Translational reprogramming as a driver of antimony-drug resistance in Leishmania.

Leishmania is a unicellular protozoan that has a limited transcriptional control and mostly uses post-transcriptional regulation of gene expression, although the molecular mechanisms of the process are still poorly understood. Treatments of leishmaniasis, pathologies associated with Leishmania infections, are limited due to drug resistance. Here, we report dramatic differences in mRNA translation in antimony drug-resistant and sensitive strains at the full translatome level. The major differences (2431 differentially translated transcripts) were demonstrated in the absence of the drug pressure supporting that complex preemptive adaptations are needed to efficiently compensate for the loss of biological fitness once they are exposed to the antimony. In contrast, drug-resistant parasites exposed to antimony activated a highly selective translation of only 156 transcripts. This selective mRNA translation is associated with surface protein rearrangement, optimized energy metabolism, amastins upregulation, and improved antioxidant response. We propose a novel model that establishes translational control as a major driver of antimony-resistant phenotypes in Leishmania.

Retraction: Rendon-Marin et al. Canine Morbillivirus from Colombian Lineage Exhibits In Silico and In Vitro Potential to Infect Human Cells. Pathogens 2021, 10, 1199.

The authors retract the article "Canine Morbillivirus from Colombian Lineage Exhibits In Silico and In Vitro Potential to Infect Human Cells" [...].

Farm management practices, biosecurity and influenza a virus detection in swine farms: a comprehensive study in colombia.

Biosecurity protocols (BP) and good management practices are key to reduce the risk of introduction and transmission of infectious diseases into the pig farms. In this observational cross-sectional study, survey data were collected from 176 pig farms with inventories over 100 sows in Colombia. We analyzed a complex survey dataset to explore the structure and identify clustering patterns using Multiple Correspondence Analysis (MCA) of swine farms in Colombia, and estimated its association with Influenza A virus detection. Two principal dimensions contributed to 27.6% of the dataset variation. Farms with highest contribution to dimension 1 were larger farrow-to-finish farms, using self-replacement of gilts and implementing most of the measures evaluated. In contrast, farms with highest contribution to dimension 2 were medium to large farrow-to-finish farms, but implemented biosecurity in a lower degree. Additionally, two farm clusters were identified by Hierarchical Cluster Analysis (HCA), and the odds of influenza A virus detection was statistically different between clusters (OR 7.29, CI: 1.7,66, p = < 0.01). Moreover, after logistic regression analysis, three important variables were associated with higher odds of influenza detection: (1) "location in an area with a high density of pigs", (2) "farm size", and (3) "after cleaning and disinfecting, the facilities are allowed to dry before use". Our results revealed two clustering patterns of swine farms. This systematic analysis of complex survey data identified relationships between biosecurity, husbandry practices and influenza status. This approach helped to identify gaps on biosecurity and key elements for designing successful strategies to prevent and control swine respiratory diseases in the swine industry.

Electrochemical genosensor for the specific detection of SARS-CoV-2.

Infection caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is responsible for the Coronavirus disease (COVID-19) and the current pandemic. Its mortality rate increases, demonstrating the imperative need for acute and rapid diagnostic tools as an alternative to current serological tests and molecular techniques. Features of electrochemical genosensor devices make them amenable for fast and accurate testing closer to the patient. This work reports on a specific electrochemical genosensor for SARS-CoV-2 detection and discrimination against homologous respiratory viruses. The electrochemical biosensor was assembled by immobilizing thiolated capture probes on top of maleimide-coated magnetic particles, followed by specific target hybridization between the capture and biotinylated signaling probes in a sandwich-type manner. The probes were rigorously designed bioinformatically and tested in vitro. Enzymatic complexes based on streptavidin-horseradish peroxidase linked the biotinylated signaling probe to render the biosensor electrochemical response. The genosensor showed to reach a sensitivity of 174.4 µA fM-1 and a limit of detection of 807 fM when using streptavidin poly-HRP20 enzymatic complex, detected SARS-CoV-2 specifically and discriminated it against homologous viruses in spiked samples and samples from SARS-CoV-2 cell cultures, a step forward to detect SARS-CoV-2 closer to the patient as a promising way for diagnosis and surveillance of COVID-19.

Differential detection of zika virus based on PCR.

Tropical countries are highly prone to infectious diseases such as the one caused by zika virus. Infection by zika is clinically and epidemiologically highly relevant. For example, when women are infected by zika during the first trimester of pregnancy, the child incurs a high risk of microcephaly and acute neurological syndromes. In adults, the virus is associated with the Guillain-Barré syndrome and other disorders. The worldwide emergency caused by zika in 2013/14 demonstrated the need for rapid and accurate diagnostic tools for the virus. Current diagnostic methods include virus isolation, serological tests, and molecular assays. However, virus isolation requires labor-intensive and time-consuming cell culture; serological detection suffers from cross-reactivity caused by previous exposure to homologous arboviruses that cause symptoms like those caused by zika, while molecular tools commonly are not designed for differential zika detection. This work reports on developing a specific molecular detection method based on phylogenetically conserved primers designed for the specific diagnosis of the zika virus. The zika primers were systematically selected through a rigorous bioinformatic analysis and demonstrated the capability to be highly specific. We tested our primers on synthetic DNA, cell cultures and samples from patients infected with zika, dengue and chikungunya and found that they detected zika with specificity high enough for differential virus diagnosis.

Filling the gaps in Leishmania naiffi and Leishmania guyanensis genome plasticity.

Insufficient and irregular data reports on Leishmaniasis, issuing from the developing world, have left much to be desired in terms of understanding the molecular signatures producing distinct infectious phenotypes of the disease. Herein, we report on the complete genome sequencing of Leishmania naiffi and Leishmania guyanensis, sampled from patients in regions of Colombia and Venezuela. In this study, the isolates of cutaneous lesions from both species presented limited structural variation at the chromosomal level, low gene copy number variation, and high genetic heterogeneity. We compared these sequences to the reference genomes hitherto related from Brazil and French Guyana. Although of the same species, we note a consequential genomic disparity between the Venezuelan and French Guyanese isolates of L. guyanensis. Although less significant on the global schema of cutaneous and mucosal disease, such genomic studies of L. naiffi and L. guyanensis substantiate the gaps in understanding of the molecular architecture and multivariate clinical pictures of Leishmaniasis, on an international scale.

Prediction of potential cysteine synthase inhibitors of Leishmania braziliensis and Leishmania major parasites by computational screening.

Leishmaniasis is a neglected tropical disease considered a public health problem that requires innovative strategies for its chemotherapeutic control. In the present investigation, a molecular docking approach was carried out using the protein cysteine synthase (CS) of Leishmania braziliensis (CSLb) and Leishmania major (CSLm) parasites to identify new compounds as potential candidates for the development of selective leishmaniasis therapy. CS protein sequence similarity, active site, structural modeling, molecular docking, and ADMET properties of compounds were analyzed using bioinformatics tools. Molecular docking analyses identified 1000 ligands with highly promising binding affinity scores for both CS proteins. A total of 182 compounds for CSLb and 173 for CSLm were selected for more detailed characterization based on the binding energy and frequency values and ADMET properties. Based on Principal Component Analysis (PCA) and K-means clusterization for both CS proteins, we classified compounds into 5 clusters for CSLb and 7 for CSLm, thus providing an excellent starting point for verification of enzyme inhibition in in vitro studies. We found the ZINC16524774 compound predicted to have a high affinity and stability for both CSLb and CSLm proteins, which was also evaluated through molecular dynamics simulations. Compounds within each of the five clusters also displayed pharmacological and structural properties that make them attractive drug candidates for the development of selective cutaneous leishmaniasis chemotherapy.

Is It Possible to Differentiate Pneumocystis jirovecii Pneumonia and Colonization in the Immunocompromised Patients with Pneumonia?

Respiratory sample staining is a standard tool used to diagnose Pneumocystis jirovecii pneumonia (PjP). Although molecular tests are more sensitive, their interpretation can be difficult due to the potential of colonization. We aimed to validate a Pneumocystis jirovecii (Pj) real-time PCR (qPCR) assay in bronchoscopic bronchoalveolar lavage (BAL) and oropharyngeal washes (OW). We included 158 immunosuppressed patients with pneumonia, 35 lung cancer patients who underwent BAL, and 20 healthy individuals. We used a SYBR green qPCR assay to look for a 103 bp fragment of the Pj mtLSU rRNA gene in BAL and OW. We calculated the qPCR cut-off as well as the analytical and diagnostic characteristics. The qPCR was positive in 67.8% of BAL samples from the immunocompromised patients. The established cut-off for discriminating between disease and colonization was Ct 24.53 for BAL samples. In the immunosuppressed group, qPCR detected all 25 microscopy-positive PjP cases, plus three additional cases. Pj colonization in the immunocompromised group was 66.2%, while in the cancer group, colonization rates were 48%. qPCR was ineffective at diagnosing PjP in the OW samples. This new qPCR allowed for reliable diagnosis of PjP, and differentiation between PjP disease and colonization in BAL of immunocompromised patients with pneumonia.

Canine Morbillivirus from Colombian Lineage Exhibits In Silico and In Vitro Potential to Infect Human Cells.

Canine morbillivirus (CDV) is a viral agent that infects domestic dogs and a vast array of wildlife species. It belongs to the Paramyxoviridae family, genus Morbillivirus, which is shared with the Measles virus (MeV). Both viruses employ orthologous cellular receptors, SLAM in mononuclear cells and Nectin-4 in epithelial cells, to enter the cells. Although CDV and MeV hemagglutinin (H) have similar functions in viral pathogenesis and cell tropism, the potential interaction of CDV-H protein with human cellular receptors is still uncertain. Considering that CDV is classified as a multi-host pathogen, the potential risk of CDV transmission to humans has not been fully discarded. In this study, we aimed to evaluate both in silico and in vitro, whether there is a cross-species transmission potential from CDV to humans. To accomplish this, the CDV-H protein belonging to the Colombian lineage was modelled. After model validations, molecular docking and molecular dynamics simulations were carried out between Colombian CDV-H protein and canine and human cellular receptors to determine different aspects of the protein-protein interactions. Moreover, cell lines expressing orthologous cellular receptors, with both reference and wild-type CDV strains, were conducted to determine the CDV cross-species transmission potential from an in vitro model. This in silico and in vitro approach suggests the possibility that CDV interacts with ortholog human SLAM (hSLAM) and human Nectin-4 receptors to infect human cell lines, which could imply a potential cross-species transmission of CDV from dogs to humans.

Revisiting the heterogeneous global genomic population structure of Leishmania infantum.

Leishmania infantum is the main causative agent responsible for visceral leishmaniasis (VL), a disease with global distribution. The genomic structure and genetic variation of this species have been widely studied in different parts of the world. However, in some countries, this information is still yet unknown, as is the genomic behaviour of the main antigens used in VL diagnosis (rK39 and rK28), which have demonstrated variable sensitivity and specificity in a manner dependent on the geographic region analysed. The objective of this study was to explore the genomic architecture and diversity of four Colombian L. infantum isolates obtained in this study and to compare these results with the genetic analysis of 183 L. infantum isolates from across the world (obtained from public databases), as well as to analyse the whole rK39 and rK28 antigen sequences in our dataset. The results showed that, at the global level, L. infantum has high genetic homogeneity and extensive aneuploidy. Furthermore, we demonstrated that there are distinct populations of L. infantum circulating in various countries throughout the globe and that populations of distant countries have close genomic relationships. Additionally, this study demonstrated the high genetic variability of the rK28 antigen worldwide. In conclusion, our study allowed us to (i) expand our knowledge of the genomic structure of global L. infantum; (ii) describe the intra-specific genomic variability of this species; and (iii) understand the genomic characteristics of the main antigens used in the diagnosis of VL. Additionally, this is the first study to report whole-genome sequences of Colombian L. infantum isolates.

Identification of Potential Kinase Inhibitors within the PI3K/AKT Pathway of Leishmania Species.

Leishmaniasis is a public health disease that requires the development of more effective treatments and the identification of novel molecular targets. Since blocking the PI3K/AKT pathway has been successfully studied as an effective anticancer strategy for decades, we examined whether the same approach would also be feasible in Leishmania due to their high amount and diverse set of annotated proteins. Here, we used a best reciprocal hits protocol to identify potential protein kinase homologues in an annotated human PI3K/AKT pathway. We calculated their ligandibility based on available bioactivity data of the reported homologues and modelled their 3D structures to estimate the druggability of their binding pockets. The models were used to run a virtual screening method with molecular docking. We found and studied five protein kinases in five different Leishmania species, which are AKT, CDK, AMPK, mTOR and GSK3 homologues from the studied pathways. The compounds found for different enzymes and species were analysed and suggested as starting point scaffolds for the design of inhibitors. We studied the kinases' participation in protein-protein interaction networks, and the potential deleterious effects, if inhibited, were supported with the literature. In the case of Leishmania GSK3, an inhibitor of its human counterpart, prioritized by our method, was validated in vitro to test its anti-Leishmania activity and indirectly infer the presence of the enzyme in the parasite. The analysis contributes to improving the knowledge about the presence of similar signalling pathways in Leishmania, as well as the discovery of compounds acting against any of these kinases as potential molecular targets in the parasite.

The performance of the recombinase polymerase amplification test for detecting Leishmania deoxyribonucleic acid from skin lesions of patients with clinical or epidemiological suspicion of cutaneous leishmaniasis.

BACKGROUND: The diagnosis of cutaneous leishmaniasis (CL) is based on demonstration of the presence of the parasite in samples obtained from the lesions by direct examination (DE), culture or polymerase chain reaction (PCR)-based molecular tests. Recombinase polymerase amplification (RPA) represents an isothermal version of the conventional PCR (cPCR) technique, being ideal for detecting Leishmania DNA, especially in field conditions. METHODS: A prospective and cross-sectional study was conducted to evaluate the diagnostic performance of RPA in the health centres of rural endemic sites or the evaluation centre (EC) of 11 Colombian municipalities and in a reference centre (RC). RESULTS: Samples of 128 patients with suspected CL were included and processed for analysis by RPA vs DE in the EC and RPA vs DE and cPCR in the RC. The RPA performed at the EC was more sensitive (90.4% [95% confidence interval {CI} 81.9 to 95.7]) than the DE (42-67%) and the specificity was 72.7% (95% CI 57.2 to 85.0). Both the sensitivity and specificity increased to 100% when adjusting by the imperfect reference standard analysis method. In the RC, the sensitivity of RPA vs cPCR was 72% and the specificity was 69.8%, while the sensitivity of cPCR vs the DE test was 78.8% and the specificity was 81%. CONCLUSIONS: The higher sensitivity and specificity shown by RPA in the EC, but also its ease and speed of use, justify performing RPA in the health centres of rural endemic sites. In addition, RPA eliminates the subjectivity inherent in the traditional DE.

Metabolite Biomarkers of Leishmania Antimony Resistance.

Leishmania parasites cause leishmaniasis, one of the most epidemiologically important neglected tropical diseases. Leishmania exhibits a high ability of developing drug resistance, and drug resistance is one of the main threats to public health, as it is associated with increased incidence, mortality, and healthcare costs. The antimonial drug is the main historically implemented drug for leishmaniasis. Nevertheless, even though antimony resistance has been widely documented, the mechanisms involved are not completely understood. In this study, we aimed to identify potential metabolite biomarkers of antimony resistance that could improve leishmaniasis treatment. Here, using L. tropica promastigotes as the biological model, we showed that the level of response to antimony can be potentially predicted using 1H-NMR-based metabolomic profiling. Antimony-resistant parasites exhibited differences in metabolite composition at the intracellular and extracellular levels, suggesting that a metabolic remodeling is required to combat the drug. Simple and time-saving exometabolomic analysis can be efficiently used for the differentiation of sensitive and resistant parasites. Our findings suggest that changes in metabolite composition are associated with an optimized response to the osmotic/oxidative stress and a rearrangement of carbon-energy metabolism. The activation of energy metabolism can be linked to the high energy requirement during the antioxidant stress response. We also found that metabolites such as proline and lactate change linearly with the level of resistance to antimony, showing a close relationship with the parasite's efficiency of drug resistance. A list of potential metabolite biomarkers is described and discussed.

Drug-Induced Lipid Remodeling in Leishmania Parasites.

Leishmania parasites efficiently develop resistance against several types of drugs including antimonials, the primary antileishmanial drug historically implemented. The resistance to antimonials is considered to be a major risk factor for effective leishmaniasis treatment. To detect biomarkers/biopatterns for the differentiation of antimony-resistant Leishmania strains, we employed untargeted global mass spectrometry to identify intracellular lipids present in antimony sensitive and resistant parasites before and after antimony exposure. The lipidomic profiles effectively differentiated the sensitive and resistant phenotypes growing with and without antimony pressure. Resistant phenotypes were characterized by significant downregulation of phosphatidylcholines, sphingolipid decrease, and lysophosphatidylcholine increase, while sensitive phenotypes were characterized by the upregulation of triglycerides with long-chain fatty acids and a tendency toward the phosphatidylethanolamine decrease. Our findings suggest that the changes in lipid composition in antimony-resistant parasites contribute to the physiological response conducted to combat the oxidative stress unbalance caused by the drug. We have identified several lipids as potential biomarkers associated with the drug resistance.

Genomic Diversification, Structural Plasticity, and Hybridization in Leishmania (Viannia) braziliensis.

Leishmania (Viannia) braziliensis is an important Leishmania species circulating in several Central and South American countries. Among Leishmania species circulating in Brazil, Argentina and Colombia, L. braziliensis has the highest genomic variability. However, genomic variability at the whole genome level has been only studied in Brazilian and Peruvian isolates; to date, no Colombian isolates have been studied. Considering that in Colombia, L. braziliensis is a species with great clinical and therapeutic relevance, as well as the role of genetic variability in the epidemiology of leishmaniasis, we analyzed and evaluated intraspecific genomic variability of L. braziliensis from Colombian and Bolivian isolates and compared them with Brazilian isolates. Twenty-one genomes were analyzed, six from Colombian patients, one from a Bolivian patient, and 14 Brazilian isolates downloaded from public databases. The results obtained of Phylogenomic analysis showed the existence of four well-supported clades, which evidenced intraspecific variability. The whole-genome analysis revealed structural variations in the somy, mainly in the Brazilian genomes (clade 1 and clade 3), low copy number variations, and a moderate number of single-nucleotide polymorphisms (SNPs) in all genomes analyzed. Interestingly, the genomes belonging to clades 2 and 3 from Colombia and Brazil, respectively, were characterized by low heterozygosity (~90% of SNP loci were homozygous) and regions suggestive of loss of heterozygosity (LOH). Additionally, we observed the drastic whole genome loss of heterozygosity and possible hybridization events in one genome belonging to clade 4. Unique/shared SNPs between and within the four clades were identified, revealing the importance of some of them in biological processes of L. braziliensis. Our analyses demonstrate high genomic variability of L. braziliensis in different regions of South America, mainly in Colombia and suggest that this species exhibits striking genomic diversity and a capacity of genomic hybridization; additionally, this is the first study to report whole-genome sequences of Colombian L. braziliensis isolates.

A new flatworm species of Temnocephala (Rhabdocoela, Temnocephalidae) ectosymbiont on the freshwater crab Valdivia serrata (Decapoda, Trichodactylidae) from Amazonas, Colombia.

A new species of temnocephalan is described from the branchial chambers of Valdivia serrata in Colombia as Temnocephala ivandarioi sp. nov. The most distinctive characters of the new species are in the cirrus and the epidermal 'excretory' syncytial plates. In the present study, the terminology to describe the cirrus of species of Temnocephala is updated. Comparison between the shape of the cirrus of the temnocephalans associated with trichodactylid crabs is also provided.

Intraspecific Genomic Divergence and Minor Structural Variations in Leishmania (Viannia) panamensis.

Leishmania (Viannia) panamensis is one of the most important Leishmania species associated with cutaneous leishmaniasis (CL) in Latin America. Despite its wide geographic distribution and pathogenic potential in humans and animals, the genomic variability of this species is low compared with other Leishmania species circulating in the same geographical area. No studies have reported a detailed analysis of the whole genome of L. panamensis from clinical isolates using DNA high-throughput sequencing to clarify its intraspecific genomic variability or plausible divergence. Therefore, this study aimed to evaluate the intraspecific genomic variability of L. panamensis from Colombia and Panama. A total of 22 genomes were analyzed, 19 from Colombian patients with CL and three genomes from Panama obtained from public databases. The phylogenomic analysis revealed the potential existence of three well-supported clades as evidence of intraspecific divergence. Additionally, the whole-genome analysis showed low structural variations in terms of ploidy, copy number variations, and single-nucleotide polymorphisms (SNPs). SNPs shared among all clades were identified, revealing their importance in different biological processes of L. panamensis. The findings not only expand our knowledge of intraspecific genomic variability of one of the most important Leishmania species in South America but also highlights the possible existence of different clades/lineages/subpopulations across a geographic scale.

Genosensors for differential detection of Zika virus.

Zika virus (ZIKV) is considered an emerging infectious disease of high clinical and epidemiological relevance. The epidemiological emergency generated by the virus in Latin America and Southeast Asia in 2014 evidenced the urgent need for rapid and acute diagnostic tools. The current laboratory diagnosis of ZIKV is based on molecular and serological methods. However, molecular tools need expensive and sophisticated equipment and trained personnel; and serological detection may suffer from cross-reactivity. In this context, genosensors offer an attractive alternative for field-ready, early and accurate diagnosis of ZIKV. This work reports on the development of genosensors for the differential detection of ZIKV and its discrimination from dengue (DENV) and chikungunya (CHIKV) homologous arboviruses. We designed specific capture and signal probes by bioinformatics, and prove their specificity to amplify the target genetic material by the polymerase chain reaction (PCR). The designed biotinylated capture and digoxigenin (Dig)-labeled signal probes hybridized the target in a sandwich-type format. An anti-Dig antibody labeled with the horseradish peroxidase (HRP) enzyme allowed for both optical and electrochemical detection. The genosensors detected the ZIKV genetic material in spiked serum, urine, and saliva samples and cDNA from infected patients, discriminating them from the DENV and ZIKV genetic material. The proposed system offers a step forward to the differential diagnosis of the ZIKV, closer to the patient, very promising for diagnosis and surveillance of this rapidly emerging disease.

Test accuracy of polymerase chain reaction methods against conventional diagnostic techniques for Cutaneous Leishmaniasis (CL) in patients with clinical or epidemiological suspicion of CL: Systematic review and meta-analysis.

BACKGROUND: Molecular diagnostic tests, notably polymerase chain reaction (PCR), are highly sensitive test for Leishmania detection, which is especially relevant in chronic cutaneous lesion with lower parasite load. An accurate diagnosis is essential because of the high toxicity of the medications for the disease. Nevertheless, diagnosis of cutaneous leishmaniasis (CL) is hampered by the absence of a reference standard. Assuming that the PCR-based molecular tools are the most accurate diagnostic method, the objective of this systematic review was to assess the diagnostic accuracy of PCR-based molecular tools in a meta-analysis of the published literature. METHODOLOGY/PRINCIPAL FINDINGS: A search of the published literature found 142 papers of which only 13 studies met the selection criteria, including conventional PCR, real-time PCR, Loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), polymorphism-specific PCR (PS-PCR). The sensitivities of the individual studies ranged from 61% to 100%, and specificities ranged from 11% to 100%. The pooled sensitivities of PCR in smears were 0.95 (95% CI, 0.90 to 0.98), and the specificity was 0.91(95% CI, 0.70 to 0.98). In general population, estimates were lower in aspirates, skin biopsies and swab samples with 0.90 (95% CI, 0.80 to 0.95) and 0.87 (95% CI, 0.76 to 0.94) for sensitivity and specificity, respectively. The specificity was lower in consecutive studies, at 0.88 (95% CI, 0.59 to 0.98) and its CI were wider. CONCLUSIONS/SIGNIFICANCE: No statistically significant differences between the accuracy in smears, aspirate, skin biopsies or swabs samples were observed. Therefore, a simple smear sample run by PCR, instead more invasive samples, may be enough to obtain a positive diagnosis of CL. The results for PCR in all samples type confirm previous reports that consider PCR as the most accurate method for the diagnosis of CL.