Comparative genomic analysis of Leishmania (Viannia) peruviana and Leishmania (Viannia) braziliensis

BACKGROUND: The Leishmania (Viannia) braziliensis complex is responsible for most cases of New World tegumentary leishmaniasis. This complex includes two closely related species but with different geographic distribution and disease phenotypes, L. (V.) peruviana and L. (V.) braziliensis. However, the genetic basis of these differences is not well understood and the status of L. (V.) peruviana as distinct species has been questioned by some. Here we sequenced the genomes of two L. (V.) peruviana isolates (LEM1537 and PAB-4377) using Illumina high throughput sequencing and performed comparative analyses against the L. (V.) braziliensis M2904 reference genome. Comparisons were focused on the detection of Single Nucleotide Polymorphisms (SNPs), insertions and deletions (INDELs), aneuploidy and gene copy number variations. RESULTS: We found 94,070 variants shared by both L. (V.) peruviana isolates (144,079 in PAB-4377 and 136,946 in LEM1537) against the L. (V.) braziliensis M2904 reference genome while only 26,853 variants separated both L. (V.) peruviana genomes. Analysis in coding sequences detected 26,750 SNPs and 1,513 indels shared by both L. (V.) peruviana isolates against L. (V.) braziliensis M2904 and revealed two L. (V.) braziliensis pseudogenes that are likely to have coding potential in L. (V.) peruviana. Chromosomal read density and allele frequency profiling showed a heterogeneous pattern of aneuploidy with an overall disomic tendency in both L. (V.) peruviana isolates, in contrast with a trisomic pattern in the L. (V.) braziliensis M2904 reference. Read depth analysis allowed us to detect more than 368 gene expansions and 14 expanded gene arrays in L. (V.) peruviana, and the likely absence of expanded amastin gene arrays. CONCLUSIONS: The greater numbers of interspecific SNP/indel differences between L. (V.) peruviana and L. (V.) braziliensis and the presence of different gene and chromosome copy number variations support the classification of both organisms as closely related but distinct species. The extensive nucleotide polymorphisms and differences in gene and chromosome copy numbers in L. (V.) peruviana suggests the possibility that these may contribute to some of the unique features of its biology, including a lower pathology and lack of mucosal development. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12864-015-1928-z) contains supplementary material, which is available to authorized users

Diversity, distribution and natural Leishmania infection of sand flies from communities along the Interoceanic Highway in the Southeastern Peruvian Amazon

The Peruvian-Brazilian border is a highly endemic tegumentary leishmaniasis region in South America. The interoceanic highway is a commercial route that connects Peru and Brazil through Madre de Dios and has raised concerns about its impact on previously undisturbed areas. In order to assess leishmaniasis transmission risk along this highway, we conducted a surveillance study of the sand fly populations in this area. Sand flies were collected between 2009 and 2010 along transects at 200 m, 600 m and 1000 m from six study sites located along the highway (Iberia, La Novia, Alto Libertad, El Carmen, Florida Baja, Mazuko and Mavila) and an undisturbed area (Malinowski). Collected specimens were identified based on morphology and non-engorged females of each species were pooled and screened by kinetoplast PCR to detect natural Leishmania infections. A total of 9,023 specimens were collected belonging to 54 different Lutzomyia species including the first report of Lu. gantieri in Peru. Four species accounted for 50% of all specimens (Lutzomyia carrerai carrerai, Lu. davisi, Lu. shawi and Lu. richardwardi). El Carmen, Alto Libertad, Florida Baja and Malinowski presented higher Shannon diversity indexes (H = 2.36, 2.30, 2.17 and 2.13, respectively) than the most human disturbed sites of Mazuko and La Novia (H = 1.53 and 1.06, respectively). PCR detected 10 positive pools belonging to Lu. carrerai carrerai, Lu. yuilli yuilli, Lu. hirsuta hirsuta, Lu. (Trichophoromyia) spp., and Lu. (Lutzomyia) spp. Positive pools from 1,000 m transects had higher infectivity rates than those from 600 m and 200 m transects (9/169 = 5.3% vs 0/79 = 0% and 1/127 = 0.8%, p = 0.018). El Carmen, accounted for eight out of ten positives whereas one positive was collected in Florida Baja and Mazuko each. Our study has shown differences in sand fly diversity, abundance and species composition across and within sites. Multiple clustered Lutzomyia pools with natural Leishmania infection suggest a complex, diverse and spotty role in leishmaniasis transmission in Madre de Dios, with increased risk farther from the highway

Comparative genomics of canine-isolated Leishmania (Leishmania) amazonensis from an endemic focus of visceral leishmaniasis in Governador Valadares, southeastern Brazil

Leishmaniasis is a highly diverse group of diseases caused by kinetoplastid of the genus Leishmania. These parasites are taxonomically diverse, with human pathogenic species separated into two subgenera according to their development site inside the alimentary tract of the sand fly insect vector. The disease encompasses a variable spectrum of clinical manifestations with tegumentary or visceral symptoms. Among the causative species in Brazil, Leishmania (Leishmania) amazonensis is an important etiological agent of human cutaneous leishmaniasis that accounts for more than 8% of all cases in endemic regions. L. (L.) amazonensis is generally found in the north and northeast regions of Brazil. Here, we report the first isolation of L. (L.) amazonensis from dogs with clinical manifestations of visceral leishmaniasis in Governador Valadares, an endemic focus in the southeastern Brazilian State of Minas Gerais where L. (L.) infantum is also endemic. These isolates were characterized in terms of SNPs, chromosome and gene copy number variations, confirming that they are closely related to a previously sequenced isolate obtained in 1973 from the typical Northern range of this species. The results presented in this article will increase our knowledge of L. (L.) amazonensis-specific adaptations to infection, parasite survival and the transmission of this Amazonian species in a new endemic area of Brazil

Comparative genomics of canine-isolated Leishmania (Leishmania) amazonensis from an endemic focus of visceral leishmaniasis in Governador Valadares, southeastern Brazil.

Leishmaniasis is a highly diverse group of diseases caused by kinetoplastid of the genus Leishmania. These parasites are taxonomically diverse, with human pathogenic species separated into two subgenera according to their development site inside the alimentary tract of the sand fly insect vector. The disease encompasses a variable spectrum of clinical manifestations with tegumentary or visceral symptoms. Among the causative species in Brazil, Leishmania (Leishmania) amazonensis is an important etiological agent of human cutaneous leishmaniasis that accounts for more than 8% of all cases in endemic regions. L. (L.) amazonensis is generally found in the north and northeast regions of Brazil. Here, we report the first isolation of L. (L.) amazonensis from dogs with clinical manifestations of visceral leishmaniasis in Governador Valadares, an endemic focus in the southeastern Brazilian State of Minas Gerais where L. (L.) infantum is also endemic. These isolates were characterized in terms of SNPs, chromosome and gene copy number variations, confirming that they are closely related to a previously sequenced isolate obtained in 1973 from the typical Northern range of this species. The results presented in this article will increase our knowledge of L. (L.) amazonensis-specific adaptations to infection, parasite survival and the transmission of this Amazonian species in a new endemic area of Brazil

Human migration and the spread of malaria parasites to the New World

We examined the mitogenomes of a large global collection of human malaria parasites to explore how and when Plasmodium falciparum and P. vivax entered the Americas. We found evidence of a significant contribution of African and South Asian lineages to present-day New World malaria parasites with additional P. vivax lineages appearing to originate from Melanesia that were putatively carried by the Australasian peoples who contributed genes to Native Americans. Importantly, mitochondrial lineages of the P. vivax-like species P. simium are shared by platyrrhine monkeys and humans in the Atlantic Forest ecosystem, but not across the Amazon, which most likely resulted from one or a few recent human-to-monkey transfers. While enslaved Africans were likely the main carriers of P. falciparum mitochondrial lineages into the Americas after the conquest, additional parasites carried by Australasian peoples in pre-Columbian times may have contributed to the extensive diversity of extant local populations of P. vivax

Field Evaluation of a Hemozoin-Based Malaria Diagnostic Device in Puerto Lempira, Honduras

The diagnosis of malaria in Honduras is based mainly on microscopic observation of the parasite in thick smears or the detection of parasite antigens through rapid diagnostic tests when microscopy is not available. The specific treatment of the disease depends exclusively on the positive result of one of these tests. Given the low sensitivity of conventional methods, new diagnostic approaches are needed. This study evaluates the in-field performance of a device (Gazelle™) based on the detection of hemozoin. This was a double-blind study evaluating symptomatic individuals with suspected malaria in the department of Gracias a Dios, Honduras, using blood samples collected from 2021 to 2022. The diagnostic performance of Gazelle™ was compared with microscopy and nested 18ssr PCR as references. The sensitivity and specificity of Gazelle™ were 59.7% and 98.6%, respectively, while microscopy had a sensitivity of 64.9% and a specificity of 100%. The kappa index between microscopy and Gazelle™ was 0.9216 using microscopy as a reference. Both methods show similar effectiveness and predictive values. No statistical differences were observed between the results of the Gazelle™ compared to light microscopy (p = 0.6831). The turnaround time was shorter for Gazelle™ than for microscopy, but the cost per sample was slightly higher for Gazelle™. Gazelle™ showed more false-negative cases when infections were caused by Plasmodium falciparum compared to P. vivax. Conclusions: The sensitivity and specificity of Gazelle™ are comparable to microscopy. The simplicity and ease of use of the Gazelle™, the ability to run on batteries, and the immediacy of its results make it a valuable tool for malaria detection in the field. However, further development is required to differentiate Plasmodium species, especially in those regions requiring differentiated treatment

Comparison of real time and malachite-green based loop-mediated isothermal amplification assays for the detection of Plasmodium vivax and P. falciparum.

The current context of malaria elimination requires urgent development and implementation of highly sensitive and specific methods for prompt detection and treatment of malaria parasites. Such methods should overcome current delays in diagnosis, allow the detection of low-density infections and address the difficulties in accessing remote endemic communities. In this study, we assessed the performance of the RealAmp and malachite-green loop mediated isothermal amplification (MG-LAMP) methodologies, using microscopy and conventional nested-PCR as reference techniques. Both LAMP techniques were performed for Plasmodium genus, P. falciparum, and P. vivax identification using 136 whole blood samples collected from three communities located in the Peruvian Amazon basin. Turnaround time and costs of performing the LAMP assays were estimated and compared to that of microscopy and nested-PCR. Using nested-PCR as reference standard, we calculated the sensitivity, specificity and 95% confidence interval (CI) for all methods. RealAmp had a sensitivity of 92% (95% CI: 85-96.5%) and specificity of 100% (95% CI: 89.1-100%) for species detection; sensitivity and specificity of MG-LAMP were 94% (95% CI: 87.5-97.8%) and 100% (89.1-100%), respectively. Whereas microscopy showed 88.1% sensitivity (95% CI: 80.2-93.7%) and 100% specificity (95%: 89.1-100%). The turnaround time and costs of performing the LAMP assays were lower compared to those associated with nested-PCR but higher than those associated with microscopy. The two LAMP assays were shown to be more sensitive and simple to implement than microscopy. Both LAMP methodologies could be used as large-scale screening tests, but the MG-LAMP assay uses a simple, portable heat-block while the RealAmp requires a RealAmp machine or a real-time PCR machine. This makes the MG-LAMP an appropriate choice for malaria surveillance studies in endemic sites. Use of LAMP tests in active case detection of Plasmodium parasites could help to detect positive malaria cases early

Genetic diversity and population structure of Leishmania (Viannia) braziliensis in the Peruvian jungle.

BackgroundHuman cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis is highly prevalent in the Peruvian jungle, where it affects military forces deployed to fight against drug trafficking and civilian people that migrate from the highland to the lowland jungle for economic activities such as mining, agriculture, construction, and chestnut harvest. We explored the genetic diversity and population structure of 124 L. (V.) braziliensis isolates collected from the highland (Junín, Cusco, and Ayacucho) and lowland Peruvian jungle (Loreto, Ucayali, and Madre de Dios). All samples were genotyped using Multilocus Microsatellite Typing (MLMT) of ten highly polymorphic markers.Principal findingsHigh polymorphism and genetic diversity were found in Peruvian isolates of L. (V.) braziliensis. Most markers are not in Hardy-Weinberg equilibrium; this deviation is most likely caused by local inbreeding, as shown by the positive FIS values. Linkage Disequilibrium in subpopulations was not strong, suggesting the reproduction was not strictly clonal. Likewise, for the first time, two genetic clusters of this parasite were determined, distributed in both areas of the Peruvian jungle, which suggested a possible recent colonization event of the highland jungle from the lowland jungle.ConclusionsL. (V.) braziliensis exhibits considerable genetic diversity with two different clusters in the Peruvian jungle. Migration analysis suggested a colonization event between geographical areas of distribution. Although no human migration was observed at the time of sampling, earlier displacement of humans, reservoirs, or vectors could have been responsible for the parasite spread in both regions

The Leishmania metaphylome: a comprehensive survey of Leishmania protein phylogenetic relationships

Background. Leishmaniasis is a neglected parasitic disease with diverse clinical manifestations and a complex epidemiology. It has been shown that its parasite-related traits vary between species and that they modulate infectivity, pathogenicity, and virulence. However, understanding of the species-specific adaptations responsible for these features and their evolutionary background is limited. To improve our knowledge regarding the parasite biology and adaptation mechanisms of different Leishmania species, we conducted a proteome-wide phylogenomic analysis to gain insights into Leishmania evolution./nResults. The analysis of the reconstructed phylomes (totaling 45,918 phylogenies) allowed us to detect genes that are shared in pathogenic Leishmania species, such as calpain-like cysteine peptidases and 3'a2rel-related proteins, or genes that could be associated with visceral or cutaneous development. This analysis also established the phylogenetic relationship of several hypothetical proteins whose roles remain to be characterized. Our findings demonstrated that gene duplication constitutes an important evolutionary force in Leishmania, acting on protein families that mediate host-parasite interactions, such as amastins, GP63 metallopeptidases, cathepsin L-like proteases, and our methods permitted a deeper analysis of their phylogenetic relationships./nConclusions. Our results highlight the importance of proteome wide phylogenetic analyses to detect adaptation and evolutionary processes in different organisms and underscore the need to characterize the role of expanded and species-specific proteins in the context of Leishmania evolution by providing a framework for the phylogenetic relationships of Leishmania proteins.We thank Leszek P. Pryszcz for his assistance with MetaPhOrs. DB group is funded by The National Institute of Science and Technology for Vaccines (Brazil) (MCT/CNPq, grant CNPq 573547/2008-4), Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG, grant # APQ-04073-10, PPM-00219-13) and Coordenação de Aperfeiçoamento de Pessoal de Nivel Superior (CAPES, grant # 051/2013). TG group research is funded in part by a grant from the Spanish ministry of Economy and Competitiveness (BIO2012-37161), a Grant from the Qatar National Research Fund grant (NPRP 5-298-3-086), and a grant from the European Research Council under the European Union's Seventh Framework Programme (FP/2007-2013) / ERC (Grant Agreement n. ERC-2012-StG-310325). GO group was funded by NIH-Fogarty (TW007012), Fundação de Amparo à Pesquisa do Estado de Minas Gerais (REDE-56/11, RED-00014-14) and Coordenação de Aperfeiçoamento de Pessoal de Nivel Superior (309312/2012-4)