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.

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

The dynamics of severe acute respiratory syndrome coronavirus 2 (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-BA.1 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 United States became increasingly significant. The number of detected introductions varied from 96 and 101 for Alpha and Delta to 39 for Omicron-BA.1. Most of these introductions left a low number of descendants (<10), suggesting a limited impact on the evolution of the pandemic in Galicia. 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.

Deregulated immune cell recruitment orchestrated by c-MET impairs pulmonary inflammation and fibrosis.

BACKGROUND: Pulmonary fibrosis (PF) represents the pathologic end stage of several interstitial lung diseases (ILDs) associated with high morbidity and mortality rates. However, current treatments can only delay disease progression rather than provide a cure. The role of inflammation in PF progression is well-established, but new insights into immune regulation are fundamental for developing more efficient therapies. c-MET signaling has been implicated in the migratory capacity and effector functions of immune cells. Nevertheless, the role of this signaling pathway in the context of PF-associated lung diseases remains unexplored. METHODS: To determine the influence of c-MET in immune cells in the progression of pulmonary fibrosis, we used a conditional deletion of c-Met in immune cells. To induce pulmonary fibrosis mice were administered with bleomycin (BLM) intratracheally. Over the course of 21 days, mice were assessed for weight change, and after euthanasia at different timepoints, bronchoalveolar lavage fluid cells and lung tissue were assessed for inflammation and fibrosis. Furthermore, c-MET expression was assessed in cryobiopsy sections, bronchoalveolar lavage fluid cells samples and single cell RNA-sequencing dataset from human patients with distinct interstitial lung diseases. RESULTS: c-MET expression was induced in lung immune cells, specifically in T cells, interstitial macrophages, and neutrophils, during the inflammatory phase of BLM-induced PF mouse model. Deletion of c-Met in immune cells correlated with earlier weight recovery and improved survival of BLM-treated mice. Moreover, the deletion of c-Met in immune cells was associated with early recruitment of the immune cell populations, normally found to express c-MET, leading to a subsequent attenuation of the cytotoxic and proinflammatory environment. Consequently, the less extensive inflammatory response, possibly coupled with tissue repair, culminated in less exacerbated fibrotic lesions. Furthermore, c-MET expression was up-regulated in lung T cells from patients with fibrosing ILD, suggesting a potential involvement of c-MET in the development of fibrosing disease. CONCLUSIONS: These results highlight the critical contribution of c-MET signaling in immune cells to their enhanced uncontrolled recruitment and activation toward a proinflammatory and profibrotic phenotype, leading to the exacerbation of lung injury and consequent development of fibrosis.

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.

Analysis of a local HIV-1 epidemic in portugal highlights established transmission of non-B and non-G subtypes.

The existing data support Portugal as the western European country with the highest HIV-1 subtype diversity. However, detailed phylogenetic studies of Portuguese HIV-1 epidemics are still scarce. Thus, our main goal was to analyze the phylodynamics of a local HIV-1 infection in the Portuguese region of Minho. Molecular epidemiological analysis was applied to data from 289 HIV-1-infected individuals followed at the reference hospital of the province of Minho, Portugal, at which isolated viruses had been sequenced between 2000 and 2012. Viruses of the G (29.1%) and B (27.0%) subtypes were the most frequent, followed by recombinant forms (17.6%) and the C (14.5%), F1 (7.3%), and A1 (4.2%) subtypes. Multinomial logistic regression revealed that the odds of being infected with the A1 and F1 subtypes increased over the years compared with those with B, G, or C subtypes or recombinant viruses. As expected, polyphyletic patterns suggesting multiple and old introductions of the B and G subtypes were found. However, transmission clusters of non-B and non-G viruses among native individuals were also found, with the dates of the most recent common ancestor estimated to be in the early 2000s. Our study supports that the HIV-1 subtype diversity in the Portuguese region of Minho is high and has been increasing in a manner that is apparently driven by factors other than immigration and international travel. Infections with A1 and F1 viruses in the region of Minho are becoming established and are mainly found in sexually transmitted clusters, reinforcing the need for more efficacious control measures targeting this infection route.

Evidence for diversifying selection in a set of Mycobacterium tuberculosis genes in response to antibiotic- and nonantibiotic-related pressure.

Tuberculosis (TB) is a global health problem estimated to kill 1.4 million people per year. Recent advances in the genomics of the causative agents of TB, bacteria known as the Mycobacterium tuberculosis complex (MTBC), have allowed a better comprehension of its population structure and provided the foundation for molecular evolution analyses. These studies are crucial for a better understanding of TB, including the variation of vaccine efficacy and disease outcome, together with the emergence of drug resistance. Starting from the analysis of 73 publicly available genomes from all the main MTBC lineages, we have screened for evidences of positive selection, a set of 576 genes previously associated with drug resistance or encoding membrane proteins. As expected, because antibiotics constitute strong selective pressure, some of the codons identified correspond to the position of confirmed drug-resistance-associated substitutions in the genes embB, rpoB, and katG. Furthermore, we identified diversifying selection in specific codons of the genes Rv0176 and Rv1872c coding for MCE1-associated transmembrane protein and a putative l-lactate dehydrogenase, respectively. Amino acid sequence analyses showed that in Rv0176, sites undergoing diversifying selection were in a predicted antigen region that varies between "modern" lineages and "ancient" MTBC/BCG strains. In Rv1872c, some of the sites under selection are predicted to impact protein function and thus might result from metabolic adaptation. These results illustrate that diversifying selection in MTBC is happening as a consequence of both antibiotic treatment and other evolutionary pressures.

Complex polymorphisms in the Plasmodium falciparum multidrug resistance protein 2 gene and its contribution to antimalarial response.

Plasmodium falciparum has the capacity to escape the actions of essentially all antimalarial drugs. ATP-binding cassette (ABC) transporter proteins are known to cause multidrug resistance in a large range of organisms, including the Apicomplexa parasites. P. falciparum genome analysis has revealed two genes coding for the multidrug resistance protein (MRP) type of ABC transporters: Pfmrp1, previously associated with decreased parasite drug susceptibility, and the poorly studied Pfmrp2. The role of Pfmrp2 polymorphisms in modulating sensitivity to antimalarial drugs has not been established. We herein report a comprehensive account of the Pfmrp2 genetic variability in 46 isolates from Thailand. A notably high frequency of 2.8 single nucleotide polymorphisms (SNPs)/kb was identified for this gene, including some novel SNPs. Additionally, we found that Pfmrp2 harbors a significant number of microindels, some previously not reported. We also investigated the potential association of the identified Pfmrp2 polymorphisms with altered in vitro susceptibility to several antimalarials used in artemisinin-based combination therapy and with parasite clearance time. Association analysis suggested Pfmrp2 polymorphisms modulate the parasite's in vitro response to quinoline antimalarials, including chloroquine, piperaquine, and mefloquine, and association with in vivo parasite clearance. In conclusion, our study reveals that the Pfmrp2 gene is the most diverse ABC transporter known in P. falciparum with a potential role in antimalarial drug resistance.

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.

Antibiotic resistance: Assessing knowledge and misconceptions among Portuguese students and science teachers.

Antimicrobial resistance poses one of the most significant medical challenges for humanity. The current burden is overwhelming and is projected to escalate rapidly, with predictions for 2050 indicating 10 million deaths per year due to antibiotic-resistant microorganisms. Enhancing public awareness and education on this topic is crucial in efforts to mitigate this issue. In our study, we translated an existing questionnaire on antimicrobial resistance into Portuguese, validated it, and applied it between December 2020 and March 2021 to a group of Portuguese students (n = 112) and science teachers (n = 95). A majority of the students surveyed (65.1%) incorrectly believed that antibiotics could treat colds/flus. As anticipated, the teachers outperformed the students in the questionnaire. However, difficulties with this topic were evident in both groups. Most notably, the misconception that the human body becomes resistant to antibiotics was prevalent among most participants (77.0% of students and 68.4% of teachers). Consistent with previous studies in other populations and geographic locations, our research reveals a worrying lack of knowledge about antimicrobial resistance among Portuguese students and science teachers. Consequently, it is deemed urgent to implement effective measures to raise awareness and educate on this topic.

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.

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.

Morphological heterogeneity of Paracoccidioides brasiliensis: relevance of the Rho-like GTPase PbCDC42.

Paracoccidioides brasiliensis budding pattern and polymorphic growth were previously shown to be closely linked to the expression of PbCDC42 and to influence the pathogenesis of the fungus. In this work we conducted a detailed morphogenetic evaluation of the yeast-forms of 11 different clinical and environmental P. brasiliensis isolates comprising four phylogenetic lineages (S1, PS2, PS3 and Pb01-like), as well as a PbCDC42 knock-down strain. High variations in the shape and size of mother and bud cells of each isolate were observed but we did not find a characteristic morphologic profile for any of the phylogenetic groups. In all isolates studied, the bud size and shape were demonstrated to be highly dependent on the mother cell. Importantly, we found strong correlations between PbCDC42 expression and both the shape of mother and bud cells and the size of the buds in all isolates and the knock-down strain. Our results suggested that PbCDC42 expression can explain approximately 80% of mother and bud cell shape and 19% of bud cell size. This data support PbCDC42 expression level as being a relevant predictor of P. brasiliensis morphology. Altogether, these findings quantitatively describe the polymorphic nature of the P. brasiliensis yeast form and provide additional support for the key role of PbCDC42 expression on yeast cell morphology.

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.

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.

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.

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 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.

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.