Improving the sensitivity of an hsp20-based PCR for genus detection of Leishmania parasites in cutaneous clinical samples: a proof of concept.

Leishmaniasis is a parasitic disease of medical importance widely distributed around the world. Several methods are available for diagnosis but molecular approaches are highly recommended. To improve the sensitivity of an existing hsp20 gene based-PCR protocol to detect Leishmania parasites, primers were redesigned to amplify a shorter fragment using a new PCR variant (PCR-hsp20S). In this study, we aimed at characterizing the performance of the new method on cutaneous clinical samples and compare it with the former PCR-hsp20. The analytical sensitivity of the PCR-hsp20S was evaluated using DNA dilutions (100-0.1 pg) from Leishmania donovani and resulted in the detection of 10 fg of parasitic DNA, the equivalent to 0.05 parasite genome. For the diagnostic evaluation, a panel of 127 human clinical samples was used to calculate the parameters of sensitivity, specificity, accuracy, and positive and negative predictive values of the PCR-hsp20S. Diagnostic sensitivity was 94% (CI, 89.1-99.7%) and the specificity of 100% (CI, 98.6-100%). The same panel was also evaluated with the PCR-hsp20 to calculate the agreement between both molecular assays and to compare their performances. While both hsp20-based PCRs showed a good agreement coefficient (kappa index = 0.6), the performance of the novel variant, PCR-hsp20S, was significantly higher in terms of sensitivity (P = 0.0001) allowing the accurate detection of a higher number of Leishmania-positive clinical samples. We endorse the use of the PCR-hsp20S over the former protocol for the detection of Leishmania parasites from cutaneous clinical samples. In addition, as an improved sensitivity was achieved with the new method merely through the amplification of a shorter gene fragment, this investigation constitutes an experimental proof of this concept.

Repurposing of known drugs for leishmaniasis treatment using bioinformatic predictions, in vitro validations and pharmacokinetic simulations.

Leishmaniasis is a neglected tropical disease caused by Leishmania parasites and is associated to more than 1.3 million cases annually. Some of the pharmacological options for treating the disease are pentavalent antimonials, pentamidine, miltefosine, and amphotericin B. However, all are associated with a wide range of adverse effects and contraindications, as well as resistance from the parasite. In the present study, we looked for pharmacological alternatives to treat leishmaniasis, with a focus on drug repurposing. This was done by detecting potential homologs between proteins targeted by approved drugs and proteins of the parasite. The proteins were analyzed using an interaction network, and the drugs were subjected to in vitro evaluations and pharmacokinetics simulations to compare probable plasma concentrations with the effective concentrations detected experimentally. This strategy yielded a list of 33 drugs with potential anti-Leishmania activity, and more than 80 possible protein targets in the parasite. From the drugs tested, two reported high in vitro activity (perphenazine EC50 = 1.2 µg/mL and rifabutin EC50 = 8.5 µg/mL). These results allowed us to propose these drugs as candidates for further in vivo studies and evaluations of the effectiveness on their topical forms.

Development of a serosurveillance assay for detection of Necoclí virus exposure.

Hantavirus cardiopulmonary syndrome (HPS) has gained importance in Latin America as an emerging disease, with reports of about 4000 HPS cases; however, this is probably an underestimate because of limited surveillance programs and diagnostic tools to confirm HPS. In order to address this issue and develop better serosurveillance capability, we evaluated three recombinant peptides from the Necoclí virus (NECV) nucleocapsid in antibody-capture ELISA. We cloned and expressed antigens representing the whole NECV nucleocapsid protein (NECV-rN), the immunodominant domain (NECV-rN100), and a serospecific domain (NECV-rN428), and then we compared these antigens in ELISA to detect IgG antibodies to NECV in human sera. We evaluated human sera collected during two epidemiological studies from the area where NECV was discovered. The first group included 609 sera from healthy individuals, and the second one included 89 samples from patients with undifferentiated febrile illness. In these two groups, hantavirus infection had previously been determined by the presence of IgG to Maciel virus (MCLV), a hantavirus closely related to NECV. The number of IgG-positive sera was higher using the Necoclí ELISA with the rN100 protein, which detected antibodies in a higher percentage of healthy individuals, 129/609 (21.2%), as well as in febrile patients, 11/89 (12.3%). In contrast, using MCLV ELISA, 8 of 609 (1.3%) and 4 of 89 (4.5%) samples from healthy and febrile patients, respectively, were seropositive. The agreement between the NECV and MCLV ELISA assays was ≥ 82.3%; however, the kappa indices were weak but statistically significant for rN (0.251 CI; 0.138-0.365) and rN100rN (0.153 CI; 0.084-0.223). The weak kappa indices were attributed to decreased MCLV ELISA assay sensitivity. These results suggest that NECV rN and rN100 have increased specificity and could be further validated for improved diagnosis of hantavirus infections.

Detection and identification of Leishmania spp.: application of two hsp70-based PCR-RFLP protocols to clinical samples from the New World.

Leishmaniasis is highly prevalent in New World countries, where several methods are available for detection and identification of Leishmania spp. Two hsp70-based PCR protocols (PCR-N and PCR-F) and their corresponding restriction fragment length polymorphisms (RFLP) were applied for detection and identification of Leishmania spp. in clinical samples recruited in Colombia, Guatemala, and Honduras. A total of 93 cases were studied. The samples were classified into positive or suspected of leishmaniasis according to parasitological criteria. Molecular amplification of two different hsp70 gene fragments and further RFLP analysis for identification of Leishmania species was done. The detection in parasitologically positive samples was higher using PCR-N than PCR-F. In the total of samples studied, the main species identified were Leishmania panamensis, Leishmania braziliensis, and Leishmania infantum (chagasi). Although RFLP-N was more efficient for the identification, RFLP-F is necessary for discrimination between L. panamensis and Leishmania guyanesis, of great importance in Colombia. Unexpectedly, one sample from this country revealed an RFLP pattern corresponding to Leishmania naiffi. Both molecular variants are applicable for the study of clinical samples originated in Colombia, Honduras, and Guatemala. Choosing the better tool for each setting depends on the species circulating. More studies are needed to confirm the presence of L. naiffi in Colombian territory.

Drug search for leishmaniasis: a virtual screening approach by grid computing.

The trypanosomatid protozoa Leishmania is endemic in ~100 countries, with infections causing ~2 million new cases of leishmaniasis annually. Disease symptoms can include severe skin and mucosal ulcers, fever, anemia, splenomegaly, and death. Unfortunately, therapeutics approved to treat leishmaniasis are associated with potentially severe side effects, including death. Furthermore, drug-resistant Leishmania parasites have developed in most endemic countries. To address an urgent need for new, safe and inexpensive anti-leishmanial drugs, we utilized the IBM World Community Grid to complete computer-based drug discovery screens (Drug Search for Leishmaniasis) using unique leishmanial proteins and a database of 600,000 drug-like small molecules. Protein structures from different Leishmania species were selected for molecular dynamics (MD) simulations, and a series of conformational "snapshots" were chosen from each MD trajectory to simulate the protein's flexibility. A Relaxed Complex Scheme methodology was used to screen ~2000 MD conformations against the small molecule database, producing >1 billion protein-ligand structures. For each protein target, a binding spectrum was calculated to identify compounds predicted to bind with highest average affinity to all protein conformations. Significantly, four different Leishmania protein targets were predicted to strongly bind small molecules, with the strongest binding interactions predicted to occur for dihydroorotate dehydrogenase (LmDHODH; PDB:3MJY). A number of predicted tight-binding LmDHODH inhibitors were tested in vitro and potent selective inhibitors of Leishmania panamensis were identified. These promising small molecules are suitable for further development using iterative structure-based optimization and in vitro/in vivo validation assays.

Comparison of serological methods with PCR-based methods for the diagnosis of community-acquired pneumonia caused by atypical bacteria.

BACKGROUND: The diagnosis of community-acquired pneumonia (CAP) caused by Legionella pneumophila, Mycoplasma pneumoniae, and Chlamydophila pneumoniae is traditionally based on cultures and serology, which have special requirements, are time-consuming, and offer delayed results that limit their clinical usefulness of these techniques. We sought to develop a multiplex PCR (mPCR) method to diagnosis these bacterial infections in CAP patients and to compare the diagnostic yields obtained from mPCR of nasopharyngeal aspirates (NPAs), nasopharyngeal swabs (NPSs), and induced sputum (IS) with those obtained with specific PCR commercial kits, paired serology, and urinary antigen. RESULTS: A total of 225 persons were included. Of these, 10 patients showed serological evidence of L. pneumophila infection, 30 of M. pneumoniae, and 18 of C. pneumoniae; 20 individuals showed no CAP. The sensitivities were mPCR-NPS = 23.1%, mPCR-IS = 57.1%, Seeplex®-IS = 52.4%, and Speed-oligo®-NPA/NPS = 11.1%, and the specificities were mPCR-NPS = 97.1%, mPCR-IS = 77.8%, Seeplex®-IS = 92.6%, and Speed-oligo®-NPA/NPS = 96.1%. The concordance between tests was poor (kappa <0.4), except for the concordance between mPCR and the commercial kit in IS (0.67). In individuals with no evidence of CAP, positive reactions were observed in paired serology and in all PCRs. CONCLUSIONS: All PCRs had good specificity but low sensitivity in nasopharyngeal samples. The sensitivity of mPCR and Seeplex® in IS was approximately 60%; thus, better diagnostic techniques for these three bacteria are required.

A comparison of methicillin-resistant and methicillin-susceptible Staphylococcus aureus reveals no clinical and epidemiological but molecular differences.

Most studies on Staphylococcus aureus have focused on the molecular epidemiology of methicillin-resistant S. aureus (MRSA) infections. In contrast, little information is available regarding the molecular epidemiology of currently circulating methicillin-susceptible S. aureus (MSSA) isolates in hospital settings, an epoch when the epidemiology of S. aureus has undergone significant changes. We conducted a cross-sectional study to compare the clinical, epidemiological, and genetic characteristics of MSSA and MRSA isolates at 3 tertiary-care hospitals in Medellín, Colombia, from February 2008 to June 2010. The infections were classified according to the Centers for Disease Control and Prevention (CDC) definitions. Genotypic analysis included spa typing, multilocus sequence typing (MLST) and staphylococcal cassette chromosome (mec) (SCCmec) typing. A total of 810 patients was enrolled. One hundred infections (12.3%) were classified as community-associated (31 CA-MSSA, 69 CA-MRSA), 379 (46.8%) as healthcare-associated community-onset (136 HACO-MSSA, 243 HACO-MRSA), and 331 (40.9%) as healthcare-associated hospital-onset (104 HAHO-MSSA, 227 HAHO-MRSA). Genotype analyses showed a higher diversity and a more varied spa type repertoire in MSSA than in MRSA strains. Most of the clinical-epidemiological characteristics and risk factors evaluated did not allow for discriminating MRSA- from MSSA-infected patients. The lack of equivalence among the genetic backgrounds of the major MSSA and MRSA clones would suggest that the MRSA clones are imported instead of arising from successful MSSA clones. This study emphasizes the importance of local surveillance to create public awareness on the changing S. aureus epidemiology.

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.

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.

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.

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.

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.

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" [...].

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.

Transfusion-transmitted visceral leishmaniasis caused by Leishmania (Leishmania) mexicana in an immunocompromised patient: a case report.

BACKGROUND: Transfusion-transmitted leishmaniasis is an increasing problem in areas where visceral and cutaneous leishmaniases are endemic. CASE REPORT: This article reports a case of transfusion-transmitted fatal visceral leishmaniasis (VL) caused by Leishmania (Leishmania) mexicana in a 42-year-old male resident of northwestern Colombia who after developing a terminal renal failure due to lupus nephritis received a renal transplant and multiple transfusions. RESULTS: Multiple intracellular Leishmania amastigotes were demonstrated in both renal biopsy and marrow aspirates. Cultures of the parasite were obtained in NNN medium and the identification of the species was done both by direct immunofluorescence and polymerase chain reaction-restriction fragment length polymorphism. CONCLUSIONS: This is the first report of a VL case produced by L. (L.) mexicana in Colombia, which usually is a dermotropic species. Our report suggests that although leishmaniasis is transmitted by the bite of phlebotomine sand flies, Leishmania parasite may be transmitted by blood transfusion, complicating the clinical course of organ transplant and being fatal.

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.

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.

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.

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.

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.