Dispersal history of SARS-CoV-2 in Galicia, Spain.

The dynamics of SARS-CoV-2 transmission are influenced by a variety of factors, including social restrictions and the emergence of distinct variants. In this study, we delve into the origins and dissemination of the Alpha, Delta, and Omicron variants of concern in Galicia, northwest Spain. For this, we leveraged genomic data collected by the EPICOVIGAL Consortium and from the GISAID database, along with mobility information from other Spanish regions and foreign countries. Our analysis indicates that initial introductions during the Alpha phase were predominantly from other Spanish regions and France. However, as the pandemic progressed, introductions from Portugal and the USA became increasingly significant. Notably, Galicia's major coastal cities emerged as critical hubs for viral transmission, highlighting their role in sustaining and spreading the virus. This research emphasizes the critical role of regional connectivity in the spread of SARS-CoV-2 and offers essential insights for enhancing public health strategies and surveillance measures.

Demystifying dimensionality reduction techniques in the 'omics' era: A practical approach for biological science students.

Dimensionality reduction techniques are essential in analyzing large 'omics' datasets in biochemistry and molecular biology. Principal component analysis, t-distributed stochastic neighbor embedding, and uniform manifold approximation and projection are commonly used for data visualization. However, these methods can be challenging for students without a strong mathematical background. In this study, intuitive examples were created using COVID-19 data to help students understand the core concepts behind these techniques. In a 4-h practical session, we used these examples to demonstrate dimensionality reduction techniques to 15 postgraduate students from biomedical backgrounds. Using Python and Jupyter notebooks, our goal was to demystify these methods, typically treated as "black boxes", and empower students to generate and interpret their own results. To assess the impact of our approach, we conducted an anonymous survey. The majority of the students agreed that using computers enriched their learning experience (67%) and that Jupyter notebooks were a valuable part of the class (66%). Additionally, 60% of the students reported increased interest in Python, and 40% gained both interest and a better understanding of dimensionality reduction methods. Despite the short duration of the course, 40% of the students reported acquiring research skills necessary in the field. While further analysis of the learning impacts of this approach is needed, we believe that sharing the examples we generated can provide valuable resources for others to use in interactive teaching environments. These examples highlight advantages and limitations of the major dimensionality reduction methods used in modern bioinformatics analysis in an easy-to-understand way.

Artemisinin resistance-associated gene mutations in Plasmodium falciparum: A case study of severe malaria from Mozambique.

BACKGROUND: The effectiveness of artemisinin-based combination therapies (ACT) in treating Plasmodium falciparum, is vital for global malaria control efforts, particularly in sub-Saharan Africa. The examination of imported cases from endemic areas holds implications for malaria chemotherapy on a global scale. METHOD: A 45-year-old male presented with high fever, dry cough, diarrhoea and generalized muscle pain, following a two-week trip to Mozambique. P. falciparum infection with hiperparasitemia was confirmed and the patient was treated initially with quinine and doxycycline, then intravenous artesunate. To assess drug susceptibility, ex vivo half-maximal inhibitory concentration assays were conducted, and the isolated P. falciparum genome was deep sequenced. RESULTS: The clinical isolate exhibited elevated ex vivo half-maximal inhibitory concentration values to dihydroartemisinin, lumefantrine, mefloquine and piperaquine. Genomic analysis identified a I416V mutation in the P. falciparum Kelch13 (PF3D7_1343700) gene, and several mutations at the Kelch13 interaction candidate genes, pfkics (PF3D7_0813000, PF3D7_1138700, PF3D7_1246300), including the ubiquitin carboxyl-terminal hydrolase 1, pfubp1 (PF3D7_0104300). Mutations at the drug transporters and genes linked to next-generation antimalarial drug resistance were also present. CONCLUSIONS: This case highlights the emergence of P. falciparum strains carrying mutations in artemisinin resistance-associated genes in Mozambique, couple with a reduction in ex vivo susceptibility to ACT drugs. Continuous surveillance of mutations linked to drug resistance and regular monitoring of drug susceptibility are imperative to anticipate the spread of potential resistant strains emerging in Mozambique and to maintain effective malaria control strategies.

The impact of single-cell genomics on the field of mycobacterial infection.

Genome sequencing projects of humans and other organisms reinforced that the complexity of biological systems is largely attributed to the tight regulation of gene expression at the epigenome and RNA levels. As a consequence, plenty of technological developments arose to increase the sequencing resolution to the cell dimension creating the single-cell genomics research field. Single-cell RNA sequencing (scRNA-seq) is leading the advances in this topic and comprises a vast array of different methodologies. scRNA-seq and its variants are more and more used in life science and biomedical research since they provide unbiased transcriptomic sequencing of large populations of individual cells. These methods go beyond the previous "bulk" methodologies and sculpt the biological understanding of cellular heterogeneity and dynamic transcriptomic states of cellular populations in immunology, oncology, and developmental biology fields. Despite the large burden caused by mycobacterial infections, advances in this field obtained via single-cell genomics had been comparatively modest. Nonetheless, seminal research publications using single-cell transcriptomics to study host cells infected by mycobacteria have become recently available. Here, we review these works summarizing the most impactful findings and emphasizing the different and recent single-cell methodologies used, potential issues, and problems. In addition, we aim at providing insights into current research gaps and potential future developments related to the use of single-cell genomics to study mycobacterial infection.

PMMA Microcapsules for the Inactivation of SARS-CoV-2.

Surface disinfection currently plays a decisive role in the epidemiological situation caused by the SARS-CoV-2 coronavirus. However, most disinfection products available on the market have a high evaporation rate and only an immediate action and not continuous, creating the need for a high frequency of disinfection. To overcome this limitation, in the present work, poly(methyl methacrylate) (PMMA) microcapsules were developed with an active agent (hydrogen peroxide) encapsulated, which has the ability to inactivate/neutralize the SARS-CoV-2 virus. PMMA-H2O2 microcapsules have a spherical shape and a smooth structure with low porosity and were successfully attached to nonwoven fabrics, as observed from scanning electron microscopy. The thermogravimetric analysis shows that PMMA-H2O2 microcapsules have high thermal stability and can increase the stability of H2O2. Nonfabric substrates functionalized with PMMA-H2O2 microcapsules were tested by a highly sensitive and specific reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR)-based method to evaluate antiviral activity through the degradation of SARS-CoV-2 deoxyribonucleic acids. The highest percentage of viral nucleic acid elimination was obtained when exposing the viral sample for 1 h to PMMA-H2O2 microcapsules, resulting in an elimination of >97% of the coronavirus. In addition, the microcapsules are stable over a period of three weeks and retain the ability to eliminate SARS-CoV-2. Hence, it is demonstrated that this microcapsule system is efficient for SARS-CoV-2 elimination and inherent surface disinfection.

Tuberculosis caused by Mycobacterium africanum: Knowns and unknowns.

Tuberculosis (TB), one of the deadliest threats to human health, is mainly caused by 2 highly related and human-adapted bacteria broadly known as Mycobacterium tuberculosis and Mycobacterium africanum. Whereas M. tuberculosis is widely spread, M. africanum is restricted to West Africa, where it remains a significant cause of tuberculosis. Although several differences have been identified between these 2 pathogens, M. africanum remains a lot less studied than M. tuberculosis. Here, we discuss the genetic, phenotypic, and clinical similarities and differences between strains of M. tuberculosis and M. africanum. We also discuss our current knowledge on the immune response to M. africanum and how it possibly articulates with distinct disease progression and with the geographical restriction attributed to this pathogen. Understanding the functional impact of the diversity existing in TB-causing bacteria, as well as incorporating this diversity in TB research, will contribute to the development of better, more specific approaches to tackle TB.

Development of an Ultraviolet-C Irradiation Room in a Public Portuguese Hospital for Safe Re-Utilization of Personal Protective Respirators.

Almost two years have passed since COVID-19 was officially declared a pandemic by the World Health Organization. However, it still holds a tight grasp on the entire human population. Several variants of concern, one after another, have spread throughout the world. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) omicron variant may become the fastest spreading virus in history. Therefore, it is more than evident that the use of personal protective equipment (PPE) will continue to play a pivotal role during the current pandemic. This work depicts an integrative approach attesting to the effectiveness of ultra-violet-C (UV-C) energy density for the sterilization of personal protective equipment, in particular FFP2 respirators used by the health care staff in intensive care units. It is increasingly clear that this approach should not be limited to health care units. Due to the record-breaking spreading rates of SARS-CoV-2, it is apparent that the use of PPE, in particular masks and respirators, will remain a critical tool to mitigate future pandemics. Therefore, similar UV-C disinfecting rooms should be considered for use within institutions and companies and even incorporated within household devices to avoid PPE shortages and, most importantly, to reduce environmental burdens.

The evolution of the HIV-1 protease folding stability.

The evolution of structural proteins is generally constrained by the folding stability. However, little is known about the particular capacity of viral proteins to accommodate mutations that can potentially affect the protein stability and, in general, the evolution of the protein stability over time. As an illustrative model case, here, we investigated the evolution of the stability of the human immunodeficiency virus (HIV-1) protease (PR), which is a common HIV-1 drug target, under diverse evolutionary scenarios that include (1) intra-host virus evolution in a cohort of seventy-five patients sampled over time, (2) intra-host virus evolution sampled before and after specific PR-based treatments, and (3) inter-host evolution considering extant and ancestral (reconstructed) PR sequences from diverse HIV-1 subtypes. We also investigated the specific influence of currently known HIV-1 PR resistance mutations on the PR folding stability. We found that the HIV-1 PR stability fluctuated over time within a constant and wide range in any studied evolutionary scenario, accommodating multiple mutations that partially affected the stability while maintaining activity. We did not identify relationships between change of PR stability and diverse clinical parameters such as viral load, CD4+ T-cell counts, and a surrogate of time from infection. Counterintuitively, we predicted that nearly half of the studied HIV-1 PR resistance mutations do not significantly decrease stability, which, together with compensatory mutations, would allow the protein to adapt without requiring dramatic stability changes. We conclude that the HIV-1 PR presents a wide structural plasticity to acquire molecular adaptations without affecting the overall evolution of stability.

The Neglected Contribution of Streptomycin to the Tuberculosis Drug Resistance Problem.

The airborne pathogen Mycobacterium tuberculosis is responsible for a present major public health problem worsened by the emergence of drug resistance. M. tuberculosis has acquired and developed streptomycin (STR) resistance mechanisms that have been maintained and transmitted in the population over the last decades. Indeed, STR resistant mutations are frequently identified across the main M. tuberculosis lineages that cause tuberculosis outbreaks worldwide. The spread of STR resistance is likely related to the low impact of the most frequent underlying mutations on the fitness of the bacteria. The withdrawal of STR from the first-line treatment of tuberculosis potentially lowered the importance of studying STR resistance. However, the prevalence of STR resistance remains very high, could be underestimated by current genotypic methods, and was found in outbreaks of multi-drug (MDR) and extensively drug (XDR) strains in different geographic regions. Therefore, the contribution of STR resistance to the problem of tuberculosis drug resistance should not be neglected. Here, we review the impact of STR resistance and detail well-known and novel candidate STR resistance mechanisms, genes, and mutations. In addition, we aim to provide insights into the possible role of STR resistance in the development of multi-drug resistant tuberculosis.

Evolutionary dynamics of HIV-1 subtype C in Brazil.

The extensive genetic diversity of HIV-1 is a major challenge for the prevention and treatment of HIV-1 infections. Subtype C accounts for most of the HIV-1 infections in the world but has been mainly localized in Southern Africa, Ethiopia and India. For elusive reasons, South Brazil harbors the largest HIV-1 subtype C epidemic in the American continent that is elsewhere dominated by subtype B. To investigate this topic, we collected clinical data and viral sequences from 2611 treatment-naïve patients diagnosed with HIV-1 in Brazil. Molecular epidemiology analysis supported 35 well-delimited transmission clusters of subtype C highlighting transmission within South Brazil but also from the South to all other Brazilian regions and internationally. Individuals infected with subtype C had lower probability to be deficient in CD4+ T cells when compared to subtype B. The HIV-1 epidemics in the South was characterized by high female-to-male infection ratios and women-to-child transmission. Our results suggest that HIV-1 subtype C probably takes advantage of longer asymptomatic periods to maximize transmission and is unlikely to outcompete subtype B in settings where the infection of women is relatively less relevant. This study contributes to elucidate factors possibly underlying the geographical distribution and expansion patterns of the most spread HIV-1 subtypes.

OmniSARS2: A Highly Sensitive and Specific RT-qPCR-Based COVID-19 Diagnostic Method Designed to Withstand SARS-CoV-2 Lineage Evolution.

Extensive transmission of SARS-CoV-2 during the COVID-19 pandemic allowed the generation of thousands of mutations within its genome. While several of these become rare, others largely increase in prevalence, potentially jeopardizing the sensitivity of PCR-based diagnostics. Taking advantage of SARS-CoV-2 genomic knowledge, we designed a one-step probe-based multiplex RT-qPCR (OmniSARS2) to simultaneously detect short fragments of the SARS-CoV-2 genome in ORF1ab, E gene and S gene. Comparative genomics of the most common SARS-CoV-2 lineages, other human betacoronavirus and alphacoronavirus, was the basis for this design, targeting both highly conserved regions across SARS-CoV-2 lineages and variable or absent in other Coronaviridae viruses. The highest analytical sensitivity of this method for SARS-CoV-2 detection was 94.2 copies/mL at 95% detection probability (~1 copy per total reaction volume) for the S gene assay, matching the most sensitive available methods. In vitro specificity tests, performed using reference strains, showed no cross-reactivity with other human coronavirus or common pathogens. The method was compared with commercially available methods and detected the virus in clinical samples encompassing different SARS-CoV-2 lineages, including B.1, B.1.1, B.1.177 or B.1.1.7 and rarer lineages. OmniSARS2 revealed a sensitive and specific viral detection method that is less likely to be affected by lineage evolution oligonucleotide-sample mismatch, of relevance to ensure the accuracy of COVID-19 molecular diagnostic methods.

Characterizing HIV-1 Genetic Subtypes and Drug Resistance Mutations among Children, Adolescents and Pregnant Women in Sierra Leone.

Human immunodeficiency virus (HIV) drug resistance (HIVDR) is widespread in sub-Saharan Africa. Children and pregnant women are particularly vulnerable, and laboratory testing capacity remains limited. We, therefore, used a cross-sectional design and convenience sampling to characterize HIV subtypes and resistance-associated mutations (RAMs) in these groups in Sierra Leone. In total, 96 children (age 2-9 years, 100% ART-experienced), 47 adolescents (age 10-18 years, 100% ART-experienced), and 54 pregnant women (>18 years, 72% ART-experienced) were enrolled. Median treatment durations were 36, 84, and 3 months, respectively, while the sequencing success rates were 45%, 70%, and 59%, respectively, among children, adolescents, and pregnant women. Overall, the predominant HIV-1 subtype was CRF02_AG (87.9%, 95/108), with minority variants constituting 12%. Among children and adolescents, the most common RAMs were M184V (76.6%, n = 49/64), K103N (45.3%, n = 29/64), Y181C/V/I (28.1%, n = 18/64), T215F/Y (25.0%, n = 16/64), and V108I (18.8%, n = 12/64). Among pregnant women, the most frequent RAMs were K103N (20.6%, n = 7/34), M184V (11.8%, n = 4/34), Y181C/V/I (5.9%, n = 2/34), P225H (8.8%, n = 3/34), and K219N/E/Q/R (5.9%, n = 2/34). Protease and integrase inhibitor-RAMs were relatively few or absent. Based on the genotype susceptibility score distributions, 73%, 88%, and 14% of children, adolescents, and pregnant women, respectively, were not susceptible to all three drug components of the WHO preferred first-line regimens per 2018 guidelines. These findings suggest that routine HIVDR surveillance and access to better ART choices may improve treatment outcomes in Sierra Leone.

Plasmodium falciparum transcription in different clinical presentations of malaria associates with circulation time of infected erythrocytes.

Following Plasmodium falciparum infection, individuals can remain asymptomatic, present with mild fever in uncomplicated malaria cases, or show one or more severe malaria symptoms. Several studies have investigated associations between parasite transcription and clinical severity, but no broad conclusions have yet been drawn. Here, we apply a series of bioinformatic approaches based on P. falciparum's tightly regulated transcriptional pattern during its ~48-hour intraerythrocytic developmental cycle (IDC) to publicly available transcriptomes of parasites obtained from malaria cases of differing clinical severity across multiple studies. Our analysis shows that within each IDC, the circulation time of infected erythrocytes without sequestering to endothelial cells decreases with increasing parasitaemia or disease severity. Accordingly, we find that the size of circulating infected erythrocytes is inversely related to parasite density and disease severity. We propose that enhanced adhesiveness of infected erythrocytes leads to a rapid increase in parasite burden, promoting higher parasitaemia and increased disease severity.

Nationwide Study of Drug Resistance Mutations in HIV-1 Infected Individuals under Antiretroviral Therapy in Brazil.

The success of antiretroviral treatment (ART) is threatened by the emergence of drug resistance mutations (DRM). Since Brazil presents the largest number of people living with HIV (PLWH) in South America we aimed at understanding the dynamics of DRM in this country. We analyzed a total of 20,226 HIV-1 sequences collected from PLWH undergoing ART between 2008-2017. Results show a mild decline of DRM over the years but an increase of the K65R reverse transcriptase mutation from 2.23% to 12.11%. This increase gradually occurred following alterations in the ART regimens replacing zidovudine (AZT) with tenofovir (TDF). PLWH harboring the K65R had significantly higher viral loads than those without this mutation (p < 0.001). Among the two most prevalent HIV-1 subtypes (B and C) there was a significant (p < 0.001) association of K65R with subtype C (11.26%) when compared with subtype B (9.27%). Nonetheless, evidence for K65R transmission in Brazil was found both for C and B subtypes. Additionally, artificial neural network-based immunoinformatic predictions suggest that K65R could enhance viral recognition by HLA-B27 that has relatively low prevalence in the Brazilian population. Overall, the results suggest that tenofovir-based regimens need to be carefully monitored particularly in settings with subtype C and specific HLA profiles.

Heterogeneous Streptomycin Resistance Level Among Mycobacterium tuberculosis Strains From the Same Transmission Cluster.

Widespread and frequent resistance to the second-line tuberculosis (TB) medicine streptomycin, suggests ongoing transmission of low fitness cost streptomycin resistance mutations. To investigate this hypothesis, we studied a cohort of 681 individuals from a TB epidemic in Portugal. Whole-genome sequencing (WGS) analyses were combined with phenotypic growth studies in culture media and in mouse bone marrow derived macrophages. Streptomycin resistance was the most frequent resistance in the cohort accounting for 82.7% (n = 67) of the resistant Mycobacterium tuberculosis isolates. WGS of 149 clinical isolates identified 13 transmission clusters, including three clusters containing only streptomycin resistant isolates. The biggest cluster was formed by eight streptomycin resistant isolates with a maximum of five pairwise single nucleotide polymorphisms of difference. Interestingly, despite their genetic similarity, these isolates displayed different resistance levels to streptomycin, as measured both in culture media and in infected mouse bone marrow derived macrophages. The genetic bases underlying this phenotype are a combination of mutations in gid and other genes. This study suggests that specific streptomycin resistance mutations were transmitted in the cohort, with the resistant isolates evolving at the cluster level to allow low-to-high streptomycin resistance levels without a significative fitness cost. This is relevant not only to better understand transmission of streptomycin resistance in a clinical setting dominated by Lineage 4 M. tuberculosis infections, but mainly because it opens new prospects for the investigation of selection and spread of drug resistance in general.

Correction: A Prediction Rule to Stratify Mortality Risk of Patients with Pulmonary Tuberculosis.

[This corrects the article DOI: 10.1371/journal.pone.0162797.].

Increased circulation time of Plasmodium falciparum underlies persistent asymptomatic infection in the dry season.

The dry season is a major challenge for Plasmodium falciparum parasites in many malaria endemic regions, where water availability limits mosquito vectors to only part of the year. How P. falciparum bridges two transmission seasons months apart, without being cleared by the human host or compromising host survival, is poorly understood. Here we show that low levels of P. falciparum parasites persist in the blood of asymptomatic Malian individuals during the 5- to 6-month dry season, rarely causing symptoms and minimally affecting the host immune response. Parasites isolated during the dry season are transcriptionally distinct from those of individuals with febrile malaria in the transmission season, coinciding with longer circulation within each replicative cycle of parasitized erythrocytes without adhering to the vascular endothelium. Low parasite levels during the dry season are not due to impaired replication but rather to increased splenic clearance of longer-circulating infected erythrocytes, which likely maintain parasitemias below clinical and immunological radar. We propose that P. falciparum virulence in areas of seasonal malaria transmission is regulated so that the parasite decreases its endothelial binding capacity, allowing increased splenic clearance and enabling several months of subclinical parasite persistence.

Phagosomal removal of fungal melanin reprograms macrophage metabolism to promote antifungal immunity.

In response to infection, macrophages adapt their metabolism rapidly to enhance glycolysis and fuel specialized antimicrobial effector functions. Here we show that fungal melanin is an essential molecule required for the metabolic rewiring of macrophages during infection with the fungal pathogen Aspergillus fumigatus. Using pharmacological and genetic tools, we reveal a molecular link between calcium sequestration by melanin inside the phagosome and induction of glycolysis required for efficient innate immune responses. By remodeling the intracellular calcium machinery and impairing signaling via calmodulin, melanin drives an immunometabolic signaling axis towards glycolysis with activation of hypoxia-inducible factor 1 subunit alpha (HIF-1α) and phagosomal recruitment of mammalian target of rapamycin (mTOR). These data demonstrate a pivotal mechanism in the immunometabolic regulation of macrophages during fungal infection and highlight the metabolic repurposing of immune cells as a potential therapeutic strategy.