasmodium.
METHODS: A case of PCR-confirmed P. malariae recurrence in a 52-year old Italian man was observed 5 months after a primary attack. In the interval between the two observed episodes of malaria the patient denied any further stay in endemic areas except for a visit to Libya, a country considered malaria-free. Genomic DNA of the P. malariae strain using five microsatellites (PM2, PM9, PM11, PM25, PM34) and the antigen marker of circumsporozoite (csp) was amplified and sequenced. Analysis of polymorphisms of the P. malariae csp central repeat region showed differences between the strains responsible of the first and second episode of malaria. A difference in the allele size was also observed for the sequence analysis of PM2 microsatellites.
CONCLUSIONS: Plasmodium malariae is a challenging human malaria parasite and even with the use of molecular techniques the pathogenesis of recurrent episodes cannot be precisely explained.

Plasmodium vivax infects a hundred million people annually and endangers 40% of the world\'s population. Unlike Plasmodium falciparum, P. vivax parasites can persist as a dormant stage in the liver, known as the hypnozoite, and these dormant forms can cause malaria relapses months or years after the initial mosquito bite. Here we analyze whole genome sequencing data from parasites in the blood of a patient who experienced consecutive P. vivax relapses over 33 months in a non-endemic country. By analyzing patterns of identity, read coverage, and the presence or absence of minor alleles in the initial polyclonal and subsequent monoclonal infections, we show that the parasites in the three infections are likely meiotic siblings. We infer that these siblings are descended from a single tetrad-like form that developed in the infecting mosquito midgut shortly after fertilization. In this natural cross we find the recombination rate for P. vivax to be 10 kb per centimorgan and we further observe areas of disequilibrium surrounding major drug resistance genes. Our data provide new strategies for studying multiclonal infections, which are common in all types of infectious diseases, and for distinguishing P. vivax relapses from reinfections in malaria endemic regions. This work provides a theoretical foundation for studies that aim to determine if new or existing drugs can provide a radical cure of P. vivax malaria.

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We report a 60-year-old woman with toxoplasma encephalopathy. She was being treated with prednisolone and methotrexate for rheumatoid arthritis that was diagnosed at the age of 40. In a preoperative examination of her left fifth finger ganglion, pericardial effusions, cardiomegaly, and a right atrial mass were detected. In addition, brain MRI showed nodular shadows in the right thalamus, bilateral globus pallidus, and left dentate nucleus of the cerebellum. T1 and T2 weighted images showed high intensities within those shadows; however, a T1 gadolinium enhancement image showed no contrast enhancement in the lesions. There were no positive neurological findings. Examination of the cerebrospinal fluid and cultivation tests showed nothing particular. The right atrial mass was subsequently diagnosed as malignant lymphoma and treated with radiation therapy. Toxoplasma gondii antibody titers were increased in both serum and cerebrospinal fluid. Based on IgG avidity index and nested PCR, we diagnosed toxoplasma encephalopathy with chronic T. gondii infection. The T. gondii gene product was also detected in cerebrospinal fluid by nested PCR. We consider that IgG avidity index and nested PCR were useful for the diagnosis of toxoplasma encephalopathy.

In medical sciences, a target is a broad concept to qualify a biological entity and/or a biological phenomenon, on which one aims to act as part of a therapy. It follows that a target can be defined as a phenotype, a biological process, a subcellular organelle, a protein or a protein domain. It also follows that a target cannot be defined independently of the type of intervention one considers implementing. In this brief review, we describe how in silico organization of genomic and post-genomic information of all partners involved in malaria (human patient, Plasmodium parasite and Anopheles vector), complying with knowledge of the disease in etiologic terms, appears as an efficient source of information not only to help selecting but also discarding target candidates. Some limitations in our capacity to explore the stored biological information, due to the current quality of genomic annotation, level of database integration, or to the performances of existing analytic and mining tools, are discussed. In silico strategies to assess the feasibility of bringing a target to a therapeutic development pipeline, in terms of target \"druggability\", are introduced.

This new feature in Parasitology Today will host reports from the laboratories involved in genomics of parasites, be that sequencing, mapping or \'functional genomics\' - the mining and analysis of the sequence datasets, and the development of postgenomics tools to examine gene expression, response to drugs and population variability. It will publicize new technology to wider audiences, let communities of researchers know about novel resources (particularly those available through the World Wide Web) and highlight significant advances in the understanding of parasitic genomes through functional genomics.

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