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BACKGROUND: Newcastle disease (ND) is a major threat to the poultry industry, leading to significant economic losses. The current ND vaccines, usually based on active or attenuated strains, are only partially effective and can cause adverse effects post-vaccination. Therefore, the development of safer and more efficient vaccines is necessary. Epitopes represent the antigenic portion of the pathogen and their identification and use for immunization could lead to safer and more effective vaccines. However, the prediction of protective epitopes for a pathogen is a major challenge, especially taking into account the immune system of the target species. RESULTS: In this study, we utilized an artificial intelligence algorithm to predict ND virus (NDV) peptides that exhibit high affinity to the chicken MHC-I complex. We selected the peptides that are conserved across different NDV genotypes and absent in the chicken proteome. From the filtered peptides, we synthesized the five peptides with the highest affinities for the L, HN, and F proteins of NDV. We evaluated these peptides in-vitro for their ability to elicit cell-mediated immunity, which was measured by the lymphocyte proliferation in spleen cells of chickens previously immunized with NDV. CONCLUSIONS: Our study identified five peptides with high affinity to MHC-I that have the potential to serve as protective epitopes and could be utilized for the development of multi-epitope NDV vaccines. This approach can provide a safer and more efficient method for NDV immunization.

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This study presents a draft genome sequence of a Newcastle disease virus (NDV) strain (VFAR-136) isolated from a fighting cock (Gallus gallus) in the south of Peru. Strain VFAR-136 is a new report of NDV genotype VII circulating in Peru.

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Fluorescence microscopy is an important tool for disease diagnosis, often requiring costly optical components, such as fluorescence filter cubes and high-power light sources. Due to its high cost, conventional fluorescence microscopy cannot be fully exploited in low-income settings. Smartphone-based fluorescence microscopy becomes an interesting low-cost alternative, but raises challenges in the optical system. We present the development of a low-cost inverted laser fluorescence microscope that uses a smartphone to visualize the fluorescence image of biological samples. Our fluorescence microscope uses a laser-based simplified optical filter system that provides analog optical filtering capabilities of a fluorescence filter cube. Firstly, we validated our inverted optical filtering by visualizing microbeads labeled with three different fluorescent compounds or fluorophores commonly used for disease diagnosis. Secondly, we validated the disease diagnosis capabilities by comparing the results of our device with those of a commercial fluorescence microscope. We successfully detected and visualized Trypanosoma cruzi parasites, responsible for the Chagas infectious disease and the presence of Antineutrophil cytoplasmic antibodies of the ANCA non-communicable autoimmune disease. The samples were labeled with the fluorescein isothiocyanate (FITC) fluorophore, one of the most commonly used fluorophores for disease diagnosis. Our device provides a 400× magnification and is at least one order of magnitude cheaper than conventional commercial fluorescence microscopes.

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Tuberculosis phenotypic detection assays are commonly used in low-resource countries. Therefore, reliable detection methods are crucial for early diagnosis and treatment. The microscopic observation drug susceptibility (MODS) assay is a culture-based test to detect Mycobacterium tuberculosis and characterize drug resistance in 7-10 days directly from sputum. The use of MODS is limited by the availability of supplies necessary for preparing the enriched culture. In this study, we evaluated three dry culture media that are easier to produce and cheaper than the standard one used in MODS [1]: an unsterilized powder-based mixed (Boldú et al., 2007) [2], a sterile-lyophilized medium, and (Sengstake et al., 2017) [3] an irradiated powder-based mixed. Mycobacterial growth and drug susceptibility were evaluated for rifampin, isoniazid, and pyrazinamide (PZA). The alternative cultures were evaluated using 282 sputum samples with positive acid-fast smears. No significant differences were observed in the positivity test rates. The positivity time showed high correlations (Rho) of 0.925, 0.889, and 0.866 between each of the three alternative media and the standard. Susceptibility testing for MDR and PZA showed an excellent concordance of 1 compared to the reference test. These results demonstrate that dry culture media are appropriate and advantageous for use in MODS in low-resource settings.

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Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb). Despite being considered curable and preventable, the increase of antibiotic resistance is becoming a serious public health problem. Mtb is a pathogen capable of surviving in macrophages, causing long-term latent infection where the mycobacterial serine/threonine protein kinase G (PknG) plays a protective role. Therefore, PknG is an important inhibitory target to prevent Mtb from entering the latency stage. In this study, we use a pharmacophore-based virtual screening and biochemical assays to identify the compound RO9021 (CHEMBL3237561) as a PknG inhibitor. In detail, 1.5 million molecules were screened using a scalable cloud-based setup, identifying 689 candidates, which were further subjected to additional screening employing molecular docking. Molecular docking spotted 62 compounds with estimated binding affinities of -7.54 kcal/mol (s.d. = 0.77 kcal/mol). Finally, 14 compounds were selected for in vitro experiments considering previously reported biological activities and commercial availability. In vitro assays of PknG activity showed that RO9021 inhibits the kinase activity similarly to AX20017, a known inhibitor. The inhibitory effect was found to be dose dependent with a relative IC50 value of 4.4 ± 1.1 µM. Molecular dynamics simulations predicted that the PknG-RO9021 complex is stable along the tested timescale. Altogether, our study indicates that RO9021 is a noteworthy drug candidate for further developing new anti-TB drugs that hold excellent reported pharmacokinetic parameters.

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Within the framework of the current COVID-19 pandemic, there is a race against time to find therapies for the outbreak to be controlled. Since vaccines are still tedious to develop and partially available for low-income countries, passive immunity based on egg-yolk antibodies (IgY) is presented as a suitable approach to preclude potential death of infected patients, based on its high specificity/avidity/production yield, cost-effective manufacture, and ease of administration. In the present study, IgY antibodies against a recombinant RBD protein of SARS-CoV-2 were produced in specific-pathogen-free chickens and purified from eggs using a biocompatible method. In vitro immunoreactivity was tested, finding high recognition and neutralization values. Safety was also demonstrated prior to efficacy evaluation, in which body weight, kinematics, and histopathological assessments of hamsters challenged with SARS-CoV-2 were performed, showing a protective effect administering IgY intranasally both as a prophylactic treatment or a post-infection treatment. The results of this study showed that intranasally delivered IgY has the potential to both aid in prevention and in overcoming COVID-19 infection, which should be very useful to control the advance of the current pandemic and the associated mortality.

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The coronavirus disease-19 (COVID-19) pandemic has already claimed millions of lives and remains one of the major catastrophes in the recorded history. While mitigation and control strategies provide short term solutions, vaccines play critical roles in long term control of the disease. Recent emergence of potentially vaccine-resistant and novel variants necessitated testing and deployment of novel technologies that are safe, effective, stable, easy to administer, and inexpensive to produce. Here we developed three recombinant Newcastle disease virus (rNDV) vectored vaccines and assessed their immunogenicity, safety, and protective efficacy against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in mice and hamsters. Intranasal administration of rNDV-based vaccine candidates elicited high levels of neutralizing antibodies. Importantly, the nasally administrated vaccine prevented lung damage, and significantly reduced viral load in the respiratory tract of vaccinated animal which was compounded by profound humoral immune responses. Taken together, the presented NDV-based vaccine candidates fully protected animals against SARS-CoV-2 challenge and warrants evaluation in a Phase I human clinical trial as a promising tool in the fight against COVID-19.

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Abstract Objectives: Evaluate the association between rifampicin resistance and the presence of at least one SNP in the rpoB and ponA1 genes and the spoligotype defined lineages. Material and Methods: This study analyzed two databases of 484 genomes of M. tuberculosis from strains isolated from patients in the cities of Lima and Callao, for which the odds ratio (OR) was calculated considering belonging to a certain spoligotype defined lineages as an exposure factor. Results: No statistically significant association (ρ value> 0.05) was found between the presence of at least one SNP in the rpoB gene and the lineages included in the study (LAM, Haarlem, T and Beijing). However, a statistically significant association was found between the presence of at least one SNP in the ponA1 gene and the LAM and Haarlem lineages (ρ value <0.05). An association was found between the P631S SNP in the ponA1 gene and the LAM and Haarlem lineages; and the A516T SNP, of this same gene, presented an association with the LAM lineage. Likewise, an association was found between rifampicin resistance and the LAM lineage. Conclusions: The presence of SNPs in the ponA1 gene is associated with the LAM and Haarlem lineages.

Resumen Objetivos: Evaluar la asociación entre la resistencia a rifampicina y la presencia de al menos un SNP en los genes rpoB y ponA1 y los linajes definidos por espoli gotipos. Material y Métodos: Este estudio analizó dos bases de datos de 484 genomas de M. tuberculosis de cepas aisladas de pacientes de las ciudades de Lima y Callao, para lo cual se calculó el odds ratio (OR) considerando la pertenencia a determinado linaje definido por espoligotipos como un factor de exposición. Resultados: No se encontró una asociación estadísticamente significativa (valor de ρ >0.05) entre la presencia de al menos un SNP en el gen rpoB y los linajes incluidos en el estudio (LAM, Haarlem, T y Beijing). No obstante, se halló una asociación estadísticamente significativa entre la presencia de al menos un SNP en el gen ponA1 y los linajes LAM y Haarlem (valor de ρ <0.05). Se encontró una asociación entre el SNP P631S del gen ponA1 y los linajes LAM y Haarlem; y el SNP A516T, de este mismo gen, presentó una asociación con el linaje LAM. Asimismo, se halló una asociación entre la resistencia a rifampicina y el linaje LAM. Conclusiones: La presencia de SNPs en el gen ponA1 está asociada con los linajes LAM y Haarlem.

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MODS, an assay for diagnosis of tuberculosis and drug-susceptibility, is based in the microscopic observation of the characteristic cords of Mycobacterium tuberculosis colonies grown in liquid media. An inverted optical microscope (100× magnification) is required to observe and interpret MODS cultures. Unfortunately, the cost of commercial inverted microscopes is not affordable in low resource settings. To perform a diagnosis of tuberculosis using the MODS assay, images with modest quality are enough for proper interpretation. Therefore, the use of a high cost commercial inverted optical microscope is not indispensable. In this study, we designed a prototype of an optical inverted microscope created by 3D-printing and based on a smartphone. The system was evaluated with 226 MODS TB positive and 207 MODS TB negative digital images. These images were obtained from 10 sputum samples MODS positive and 10 sputum samples MODS negative. The quality of all images was assessed by a qualified technician, in terms of adequacy to interpret and classify them as positive or negative for tuberculosis. The quality of the images was considered appropriate for MODS interpretation. All the 20 samples were correctly classified (as TB positive/negative) by reading with the prototype 3D-printed inverted microscope.

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The role of domestic cats in the dynamics of SARS-CoV-2 remains poorly characterized, especially in epidemiologic contexts of countries with high viral transmission. Here, we report the first evidence of SARS-CoV-2 Lambda variant of interest in symptomatic domestic cats whose owners were diagnosed with COVID-19 in Lima, Peru, providing evidence that transmission of this new variant in domestic cats is occurring. More epidemiological studies are required to further characterize the role of domestic animals in the transmission dynamics of SARS-CoV-2.

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SARS-CoV-2 can infect a variety of wild and domestic animals worldwide. Of these, domestic cats are highly susceptible species and potential viral reservoirs. As such, it is important to investigate disease exposure in domestic cats in areas with active community transmission and high disease prevalence. In this report we demonstrate the presence of serum neutralizing antibodies against the receptor binding-domain (RBD) of the SARS-CoV-2 in cats whose owners had been infected with SARS-CoV-2 in Lima, Peru, using a commercial competitive ELISA SARS-CoV-2 Surrogate Virus Neutralization Test. Out of 41 samples, 17.1% (7/41) and 31.7% (13/41) were positive, using the cut-off inhibition value of 30% and 20%, respectively. Not all cats living in a single house had detectable neutralizing antibodies showing heterogenous exposure and immunity among cohabiting animals. This is the first report of SARS-COV-2 exposure of domestic cats in Lima, Peru. Further studies are required to ascertain the prevalence of SARS-COV-2 exposure among domestic cats.

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SARS-CoV-2 main protease is a common target for inhibition assays due to its high conservation among coronaviruses. Since flavonoids show antiviral activity, several in silico works have proposed them as potential SARS-CoV-2 main protease inhibitors. Nonetheless, there is reason to doubt certain results given the lack of consideration for flavonoid promiscuity or main protease plasticity, usage of short library sizes, absence of control molecules and/or the limitation of the methodology to a single target site. Here, we report a virtual screening study where dorsilurin E, euchrenone a11, sanggenol O and CHEMBL2171598 are proposed to inhibit main protease through different pathways. Remarkably, novel structural mechanisms were observed after sanggenol O and CHEMBL2171598 bound to experimentally proven allosteric sites. The former drastically affected the active site, while the latter triggered a hinge movement which has been previously reported for an inactive SARS-CoV main protease mutant. The use of a curated database of 4.8 k flavonoids, combining two well-known docking software (AutoDock Vina and AutoDock4.2), molecular dynamics and MMPBSA, guaranteed an adequate analysis and robust interpretation. These criteria can be considered for future screening campaigns against SARS-CoV-2 main protease.

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SARS-CoV-2 can infect a variety of wild and domestic animals worldwide. Of these, domestic cats are highly susceptible species and potential viral reservoirs. As such, it is important to investigate disease exposure in areas with active community transmission and high disease prevalence. In this report we demonstrate the presence of serum neutralizing antibodies against the receptor binding-domain (RBD) of the SARS-CoV-2 in cats whose owners had been infected with SARS-CoV-2 in Lima, Peru, using a commercial competitive ELISA SARS-CoV-2 Surrogate Virus Neutralization Test. Out of 41 samples, 17.1% (7/41) and 31.7% (13/41) were positive, using the cut-off inhibition value of 30% and 20%, respectively. Not all cats living in a single house had detectable neutralizing antibodies showing that heterogenous exposure and immune among cohabiting animals. This is the first report of SARS-COV-2 exposure of domestic cats in Lima, Peru. Further studies are required to ascertain the prevalence of SARS-COV-2 exposure among domestic cats of Lima, Peru.

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Most children with autism spectrum disorder (ASD), in resource-limited settings (RLS), are diagnosed after the age of four. Our work confirmed and extended results of Pierce that eye tracking could discriminate between typically developing (TD) children and those with ASD. We demonstrated the initial 15 s was at least as discriminating as the entire video. We evaluated the GP-MCHAT-R, which combines the first 15 s of manually-coded gaze preference (GP) video with M-CHAT-R results on 73 TD children and 28 children with ASD, 36-99 months of age. The GP-MCHAT-R (AUC = 0.89 (95%CI: 0.82-0.95)), performed significantly better than the MCHAT-R (AUC = 0.78 (95%CI: 0.71-0.85)) and gaze preference (AUC = 0.76 (95%CI: 0.64-0.88)) alone. This tool may enable early screening for ASD in RLS.

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BACKGROUND: Pyrazinamide is an important drug against the latent stage of tuberculosis and is used in both first- and second-line treatment regimens. Pyrazinamide-susceptibility test usually takes a week to have a diagnosis to guide initial therapy, implying a delay in receiving appropriate therapy. The continued increase in multi-drug resistant tuberculosis and the prevalence of pyrazinamide resistance in several countries makes the development of assays for prompt identification of resistance necessary. The main cause of pyrazinamide resistance is the impairment of pyrazinamidase function attributed to mutations in the promoter and/or pncA coding gene. However, not all pncA mutations necessarily affect the pyrazinamidase function. OBJECTIVE: To develop a methodology to predict pyrazinamidase function from detected mutations in the pncA gene. METHODS: We measured the catalytic constant (kcat), KM, enzymatic efficiency, and enzymatic activity of 35 recombinant mutated pyrazinamidase and the wild type (Protein Data Bank ID = 3pl1). From all the 3D modeled structures, we extracted several predictors based on three categories: structural stability (estimated by normal mode analysis and molecular dynamics), physicochemical, and geometrical characteristics. We used a stepwise Akaike's information criterion forward multiple log-linear regression to model each kinetic parameter with each category of predictors. We also developed weighted models combining the three categories of predictive models for each kinetic parameter. We tested the robustness of the predictive ability of each model by 6-fold cross-validation against random models. RESULTS: The stability, physicochemical, and geometrical descriptors explained most of the variability (R2) of the kinetic parameters. Our models are best suited to predict kcat, efficiency, and activity based on the root-mean-square error of prediction of the 6-fold cross-validation. CONCLUSIONS: This study shows a quick approach to predict the pyrazinamidase function only from the pncA sequence when point mutations are present. This can be an important tool to detect pyrazinamide resistance.

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Background: Rifampicin (RIF) resistance in Mycobacterium tuberculosis is frequently caused by mutations in the rpoB gene. These mutations are associated with a fitness cost, which can be overcome by compensatory mutations in other genes, among which rpoC may be the most important. We analyzed 469 Peruvian M. tuberculosis clinical isolates to identify compensatory mutations in rpoC/rpoA associated with RIF resistance. Methods: The M. tuberculosis isolates were collected and tested for RIF susceptibility and spoligotyping. Samples were sequenced and aligned to the reference genome to identify mutations. By analyzing the sequences and the metadata, we identified a list of rpoC mutations exclusively associated with RIF resistance and mutations in rpoB. We then evaluated the distribution of these mutations along the protein sequence and tridimensional structure. Results: One hundred and twenty-five strains were RIF susceptible and 346 were resistant. We identified 35 potential new compensatory mutations, some of which were distributed on the interface surface between rpoB and rpoC, arising in clusters and suggesting the presence of hotspots for compensatory mutations. Conclusion: This study identifies 35 putative novel compensatory mutations in the ß' subunit of M. tuberculosis RNApol. Six of these (S428T, L507V, A734V, I997V, and V1252LM) are considered most likely to have a compensatory role, as they fall in the interaction zone of the two subunits and the mutation did not lead to any change in the protein's physical-chemical properties.

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Mycobacterium tuberculosis nicotinamidase-pyrazinamidase (PZAse) is a metalloenzyme that catalyzes conversion of nicotinamide-pyrazinamide to nicotinic acid-pyrazinoic acid. This study investigated whether a metallochaperone is required for optimal PZAse activity. M. tuberculosis and Escherichia coli PZAses (PZAse-MT and PZAse-EC, respectively) were inactivated by metal depletion (giving PZAse-MT-Apo and PZAse-EC-Apo). Reactivation with the E. coli metallochaperone ZnuA or Rv2059 (the M. tuberculosis analog) was measured. This was repeated following proteolytic and thermal treatment of ZnuA and Rv2059. The CDC1551 M. tuberculosis reference strain had the Rv2059 coding gene knocked out, and PZA susceptibility and the pyrazinoic acid (POA) efflux rate were measured. ZnuA (200 µM) achieved 65% PZAse-EC-Apo reactivation. Rv2059 (1 µM) and ZnuA (1 µM) achieved 69% and 34.3% PZAse-MT-Apo reactivation, respectively. Proteolytic treatment of ZnuA and Rv2059 and application of three (but not one) thermal shocks to ZnuA significantly reduced the capacity to reactivate PZAse-MT-Apo. An M. tuberculosis Rv2059 knockout strain was Wayne positive and susceptible to PZA and did not have a significantly different POA efflux rate than the reference strain, although a trend toward a lower efflux rate was observed after knockout. The metallochaperone Rv2059 restored the activity of metal-depleted PZAse in vitro Although Rv2059 is important in vitro, it seems to have a smaller effect on PZA susceptibility in vivo. It may be important to mechanisms of action and resistance to pyrazinamide in M. tuberculosis Further studies are needed for confirmation.IMPORTANCE Tuberculosis is an infectious disease caused by the bacterium Mycobacterium tuberculosis and remains one of the major causes of disease and death worldwide. Pyrazinamide is a key drug used in the treatment of tuberculosis, yet its mechanism of action is not fully understood, and testing strains of M. tuberculosis for pyrazinamide resistance is not easy with the tools that are presently available. The significance of the present research is that a metallochaperone-like protein may be crucial to pyrazinamide's mechanisms of action and of resistance. This may support the development of improved tools to detect pyrazinamide resistance, which would have significant implications for the clinical management of patients with tuberculosis: drug regimens that are appropriately tailored to the resistance profile of a patient's individual strain lead to better clinical outcomes, reduced onward transmission of infection, and reduction of the development of resistant strains that are more challenging and expensive to treat.

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Here, we report a draft genome sequence of Aeromonas veronii strain CTe-01 (4.5 Mb), a hemolytic, heavy metal-resistant bacterium isolated from a wastewater treatment plant located at Cachiche, Ica, Peru. These characteristics could be used for bioremediation of contaminated environments.

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Tuberculosis is an infectious disease that causes ill health and death in millions of people each year worldwide. Timely diagnosis and treatment is key to full patient recovery. The Microscopic Observed Drug Susceptibility (MODS) is a test to diagnose TB infection and drug susceptibility directly from a sputum sample in 7-10 days with a low cost and high sensitivity and specificity, based on the visual recognition of specific growth cording patterns of M. Tuberculosis in a broth culture. Despite its advantages, MODS is still limited in remote, low resource settings, because it requires permanent and trained technical staff for the image-based diagnostics. Hence, it is important to develop alternative solutions, based on reliable automated analysis and interpretation of MODS cultures. In this study, we trained and evaluated a convolutional neural network (CNN) for automatic interpretation of MODS cultures digital images. The CNN was trained on a dataset of 12,510 MODS positive and negative images obtained from three different laboratories, where it achieved 96.63 +/- 0.35% accuracy, and a sensitivity and specificity ranging from 91% to 99%, when validated across held-out laboratory datasets. The model's learned features resemble visual cues used by expert diagnosticians to interpret MODS cultures, suggesting that our model may have the ability to generalize and scale. It performed robustly when validated across held-out laboratory datasets and can be improved upon with data from new laboratories. This CNN can assist laboratory personnel, in low resource settings, and is a step towards facilitating automated diagnostics access to critical areas in developing countries.

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Although pyrazinamide (PZA) is a key component of first- and second-line tuberculosis treatment regimens, there is no gold standard to determine PZA resistance. Approximately 50% of multidrug-resistant tuberculosis (MDR-TB) and over 90% of extensively drug-resistant tuberculosis (XDR-TB) strains are also PZA resistant. pncA sequencing is the endorsed test to evaluate PZA susceptibility. However, molecular methods have limitations for their wide application. In this study, we standardized and evaluated a new method, MODS-Wayne, to determine PZA resistance. MODS-Wayne is based on the detection of pyrazinoic acid, the hydrolysis product of PZA, directly in the supernatant of sputum cultures by detecting a color change following the addition of 10% ferrous ammonium sulfate. Using a PZA concentration of 800 µg/ml, sensitivity and specificity were evaluated at three different periods of incubation (reading 1, reading 2, and reading 3) using a composite reference standard (MGIT-PZA, pncA sequencing, and the classic Wayne test). MODS-Wayne was able to detect PZA resistance, with a sensitivity and specificity of 92.7% and 99.3%, respectively, at reading 3. MODS-Wayne had an agreement of 93.8% and a kappa index of 0.79 compared to the classic Wayne test, an agreement of 95.3% and kappa index of 0.86 compared to MGIT-PZA, and an agreement of 96.9% and kappa index of 0.90 compared to pncA sequencing. In conclusion, MODS-Wayne is a simple, fast, accurate, and inexpensive approach to detect PZA resistance, making this an attractive assay especially for low-resource countries, where TB is a major public health problem.

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