Biology subjects

Zimic, M.

Publications and source records attributed to Zimic, M..

11 recordsLinked to original sources

bioRxiv2026

Pericystic brain transcriptomics reveals molecular signatures of immune activation and neurovascular remodelling in viable and post-treatment porcine neurocysticercosis

Neurocysticercosis (NCC), the infection of the central nervous system by Taenia solium larvae, is a leading cause of acquired epilepsy in endemic regions. While viable cysticerci can persist asymptomatically for extended periods, their spontaneous or drug-induced degradation triggers marked perilesional inflammation and severe neurological symptoms. Despite well-documented histopathological characterisation of these lesion states, the host transcriptional programmes associated with viable parasite persistence and early post-treatment lesion disruption remain poorly understood. To address this gap, we performed the first bulk RNA sequencing of pericystic brain tissue using a physiologically relevant porcine model of NCC. Comparing uninfected controls (n = 3), infected untreated pigs with intact viable cysts (n = 6), and antiparasitic-treated pigs with disrupted cysts (n = 3), we identified distinct transcriptional signatures associated with each disease state. Viable infection was associated with broad transcriptional changes (461 upregulated and 175 downregulated genes), characterised by local immune activation alongside suppression of blood-brain barrier (BBB) remodelling, vascular, and neuronal signalling molecular signatures. The post-treatment state with confirmed BBB disruption was associated with a smaller but directionally distinct response (160 upregulated and 57 downregulated genes), marked by inflammatory signalling and increased expression of genes associated with endothelial activation, vascular regulation, and BBB-associated remodelling. Together, these findings suggest that, while immune engagement is a feature shared across both lesion states, the BBB-associated transcriptional axis shifts substantially following treatment. These results provide an exploratory transcriptomic framework for understanding parasite persistence, treatment-induced neuroinflammation, and neurovascular remodelling in NCC, and highlight candidate pathways and genes for future mechanistic investigation. Author SummaryNeurocysticercosis is a major cause of epilepsy in regions where Taenia solium is endemic. Brain cysts can remain viable for long periods with limited symptoms, but parasite degeneration, whether spontaneous or drug-induced, can trigger damaging neuroinflammation. In this study, we used RNA sequencing in a pig model that closely resembles human disease to characterise how brain tissue responds to viable cysts and to early treatment-induced cyst disruption. We found that viable infection was associated with local immune activation alongside reduced expression of genes involved in blood-brain barrier function. Following antiparasitic treatment, disrupted lesions showed an increased expression of genes linked to vascular and barrier remodelling. These findings suggest that the host transcriptional environment changes substantially after parasite disruption, and highlight molecular pathways that may contribute to neuroinflammation, blood-brain barrier changes, and neurological disease in NCC. As an exploratory first transcriptomic survey in this model, these results provide a candidate framework for future studies aimed at identifying biomarkers and adjunctive therapeutic targets in NCC.

genomics↗

bioRxiv2024

Tiled Amplicon Sequencing Enables Culture-free Whole-Genome Sequencing of Pathogenic Bacteria From Clinical Specimens

Pathogen sequencing is an important tool for disease surveillance and demonstrated its high value during the COVID-19 pandemic. Viral sequencing during the pandemic allowed us to track disease spread, quickly identify new variants, and guide the development of vaccines. Tiled amplicon sequencing, in which a panel of primers is used for multiplex amplification of fragments across an entire genome, was the cornerstone of SARS-CoV-2 sequencing. The speed, reliability, and cost-effectiveness of this method led to its implementation in academic and public health laboratories across the world and adaptation to a broad range of viral pathogens. However, similar methods are not available for larger bacterial genomes, for which whole-genome sequencing typically requires in vitro culture. This increases costs, error rates and turnaround times. The need to culture poses particular problems for medically important bacteria such as Mycobacterium tuberculosis, which are slow to grow and challenging to culture. As a proof of concept, we developed two novel whole-genome amplicon panels for M. tuberculosis and Streptococcus pneumoniae. Applying our amplicon panels to clinical samples, we show the ability to classify pathogen subgroups and to reliably identify markers of drug resistance without culturing. Development of this work in clinical settings has the potential to dramatically reduce the time of diagnosis of drug resistance for multiple drugs in parallel, enabling earlier intervention for high priority pathogens.

genomics↗

bioRxiv2022

Amplicon sequencing reveals complex infection in infants congenitally infected with Trypanosoma cruzi and informs the dynamics of parasite transmission

Congenital transmission of Trypanosoma cruzi, the causative agent of Chagas disease, is an important source of new infections worldwide. The mechanisms of congenital transmission remain poorly understood, but there is evidence that parasite factors could play a role. Investigating changes in parasite strain diversity during transmission could provide insight into the parasite factors that influence the process. Here we use deep amplicon sequencing of a single copy gene in the T. cruzi genome to evaluate the diversity of infection in a collection of clinical blood samples from Chagas positive mothers and their infected infants. We found several infants and mothers infected with more than two parasite haplotypes, indicating infection with multiple parasite strains. Two haplotypes were detected exclusively in infant samples, while one haplotype was never found in infants, suggesting a relationship between the probability of transmission and parasite genotype. Finally, we found an increase in parasite population diversity in children after birth compared to their mothers, suggesting that there is no transmission bottleneck during congenital infection and that multiple parasites breach the placenta in the course of congenital transmission.

microbiology↗

bioRxiv2022

Assembly and phylogeographic analysis of novel Taenia solium mitochondrial genomes reveal further differentiation between and within Asian and African-American genotypes

BackgroundTaenia solium is a parasite that hampers human health, causing taeniasis and cysticercosis. The genetic variability in its mitochondrial genome is related to the geographical origin of the specimen. Two main genotypes have been identified: The Asian and the African-American. The geographic genetic variability is expected to cause different clinical manifestations. Thus, characterizing differences between and within genotypes is crucial for completing the epidemiology of T. solium diseases. Methods/Principal FindingsHere, two Peruvian (one complete and one partial; 7,811X and 42X of coverage, respectively) and one Mexican (complete, 3,395X) T. solium mitochondrial genomes were assembled using the Chinese reference. Variant calling with respect to the reference was performed. Thirteen SNPs that involved a change in the amino acid physicochemical nature were identified. Those were present in all the assembled genomes and might be linked to differences in aerobic respiration efficiency between Latin American (African-American) and Asian genotypes. Then, phylogeographic studies were conducted using Cytochrome C oxidase subunit I and cytochrome B from these genomes and other isolates. The analysis showed that Indonesian samples are the most ancient and related to the modern T. solium ancestor of the Asian genotype. Finally, a consistent subdivision of the African-American genotype into two subgroups was found. One subgroup relates to East African countries, while the other is West Africa. The East African linage suggests a previously unnoticed influence of the Indian Ocean trade in the genetic structure of Latin America T. solium. Conclusions/SignificanceOverall, this study reports novel mitochondrial genomes valuable for further studies. New Latin American SNPs were identified and suggest metabolic differences between parasites of the Asian and African-American genotypes. Moreover, the phylogeographic analysis revealed differences within each genotype that shed light on T. soliums historical spread. Overall, the results represent an important step in completing T. solium genetic epidemiology. Author SummaryTaenia solium is a human parasite that causes taeniasis and cysticercosis. Eradicated from developed countries, they are still a public health problem in developing nations. T. solium differences in the mitochondrial genetic material depend on its geographical origin. This is expected to cause different clinical manifestations. Despite the importance of genetics to the epidemiology of T. solium diseases, few efforts have been made to assemble and compare their genomes. We aimed to help fill this knowledge gap by assembling three mitochondrial genomes from Latin America and comparing them to the Chinese reference. Additionally, two genes from the Latin American genomes and from other isolates were employed to assess T. solium genetic distribution. We found thirteen mutations with respect to the Chinese genome present in all Latin American samples, which involved a change in the amino acid physicochemical nature. Those might be causing metabolic differences between Asian and Latin American parasites that could change their affinity to specific human tissues. Moreover, we determined that Indonesian samples are the most ancient and related to the modern T. solium ancestor. Finally, we identified a previously unnoticed influence of East African countries in T. solium phylogeny, with which our assembled genomes are closely related.

genetics↗

bioRxiv2021

A recombinant SARS-CoV-2 RBD antigen expressed in insect cells elicits immunogenicity and confirms safety in animal models

COVID-19 pandemic has accelerated the development of vaccines against its etiologic agent, SARS-CoV-2. However, the emergence of new variants of the virus requires new immunization strategies in addition to the current vaccines approved for human administration. In the present report, the immunological and safety evaluation in mice and hamsters of a subunit vaccine based on the RBD sub-domain with two adjuvants of oil origin is described. The RBD protein was expressed in insect cells and purified by chromatography until >95% purity. The protein was shown to have the appropriate folding as determined by ELISA and flow cytometry binding assays to its receptor, as well as by its detection by hamster immune anti-S1 sera under non-reducing conditions. In immunization assays in mice and hamsters, the purified RBD formulated with adjuvants based on oil-water emulsifications and squalene was able to stimulate specific neutralizing antibodies and confirm the secretion of IFN-{gamma} after stimulating spleen cells with the purified RBD. The vaccine candidate was shown to be safe, as demonstrated by the histopathological analysis in lungs, liver and kidney. These results demonstrate the potential of the purified RBD administered with adjuvants through an intramuscular route, to be evaluated in a challenge against SARS-CoV-2 and determine its ability to confer protection against infection.

immunology↗

bioRxiv2021

Silencing of an efflux pump coding gene decreases the efflux rate of pyrazinoic acid in Mycobacterium smegmatis

BackgroundTuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (MTB). The recommended treatment for TB is based on the use of first-line drugs, including pyrazinamide (PZA). PZA is also a drug used in the treatment of multidrug-resistant TB (MDR-TB) because of its main effect against the latent stage. The main cause of resistance to PZA is mutations in the pncA gene, which compromise the activity of the encoded enzyme pyrazinamidase (PZAse), which hydrolyzes PZA into POA, the active antituberculosis molecule. The mechanism of action of PZA requires that POA is expelled from the bacterium by an efflux mechanism. After that, if the extracellular medium is sufficiently acidic, POA is protonated and returns to the cytosol, releasing the proton and repeating the cycle, resulting lethal to the bacteria. The efflux pump responsible for extruding the POA to the extracellular environment is not yet known. Mycobacterium smegmatis is naturally resistant to PZA and has a 900-fold faster POA efflux rate than MTB, and has the advantage to be a faster growing mycobacterium. MethodsIn the present study we have silenced the transcription of several genes encoding efflux pumps in M. smegmatis by CRISPRi (CRISPR interference). These genes (MSMEG_0250, MSMEG_3815, MSMEG_0241, MSMEG_5046 and MSMEG_0410) were homologous to efflux pump genes in MTB. POA efflux rate was measured, and a quantitative Waynes test was performed after silencing each gene. ResultsSilencing of MSMEG_0250, resulted in approximately 5-fold decrease in the POA efflux rate in M. smegmatis (P<0.0001). None of the other silenced genes showed a notable decrease in POA efflux rate.

molecular biology↗

bioRxiv2021

Evidence of neutralizing antibodies against SARS-CoV-2 in domestic cats living with owners with a history of COVID-19 in Lima, Peru

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.

immunology↗

bioRxiv2020

Laying Hens Affected By Newcastle Virus And Laryngotracheitis Virus Show Rapid Recovery After Treatment With Ivermectin

This is an anecdotal observation of an intervention study involving laying hens from a commercial farm in the city of Chincha, Peru, who suffered an outbreak caused by Newcastle disease virus (NDV) and infectious laryngotracheitis virus (ILTV), confirmed by clinical observations, serological and molecular tests (PCR). In addition to receiving standard treatment appropriate to the state of health at the time of the operation, a group of birds were treated with a single dose of ivermectin administered subcutaneously (0.2 mL of a 1% solution equivalent to 200 {micro}g/kg body weight), with a group of control birds not receiving the treatment being reserved. The results showed a remarkable recovery of symptoms after 24 hours of treatment among the birds that received ivermectin. At 4 days after treatment, the birds that received ivermectin showed visibly greater mobility and vivacity, as well as a recovery in egg production. PCR tests after 4 days of treatment with ivermectin were negative for NDV and ILTV. These results are interesting and suggest a possible effect of ivermectin against NDV and ILTV in birds. More controlled studies are needed to confirm this hypothesis.

microbiology↗

bioRxiv2020

Low-cost 3D-printed inverted microscope to detect Mycobacterium tuberculosis in a MODS culture

BackgroundThe MODS is an important assay for early diagnosis of tuberculosis and drug susceptibility. MODS is based in the microscopic observation, underneath, of the characteristic cords of Mycobacterium tuberculosis colonies grown in liquid media. An inverted optical microscope is required to observe and interpret MODS cultures. Unfortunately, the cost of commercial inverted microscopes is not affordable in low resource settings in developing countries. MethodologyTo 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 with a 3D printer and based on a smartphone. The system was evaluated by comparison of manual interpretations of 226 TB positive MODS culture images and 207 negative MODS culture images. SignificanceThe prototype resulted in a low-cost inverted optical microscope, with simple functioning, and whose parts have been manufactured using 3D printing techniques. The quality of the images was good enough and achieved a 100% concordance between the manual inspection with the developed microscope, and the standard diagnostics of MODS.

microbiology↗

bioRxiv2020

EXPRESSION OF Mycobacterium tuberculosis RpsA IN Mycobacterium smegmatis INCREASES SUSCEPTIBILITY TO PYRAZINAMIDE

Pyrazinamide (PZA) is one of the most important drugs used in combined antituberculous therapy. After the drug enters Mycobacterium tuberculosis it is hydrolyzed by pyrazinamidase (PZAse) to the bactericidal molecule pyrazinoic acid (POA). Ribosomal protein S1 (RpsA) was recently identified as a possible target of PZA based on its binding activity to POA and capacity to inhibit trans-translation. However, its role is not completely understood. It has been proposed that Mycobacterium smegmatis RpsA is not capable of binding POA, unlike M. tuberculosis RpsA. This may be due to the different amino acid sequence in the carboxy-terminal region of the two molecules: in M. smegmatis RpsA it is much closer to the sites that may interact with POA than in M. tuberculosis RpsA. These differences could be contributing, along with the presence of highly active POA efflux, to the natural resistance to PZA in M. smegmatis. To further understand the mechanisms of action of PZA and the role of RpsA in PZA susceptibility, we evaluated the effect of complementing M. tuberculosis RpsA expression in M. smegmatis using pNIT mycobacterial non-integrative expression vector and then performed a PZA susceptibility test determining the minimum inhibitory concentration (MIC) of PZA. It was expected that chimeric ribosomes comprising M. tuberculosis RpsA may be present and may affect PZA susceptibility. Our results showed a reduction in PZA MIC in M. smegmatis complemented with overexpressed M. tuberculosis RpsA compared to non-overexpressed M. smegmatis (468 {micro}g/mL and >7500 {micro}g/mL respectively).

microbiology↗

bioRxiv2017

The relative fitness of drug resistant Mycobacterium tuberculosis: a modelling study of household transmission in Lima, Peru

The relative fitness of drug resistant versus susceptible bacteria in an environment dictates resistance prevalence. Estimates for the relative fitness of resistant Mycobacterium tuberculosis (Mtb) strains are highly heterogeneous and mostly derived from in-vitro experiments. Measuring fitness in the field allows us to determine how the environment influences resistance spread.\n\nWe designed a household structured, stochastic mathematical model to estimate the fitness costs associated with multi-drug resistance (MDR) carriage in Mtb in Lima, Peru between 2010-2013. By fitting the model to data from a large prospective cohort study of TB disease in household contacts we estimated the fitness, relative to susceptible strains with a fitness of 1, of MDR-Mtb to be 0.33 (95% credible interval: 0.17-0.54) or 0.39 (0.26-0.58), if only transmission or progression to disease, respectively, was affected. The relative fitness of MDR-Mtb increased to 0.57 (0.43-0.73) when the fitness cost influenced both transmission and progression to disease equally.\n\nWe found the average relative fitness of MDR-Mtb circulating within households in Lima, Peru between 2010-2013 to be significantly lower than concurrent susceptible-Mtb. If these fitness levels do not change, then existing TB control programmes are likely to keep MDR-TB prevalence at current levels in Lima, Peru.

epidemiology↗