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Reconstructing the evolutionary origins of Mycobacterium tuberculosis, the causative agent of human tuberculosis, has helped identify bacterial factors that have led to the tubercle bacillus becoming such a formidable human pathogen. Here we report the discovery and detailed characterization of an exceedingly slow growing mycobacterium that is closely related to M. tuberculosis for which we have proposed the species name Mycobacterium spongiae sp. nov., (strain ID: FSD4b-SM). The bacterium was isolated from a marine sponge, taken from the waters of the Great Barrier Reef in Queensland, Australia. Comparative genomics revealed that, after the opportunistic human pathogen Mycobacterium decipiens, M. spongiae is the most closely related species to the M. tuberculosis complex reported to date, with 80% shared average nucleotide identity and extensive conservation of key M. tuberculosis virulence factors, including intact ESX secretion systems and associated effectors. Proteomic and lipidomic analyses showed that these conserved systems are functional in FSD4b-SM, but that it also produces cell wall lipids not previously reported in mycobacteria. We investigated the virulence potential of FSD4b-SM in mice and found that, while the bacteria persist in lungs for 56 days after intranasal infection, no overt pathology was detected. The similarities with M. tuberculosis, together with its lack of virulence, motivated us to investigate the potential of FSD4b-SM as a vaccine strain and as a genetic donor of the ESX-1 genetic locus to improve BCG immunogenicity. However, neither of these approaches resulted in superior protection against M. tuberculosis challenge compared to BCG vaccination alone. The discovery of M. spongiae adds to our understanding of the emergence of the M. tuberculosis complex and it will be another useful resource to refine our understanding of the factors that shaped the evolution and pathogenesis of M. tuberculosis.

ABSTRACT Four species of non-tuberculous mycobacteria (NTM) rated as biosafety level 1 or 2 (BSL-1/BSL-2) organisms and showing higher genomic similarity with Mycobacterium tuberculosis (Mtb) than previous comparator species Mycobacterium kansasii and Mycobacterium marinum were subjected to genomic and phenotypic characterization. These species named Mycobacterium decipiens, Mycobacterium lacus, Mycobacterium riyadhense, and Mycobacterium shinjukuense might represent “missing links” between low-virulent mycobacterial opportunists and the highly virulent obligate pathogen Mtb. We confirmed that M. decipiens is the closest NTM species to Mtb currently known and found that it has an optimal growth temperature of 32°C–35°C and not 37°C. M. decipiens showed resistance to rifampicin, isoniazid, and ethambutol, whereas M. lacus and M. riyadhense showed resistance to isoniazid and ethambutol. M. shinjukuense was sensitive to all three first-line TB drugs, and all four species were sensitive to bedaquiline, a third-generation anti-TB drug. Our results suggest these four NTM may be useful models for the identification and study of new anti-TB molecules, facilitated by their culture under non-BSL-3 conditions as compared to Mtb. M. riyadhense was the most virulent of the four species in cellular and mouse infection models. M. decipiens also multiplied in THP-1 cells at 35°C but was growth impaired at 37°C. Genomic comparisons showed that the espACD locus, essential for the secretion of ESX-1 proteins in Mtb, was present only in M. decipiens, which was able to secrete ESAT-6 and CFP-10, whereas secretion of these antigens varied in the other species, making the four species interesting examples for studying ESX-1 secretion mechanisms.IMPORTANCEIn this work, we investigated recently identified opportunistic mycobacterial pathogens that are genomically more closely related to Mycobacterium tuberculosis (Mtb) than previously used comparator species Mycobacterium kansasii and Mycobacterium marinum. We confirmed that Mycobacterium decipiens is the currently closest known species to the tubercle bacilli, represented by Mycobacterium canettii and Mtb strains. Surprisingly, the reference strain of Mycobacterium riyadhense (DSM 45176), which was purchased as a biosafety level 1 (BSL-1)-rated organism, was the most virulent of the four species in the tested cellular and mouse infection models, suggesting that a BSL-2 rating might be more appropriate for this strain than the current BSL-1 rating. Our work establishes the four NTM species as interesting study models to obtain new insights into the evolutionary mechanisms and phenotypic particularities of mycobacterial pathogens that likely have also impacted the evolution of the key pathogen Mtb.

Cyclic (di)nucleotides act as universal second messengers endogenously produced by several pathogens. Specifically, the roles of c-di-AMP in Mycobacterium tuberculosis immunity and virulence have been largely explored, although its contribution to the safety and efficacy of live tuberculosis vaccines is less understood. In this study, we demonstrate that the synthesis of c-di-AMP is negatively regulated by the M. tuberculosis PhoPR virulence system. Accordingly, the live attenuated tuberculosis vaccine candidate M. tuberculosis vaccine (MTBVAC), based on double phoP and fadD26 deletions, produces more than 25- and 45-fold c-di-AMP levels relative to wild-type M. tuberculosis or the current vaccine bacille Calmette-Guérin (BCG), respectively. Secretion of this second messenger was exclusively detected in MTBVAC but not in M. tuberculosis or in BCG. We also demonstrate that c-di-AMP synthesis during in vitro cultivation of M. tuberculosis is a growth-phase- and medium-dependent phenotype. To uncover the role of this metabolite in the vaccine properties of MTBVAC, we constructed and validated knockout and overproducing/oversecreting derivatives by inactivating the disA or cnpB gene, respectively. All MTBVAC derivatives elicited superior interleukin-1β (IL-1β) responses compared with BCG during an in vitro infection of human macrophages. However, both vaccines failed to elicit interferon β (IFNβ) activation in this cellular model. We found that increasing c-di-AMP levels remarkably correlated with a safer profile of tuberculosis vaccines in the immunodeficient mouse model. Finally, we demonstrate that overproduction of c-di-AMP due to cnpB inactivation resulted in lower protection of MTBVAC, while the absence of c-di-AMP in the MTBVAC disA derivative maintains the protective efficacy of this vaccine in mice.

Mycobacterium tuberculosis (Mtb) modern strains emerged from a common progenitor after the loss of Mtb-specific deletion 1 region (TbD1). Here, the authors show that deletion of TbD1 correlates with enhanced Mtb virulence in animal models, mirroring the development of hypoxic granulomas in human disease progression.

Mycobacterium tuberculosis (Mtb) synthesizes a variety of atypical lipids that are exposed at the cell surface and help the bacterium infect macrophages and escape elimination by the cell's immune responses. In the present study, we investigate the mechanism of action of one family of hydrophobic lipids, the phthiocerol dimycocerosates (DIM/PDIM), major lipid virulence factors. DIM are transferred from the envelope of Mtb to host membranes during infection. Using the polarity-sensitive fluorophore C-Laurdan, we visualized that DIM decrease the membrane polarity of a supported lipid bilayer put in contact with mycobacteria, even beyond the site of contact. We observed that DIM activate the complement receptor 3, a predominant receptor for phagocytosis of Mtb by macrophages. DIM also increased the activity of membrane-permeabilizing effectors of Mtb, among which the virulence factor EsxA. This is consistent with previous observations that DIM help Mtb disrupt host cell membranes. Taken together, our data show that transferred DIM spread within the target membrane, modify its physical properties and increase the activity of host cell receptors and bacterial effectors, diverting in a non-specific manner host cell functions. We therefore bring new insight into the molecular mechanisms by which DIM increase Mtb's capability to escape the cell's immune responses.

Background: Human tuberculosis (TB) is caused by a plethora of Mycobacterium tuberculosis complex (MTBC) strains belonging to seven phylogenetic branches. Lineages 2, 3 and 4 are considered “modern” branches of the MTBC responsible for the majority of worldwide TB. Since the current BCG vaccine confers variable protection against pulmonary TB, new candidates are investigated. MTBVAC is the unique live attenuated vaccine based on M. tuberculosis in human clinical trials. Methods: MTBVAC was originally constructed by unmarked phoP and fadD26 deletions in a clinical isolate belonging to L4. Here we construct new vaccines based on isogenic gene deletions in clinical isolates of the L2 and L3 modern lineages. These three vaccine candidates were characterized at molecular level and also in animal experiments of protection and safety. Findings: Safety studies in immunocompromised mice showed that MTBVAC-L2 was less attenuated than BCG Pasteur, while the original MTBVAC was found even more attenuated than BCG and MTBVAC-L3 showed an intermediate phenotype. The three MTBVAC candidates showed similar or superior protection compared to BCG in immunocompetent mice vaccinated with each MTBVAC candidate and challenged with three representative strains of the modern lineages. Interpretation: MTBVAC vaccines, based on double phoP and fadD26 deletions, protect against TB independently of the phylogenetic linage used as template strain for their construction. Nevertheless, lineage L4 confers the best safety profile. Funding: European Commission (TBVAC2020, H2020-PHC-643381), Spanish Ministry of Science (RTI2018-097625-B-I00), Instituto de Salud Carlos III (PI18/0336), Gobierno de Aragón/Fondo Social Europeo and the French National Research Council (ANR-10-LABX-62-IBEID, ANR-16-CE35-0009, ANR-16-CE15-0003).

Basophils are effector cells that respond to protease allergens and parasites, thus contributing to allergic inflammation and Th2 differentiation. However, the molecular interactions through which pathogens promote activation as well as recruitment of these cells to sites of inflammation remain poorly understood. We found that administration of extracts from Nippostrongylus brasiliensis induced both basophil recruitment into blood and liver in vivo and IL-4 and histamine production by purified bone marrow-derived basophils in vitro. Starting from this finding, we set out to identify putative pathogen-derived molecules for their capacity to activate murine basophils, using a basophil population differentiated and expanded from bone marrow cells cultured with IL-3 and sorted as a CD49b+ c-kit– subset. Among a number of Toll-like receptor (TLR) agonists tested, we found that the lipopeptide Pam2CSK4 and lipopolysaccharide (LPS) activate basophils in terms of IL-4, IL-6 and histamine production through TLR2 and TLR4, respectively. By contrast, TLR3 or TLR7 agonists had no such effect. We further identified nitric oxide (NO) as key mediator for LPS stimulation and established that in vivo administration of LPS led to basophil recruitment into the liver. Our results reveal the important contribution of MyD88 and NO signaling to antigen recognition through TLR2 and TLR4 pathways leading to activation, degranulation and release of immunoregulatory mediators.

Summary: The pathogenic potential of Mycobacterium tuberculosis largely depends on ESX secretion systems exporting members of the multigenic Esx, Esp, and PE/PPE protein families. To study the secretion and regulation patterns of these proteins while circumventing immune cross-reactions due to their extensive sequence homologies, we developed an approach that relies on the recognition of their MHC class II epitopes by highly discriminative T cell receptors (TCRs) of a panel of T cell hybridomas. The latter were engineered so that each expresses a unique fluorescent reporter linked to specific antigen recognition. The resulting polychromatic and multiplexed imaging assay enabled us to measure the secretion of mycobacterial effectors inside infected host cells. We applied this novel technology to a large panel of mutants, clinical isolates, and host-cell types to explore the host-mycobacteria interplay and its impact on the intracellular bacterial secretome, which also revealed the unexpected capacity of phagocytes from lung granuloma to present mycobacterial antigens via MHC class II. : Sayes et al. develop an approach to express distinct fluorescent reporters that is based on the recognition of specific Mycobacterium tuberculosis MHC class II epitopes by highly discriminative T cell hybridomas. This multiplexed technology allows the study of secretion, subcellular location, and regulation patterns of these instrumental protein members. Keywords: mycobacterium tuberculosis, type VII secretion systems, intracellular bacteria, T-cell hybridomas, mycobacterial virulence factors, bacterial antigen presentation, lentiviral vectors, reporter T cells, in vivo antigen presentation, protein localization

Recent insights into the mechanisms by which Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, is recognized by cytosolic nucleotide sensors have opened new avenues for rational vaccine design. The only licensed anti-tuberculosis vaccine, Mycobacterium bovis BCG, provides limited protection. A feature of BCG is the partial deletion of the ESX-1 type VII secretion system, which governs phagosomal rupture and cytosolic pattern recognition, key intracellular phenotypes linked to increased immune signaling. Here, by heterologously expressing the esx-1 region of Mycobacterium marinum in BCG, we engineered a low-virulence, ESX-1-proficient, recombinant BCG (BCG::ESX-1Mmar) that induces the cGas/STING/TBK1/IRF-3/type I interferon axis and enhances AIM2 and NLRP3 inflammasome activity, resulting in both higher proportions of CD8+ T cell effectors against mycobacterial antigens shared with BCG and polyfunctional CD4+ Th1 cells specific to ESX-1 antigens. Importantly, independent mouse vaccination models show that BCG::ESX-1Mmar confers superior protection relative to parental BCG against challenges with highly virulent M. tuberculosis.

The borderline between virulence and efficacy in live attenuated vaccine strains is often blurred and this is also the case for the Bacillus Calmette–Guérin (BCG), the only currently licensed anti-tuberculosis vaccine used on a large, global scale, which was obtained almost 100 years ago. While BCG is more than 99% identical at the genome level to Mycobacterium tuberculosis, the causative pathogen of human tuberculosis, some important differences in virulence factors cause naturally irreversible attenuation and safety of this vaccine in the immunocompetent host. Some of these virulence factors are involved in persistence capacities of the vaccine strains and also represent strong immunogens, responsible for inducing different host signaling pathways, which have to be taken into consideration for the development of revised and new vaccine strains. Here we discuss a number of selected mycobacterial features in relation to their biological functions and potential impact on virulence and vaccine efficacy.

Tuberculosis is the deadliest infectious disease worldwide. Although the BCG vaccine is widely used, it does not efficiently protect against pulmonary tuberculosis and an improved tuberculosis vaccine is therefore urgently needed. Mycobacterium tuberculosis uses different ESX/Type VII secretion (T7S) systems to transport proteins important for virulence and host immune responses. We recently reported that secretion of T7S substrates belonging to the mycobacteria-specific Pro-Glu (PE) and Pro-Pro-Glu (PPE) proteins of the PGRS (polymorphic GC-rich sequences) and MPTR (major polymorphic tandem repeat) subfamilies required both a functional ESX-5 system and a functional PPE38/71 protein for secretion. Inactivation of ppe38/71 and the resulting loss of PE_PGRS/PPE-MPTR secretion were linked to increased virulence of M. tuberculosis strains. Here, we show that a predicted total of 89 PE_PGRS/PPE-MPTR surface proteins are not exported by certain animal-adapted strains of the M. tuberculosis complex including M. bovis. This Δppe38/71-associated secretion defect therefore also occurs in the M. bovis-derived tuberculosis vaccine BCG and could be partially restored by introduction of the M. tuberculosis ppe38-locus. Epitope mapping of the PPE-MPTR protein PPE10, further allowed us to monitor T-cell responses in splenocytes from BCG/M. tuberculosis immunized mice, confirming the dependence of PPE10-specific immune-induction on ESX-5/PPE38-mediated secretion. Restoration of PE_PGRS/PPE-MPTR secretion in recombinant BCG neither altered global antigenic presentation or activation of innate immune cells, nor protective efficacy in two different mouse vaccination-infection models. This unexpected finding stimulates a reassessment of the immunomodulatory properties of PE_PGRS/PPE-MPTR proteins, some of which are contained in vaccine formulations currently in clinical evaluation.

Mycobacterium tuberculosis (Mtb), possesses at least three type VII secretion systems, ESX-1, -3 and -5 that are actively involved in pathogenesis and host-pathogen interaction. We recently showed that an attenuated Mtb vaccine candidate (Mtb Δppe25-pe19), which lacks the characteristic ESX-5-associated pe/ppe genes, but harbors all other components of the ESX-5 system, induces CD4+ T-cell immune responses against non-esx-5-associated PE/PPE protein homologs. These T cells strongly cross-recognize the missing esx-5-associated PE/PPE proteins. Here, we characterized the fine composition of the functional cross-reactive Th1 effector subsets specific to the shared PE/PPE epitopes in mice immunized with the Mtb Δppe25-pe19 vaccine candidate. We provide evidence that the Mtb Δppe25-pe19 strain, despite its significant attenuation, is comparable to the WT Mtb strain with regard to: (i) its antigenic repertoire related to the different ESX systems, (ii) the induced Th1 effector subset composition, (iii) the differentiation status of the Th1 cells induced, and (iv) its particular features at stimulating the innate immune response. Indeed, we found significant contribution of PE/PPE-specific Th1 effector cells in the protective immunity against pulmonary Mtb infection. These results offer detailed insights into the immune mechanisms underlying the remarkable protective efficacy of the live attenuated Mtb Δppe25-pe19 vaccine candidate, as well as the specific potential of PE/PPE proteins as protective immunogens.

Mycobacterium tuberculosis (Mtb) uses efficient strategies to evade the eradication by professional phagocytes, involving--as recently confirmed--escape from phagosomal confinement. While Mtb determinants, such as the ESX-1 type VII secretion system, that contribute to this phenomenon are known, the host cell factors governing this important biological process are yet unexplored. Using a newly developed flow-cytometric approach for Mtb, we show that macrophages expressing the phagosomal bivalent cation transporter Nramp-1, are much less susceptible to phagosomal rupture. Together with results from the use of the phagosome acidification inhibitor bafilomycin, we demonstrate that restriction of phagosomal acidification is a prerequisite for mycobacterial phagosomal rupture and cytosolic contact. Using different in vivo approaches including an enrichment and screen for tracking rare infected phagocytes carrying the CD45.1 hematopoietic allelic marker, we here provide first and unique evidence of M. tuberculosis-mediated phagosomal rupture in mouse spleen and lungs and in numerous phagocyte types. Our results, linking the ability of restriction of phagosome acidification to cytosolic access, provide an important conceptual advance for our knowledge on host processes targeted by Mtb evasion strategies.

Faith-based vaccine initiatives are of growing interest to public health agencies who are looking to increase vaccine confidence among ethnoracially minoritized populations. Despite evidence that support faith-based organizations' (FBOs) partnerships with public health agencies (PHAs) to increase vaccine confidence, reviews on the scope and efforts to ensure equitable vaccination delivery for ethnoracially minoritized populations are scarce. We aimed to understand how public health agencies collaborate with FBOs or faith communities to improve vaccine confidence among minoritized communities in high-, low- and middle- income countries. We conducted a scoping review by searching OVID MEDLINE, Cochrane Library, Cumulative Index to Nursing and Allied Health Literature (CINAHL), SCOPUS, and PROQUEST from 2011 to 2023. We included case studies, news reports, observational studies, experimental, and quasi-experimental studies and multimedia content that describes PHA-FBO partnerships that created vaccine initiatives for marginalized and minoritized communities. The data was extracted, summarized, and results were described narratively. We included 167 initiatives reported in 160 publications; 83.8% of the included articles were published between 2019 to 2023. The interventions carried out by PHA-FBO partnerships attempted to increase vaccine uptake using any or all the following methods. First, the initiatives provided digital and in-person platforms for interfaith learning and established training programs to empower faith leaders to become vaccine ambassadors. Second, the initiatives designed and disseminated education and awareness materials that aimed to be sensitive to religious and gender norms. Third, PHA-FBO partnered to apply equity and faith-based frameworks and provided wrap-around support to enable equitable vaccine access. Majority of the initiatives reported that PHA-FBO partnerships improved vaccine confidence and uptake (71.3%). About 22.2% of the initiatives reported quantitative outcomes post-intervention. PHA-FBO initiatives over the past decade increased vaccine uptake and acceptance among diverse ethnoracially minoritized populations. Reporting of faith-based initiatives are subject to publication bias and can be strengthened by examining more evaluation studies and establishment of key outcome indicators to critically appraise intervention outcomes., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Song et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: There is low uptake of the pneumococcal vaccination in eligible older adults, even in high-income countries that offer routine and universal vaccination programs., Objective: To systematically characterize interventions aimed at improving pneumococcal vaccine uptake in older adults., Design: We conducted a scoping review following PRISMA-SCr guidelines of five interdisciplinary databases: Medline-Ovid, Embase, CINAHL, PsychInfo, and Cochrane Library. Databases were searched from January 2015 until April 2020. The interventions were summarized into three pillars according to the European Union Conceptional Framework for Action: information campaigns, prioritization of vaccination schemes, and primary care interventions., Results: Our scoping review included 39 studies that summarized interventions related to pneumococcal vaccine uptake for older adults, encompassing 2,481,887 study participants (945 healthcare providers and 2,480,942 older adults) across seven countries. Examples of interventions that were associated with increased pneumococcal vaccination rate included periodic health examinations, reminders and decision-making tools built into electronic medical records, inpatient vaccination protocols, preventative health checklists, and multimodal educational interventions. When comparing the three pillars, prioiritization of vaccination schemes had the highest evidence for improved rates of vaccination (n = 14 studies), followed by primary care interventions (n = 8 studies), then information campaigns (n = 5 studies)., Conclusion: Several promising interventions were associated with improved outcomes related to vaccine uptake, although controlled study designs are needed to determine which interventions are most effective., (© 2022. The Author(s).)

Background: There is a paucity of data on the burden of the full spectrum of community-acquired pneumonia (CAP) and acute otitis media (AOM) from outpatient and inpatient settings across the age spectrum., Methods: We conducted a population-based retrospective study in Ontario and British Columbia (BC), Canada, to estimate the incidence rate of CAP and AOM in children and adults over a 14-year period using health administrative databases. CAP and AOM cases were identified from outpatient physician consultation and hospitalisation data in both provinces, and from emergency department visit data in Ontario., Results: During 2005-2018, Ontario had 3 607 124 CAP, 172 290 bacterial CAP, 7814 pneumococcal pneumonia, and 8 026 971 AOM cases. The incidence rate of CAP declined from 3077/100 000 in 2005 to 2604/100 000 in 2010 before increasing to 2843/100 000 in 2018; bacterial CAP incidence rate also declined from 178/100 000 in 2005 to 112/100 000 in 2010 before increasing to 149/100 000 in 2018. The incidence rate of AOM decreased from 4192/100 000 in 2005 to 3178/100 000 in 2018. BC had 970 455 CAP, 317 913 bacterial CAP, 35 287 pneumococcal pneumonia and 2 022 871 AOM cases. The incidence rate of CAP in BC decreased from 2214/100 000 in 2005 to 1964/100 000 in 2010 before increasing to 2176/100 000 in 2018; bacterial CAP incidence rate increased from 442/100 000 in 2005 to 981/100 000 in 2018. The incidence rate of AOM decreased from 3684/100 000 in 2005 to 2398/100 000 in 2018. The incidence rate of bacterial CAP increased with age in older adults (≥65 years) with the highest burden in the oldest cohort aged ≥85 years both before and after 13-valent pneumococcal conjugate vaccine (PCV13) programme in both provinces. Hospitalised pneumococcal pneumonia decreased slightly but non-hospitalised pneumococcal pneumonia increased in BC during PCV13 period. No consistent direct benefit of PCV13 on CAP was observed in the paediatric population., Conclusions: There is a substantial burden of CAP and AOM in Ontario and BC. Indirect benefits from childhood PCV vaccination and polysaccharide vaccination of older adults have not substantially decreased the burden of pneumococcal pneumonia in older adults., Competing Interests: Competing interests: MS has been an investigator on projects funded by GlaxoSmithKline, Merck, Moderna, Pfizer, Sanofi-Pasteur, Seqirus, Symvivo and VBI Vaccines. All funds have been paid to his institute, and he has not received any personal payments. SKM is co-PI on a project funded by Pfizer that is unrelated to this manuscript, has served on ad hoc advisory boards for Pfizer and Sanofi-Pasteur, and has received speaker fees from GlaxoSmithKline. AM is an investigator on projects funded by Merck, Pfizer, Sanofi-Pasteur and Seqirus with payments to her institution, and has served on advisory boards for GlaxoSmithKline, Merck, Pfizer, Sanofi-Pasteur and Seqirus. The Centre for Vaccine Preventable Diseases is operated by the Dalla Lana School of Public Health, which receives funding from government, philanthropic, not for profit and private sector organization, including vaccine manufacturers. This includes a donation from Merck to the institution that supports the salary of SF. SF does not receive funding directly from Merck or any personal payment or direct funds from vaccine manufacturers to support her research. Other authors declare no conflicts of interest., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Competing Interests: We declare no competing interests.

Background: : microRNAs are playing important roles in the diagnosis and prognosis of pediatric acute lymphoblastic leukemia (ALL)., Methods: Expression levels of miR-100 and miR-210 were assessed in bone marrow aspirate of 85 pediatric ALL patients compared to 12 healthy control using quantitative real-time polymerase chain reaction. Data were correlated with relevant clinico-pathological features of the patients, response to treatment, disease-free survival (DFS), and overall survival (OS)., Results: miR-100 was significantly downregulated in ALL patients [median: 1.21, range: 0-434.3] compared to the control group [median: 8.41, range; 0-840.3, P  = 0.035]. miR-210 was significantly upregulated in ALL patients [median: 6.34, range: 1.16-1088.7] compared to the control group [median: 2.57, range: 0.11-709.2, P  = 0.025]. The sensitivity, specificity, and area under curve of miR-100 were (64.7%, 62.5%, and 0.642; respectively, P  = 0.035) at a cut-off 2.6 and that of miR-210 were (60%, 58.3% and 0.650; respectively, P  = 0.025) at a cut-off 3.5. miR-100 overexpression associated with shorter DFS and OS ( P  = 0.033 and 0.046; respectively). Patients with miR-100 lowexpression showed a significant incidence of late death ( P  = 0.024 ) . There was no significant association between miR-210 expression and DFS, OS, incidence of early or late death., Conclusion: : miR-100 and miR-210 could be used as potential diagnostic markers for pediatric ALL. miR-100 is a useful prognostic and predictive biomarker for childhood ALL.

Background: Many countries, including India, seek locally constructed disease burden estimates comprising mortality and loss of health to aid priority setting for the prevention and treatment of diseases. We created the National Burden Estimates (NBE) to provide transparent and understandable disease burdens at the national and subnational levels, and to identify gaps in knowledge., Methods: To calculate the NBE for India, we combined 2017 UN death totals with national and subnational mortality rates for 2010-17 and causes of death from 211 166 verbal autopsy interviews in the Indian Million Death Study for 2010-14. We calculated years of life lost (YLLs) and years lived with disability (YLDs) for 2017 using published YLD-YLL ratios from WHO Global Health Estimates. We grouped causes of death into 45 groups, including ill-defined deaths, and summed YLLs and YLDs to calculate disability-adjusted life-years (DALYs) for these causes in eight age groups covering rural and urban areas and 21 major states of India., Findings: In 2017, there were about 9·7 million deaths and 486 million DALYs in India. About three quarters of deaths and DALYs occurred in rural areas. More than a third of national DALYs arose from communicable, maternal, perinatal, and nutritional disorders. DALY rates in rural areas were at least twice those of urban areas for perinatal and nutritional conditions, chronic respiratory diseases, diarrhoea, and fever of unknown origin. DALY rates for ischaemic heart disease were greater in urban areas. Injuries caused 11·4% of DALYs nationally. The top 15 conditions that accounted for the most DALYs were mostly those causing mortality (ischaemic heart disease, perinatal conditions, chronic respiratory diseases, diarrhoea, respiratory infections, cancer, stroke, road traffic accidents, tuberculosis, and liver and alcohol-related conditions), with disability mostly due to a few conditions (nutritional deficiencies, neuropsychiatric conditions, vision and other sensory loss, musculoskeletal disorders, and genitourinary diseases). Every condition that was common in one part of India was uncommon elsewhere, suggesting state-specific priorities for disease control., Interpretation: The NBE method quantifies disease burden using transparent, intuitive, and reproducible methods. It provides a simple, locally operable tool to aid policy makers in priority setting in India and other low-income and middle-income countries. The NBE underlines the need for many more countries to collect nationally representative cause of death data, paired with focused surveys of disability., Funding: Ministry of Health and Family Welfare, Government of India., (Copyright © 2019 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Control of pneumonia and diarrhea mortality in India requires understanding of their etiologies. We combined time series analysis of seasonality, climate region, and clinical syndromes from 243,000 verbal autopsies in the nationally representative Million Death Study. Pneumonia mortality at 1 month-14 years was greatest in January (Rate ratio (RR) 1.66, 99% CI 1.51-1.82; versus the April minimum). Higher RRs at 1-11 months suggested respiratory syncytial virus (RSV) etiology. India's humid subtropical region experienced a unique summer pneumonia mortality. Diarrhea mortality peaked in July (RR 1.66, 1.48-1.85) and January (RR 1.37, 1.23-1.48), while deaths with fever and bloody diarrhea (indicating enteroinvasive bacterial etiology) showed little seasonality. Combining mortality at ages 1-59 months with prevalence surveys, we estimate 40,600 pneumonia deaths from Streptococcus pneumoniae , 20,700 from RSV, 12,600 from influenza, and 7200 from Haemophilus influenzae type b and 24,700 diarrheal deaths from rotavirus occurred in 2015. Careful mortality studies can elucidate etiologies and inform vaccine introduction., Competing Interests: DF, SA, SF, RK, AS, SF, BW, SM No competing interests declared, PJ Reviewing editor, eLife, (© 2019, Farrar et al.)

Background: India had the largest number of under-5 deaths of all countries in 2015, with substantial subnational disparities. We estimated national and subnational all-cause and cause-specific mortality among children younger than 5 years annually in 2000-15 in India to understand progress made and to consider implications for achieving the Sustainable Development Goal (SDG) child survival targets., Methods: We used a multicause model to estimate cause-specific mortality proportions in neonates and children aged 1-59 months at the state level, with causes of death grouped into pneumonia, diarrhoea, meningitis, injury, measles, congenital abnormalities, preterm birth complications, intrapartum-related events, and other causes. AIDS and malaria were estimated separately. The model was based on verbal autopsy studies representing more than 100 000 neonatal deaths globally and 16 962 deaths among children aged 1-59 months at the subnational level in India. By applying these proportions to all-cause deaths by state, we estimated cause-specific numbers of deaths and mortality rates at the state, regional, and national levels., Findings: In 2015, there were 25·121 million livebirths in India and 1·201 million under-5 deaths (under-5 mortality rate 47·81 per 1000 livebirths). 0·696 million (57·9%) of these deaths occurred in neonates. There were disparities in child mortality across states (from 9·7 deaths [Goa] to 73·1 deaths [Assam] per 1000 livebirths) and regions (from 29·7 deaths [the south] to 63·8 deaths [the northeast] per 1000 livebirths). Overall, the leading causes of under-5 deaths were preterm birth complications (0·330 million [95% uncertainty range 0·279-0·367]; 27·5% of under-5 deaths), pneumonia (0·191 million [0·168-0·219]; 15·9%), and intrapartum-related events (0·139 million [0·116-0·165]; 11·6%), with cause-of-death distributions varying across states and regions. In states with very high under-5 mortality, infectious-disease-related causes (pneumonia and diarrhoea) were among the three leading causes, whereas the three leading causes were all non-communicable in states with very low mortality. Most states had a slower decline in neonatal mortality than in mortality among children aged 1-59 months. Ten major states must accelerate progress to achieve the SDG under-5 mortality target, while 17 are not on track to meet the neonatal mortality target., Interpretation: Efforts to reduce vaccine-preventable deaths and to reduce geographical disparities should continue to maintain progress achieved in 2000-15. Enhanced policies and programmes are needed to accelerate mortality reduction in high-burden states and among neonates to achieve the SDG child survival targets in India by 2030., Funding: Bill & Melinda Gates Foundation., (Copyright © 2019 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

India comprises much of the persisting global childhood measles mortality. India implemented a mass second-dose measles immunization campaign in 2010. We used interrupted time series and multilevel regression to quantify the campaign's impact on measles mortality using the nationally representative Million Death Study (including 27,000 child deaths in 1.3 million households surveyed from 2005 to 2013). 1-59 month measles mortality rates fell more in the campaign states following launch (27%) versus non-campaign states (11%). Declines were steeper in girls than boys and were specific to measles deaths. Measles mortality risk was lower for children living in a campaign district (OR 0.6, 99% CI 0.4-0.8) or born in 2009 or later (OR 0.8, 99% CI 0.7-0.9). The campaign averted up to 41,000-56,000 deaths during 2010-13, or 39-57% of the expected deaths nationally. Elimination of measles deaths in India is feasible., Competing Interests: BW, SF, SA, AK, RK, GM No competing interests declared, PJ Prabhat Jha is a Reviewing Editor at eLife, (© 2019, Wong et al.)

Background: Breast carcinoma is the commonest malignancy in females. Folate is required for the biosynthesis of nucleotide bases, amino acids, and other cellular methylation reactions in proteins and phospholipids. The high affinity folate receptor alpha (FRα) has been shown to be expressed in several kinds of human cancers., Methods: In this descriptive-analytic study, sections from formalin-fixed paraffin-embedded tissue blocks of 50 cases of invasive ductal breast carcinoma (IDC) as well as 15 cases of non-neoplastic breast specimens were immunohistochemically stained with FRα antibody. Histopathological evaluation for various clinicopathological parameters was done and was correlated with FRα expression., Results: Positive FRα expression was more frequently detected in IDC (64%) compared to non-neoplastic breast specimens (20%). In IDC, Positive FRα expression was significantly associated with high tumor grade (p = 0.007), large tumor size (p < 0.001), high lymph node stage (p = 0.004), presence of angiolymphatic emboli (p = 0.001), presence of perineural invasion (p = 0.001). Significant association between FRα positivity and negative hormone receptors (estrogen and progesterone) (p < 0.001) and triple negative cases (p = 0.0021)., Conclusion: Our work demonstrates that FRα is over expressed in IDC compared to non-neoplastic breast tissue. Folate receptor α expression was associated with poor clinicopathological perspective. This work suggests that FRα may be an independent prognostic factor and supports the possibility of using FRα-targeted therapies of breast carcinoma. However, our work requires validation on larger cohort with correlation with survival data of patients., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Aim: To determine factors associated with a serious outcome (hospital admission or severe outcome: critical care or death) and associated with illness caused by laboratory-confirmed influenza, with a specific interest in low- and middle-income countries (LMIC)., Method: Databases were searched on 11 March 2016 for reports of influenza and factors associated with mortality or morbidity in humans, with no language restrictions. Pooled risks were estimated using random-effects models., Results: Despite the heterogeneity of results across studies, known risk factors for serious disease were associated with both hospital admission and severe outcomes (critical care and/or death). In LMIC, but not in high income countries (HIC), pregnant women, people with HIV/AIDS and children < 5 years old (compared with older children) were at increased risk of a severe outcome. Also, although all patients with neurological conditions were at higher risk of severe outcomes than those without, children were at higher risk than adults and children who lived in a LMIC were at significantly higher risk than those living in HIC. Adults were more likely than children to suffer a severe outcome if they had diabetes or a hematologic condition, were obese or had liver disease. Asthma is a risk factor for hospital admission but not for severe outcomes., Conclusion: Known risk factors for serious disease remain important predictors of hospital admission and severe outcomes with few differences between HIC and LMIC countries. These differences likely reflect differences in health-seeking behaviours and health services, but high heterogeneity between studies limits conclusions about the effect size., (© 2017 The World Health Organization. Influenza and Other Respiratory Viruses. Published by John Wiley & Sons Ltd.)

Influenza vaccination programmes are assumed to have a herd effect and protect contacts of vaccinated persons from influenza virus infection. We searched MEDLINE, EMBASE, the Cumulative Index to Nursing and Allied Health Literature (CINAHL), Global Health and the Cochrane Central Register of Controlled Trials (CENTRAL) from inception to March 2014 for studies assessing the protective effect of influenza vaccination vs no vaccination on influenza virus infections in contacts. We calculated odds ratios (ORs) and 95% confidence intervals (CIs) using a random-effects model. Of 43,082 screened articles, nine randomised controlled trials (RCTs) and four observational studies were eligible. Among the RCTs, no statistically significant herd effect on the occurrence of influenza in contacts could be found (OR: 0.62; 95% CI: 0.34-1.12). The one RCT conducted in a community setting, however, showed a significant effect (OR: 0.39; 95% CI: 0.26-0.57), as did the observational studies (OR: 0.57; 95% CI: 0.43-0.77). We found only a few studies that quantified the herd effect of vaccination, all studies except one were conducted in children, and the overall evidence was graded as low. The evidence is too limited to conclude in what setting(s) a herd effect may or may not be achieved., Competing Interests: SPK received travel grants from Pfizer and Novartis. DT received grant funding from GSK Canada. SAF, PL, JS, SAA, MS, JN, JJ, JRO, DM, ML: none declared., (This article is copyright of The Authors, 2016.)

Objective: Clinical studies demonstrate the efficacy of interventions to reduce neonatal deaths, but there are fewer studies of their real-life effectiveness. In India, women often seek facility delivery after complications arise, rather than to avoid complications. Our objective was to quantify the association of facility delivery and postnatal checkups with neonatal mortality while examining the "reverse causality" in which the mothers deliver at a health facility due to adverse perinatal events., Methods: We conducted nationally representative case-control studies of about 300,000 live births and 4,000 neonatal deaths to examine the effect of, place of delivery and postnatal checkup on neonatal mortality. We compared neonatal deaths to all live births and to a subset of live births reporting excessive bleeding or obstructed labour that were more comparable to cases in seeking care., Findings: In the larger study of 2004-8 births, facility delivery without postnatal checkup was associated with an increased odds of neonatal death (Odds ratio = 2.5; 99% CI 2.2-2.9), especially for early versus late neonatal deaths. However, use of more comparable controls showed marked attenuation (Odds ratio = 0.5; 0.4-0.5). Facility delivery with postnatal checkup was associated with reduced odds of neonatal death. Excess risks were attenuated in the earlier study of 2001-4 births., Conclusion: The combined effect of facility deliveries with postnatal checks ups is substantially higher than just facility delivery alone. Evaluation of the real-life effectiveness of interventions to reduce child and maternal deaths need to consider reverse causality. If these associations are causal, facility delivery with postnatal check up could avoid about 1/3 of all neonatal deaths in India (~100,000/year).

Background: Healthcare workers may be exposed to people with respiratory viral infections more often than other working adults. Understanding the risk and the effectiveness of different preventive measures is of great importance., Objectives: To estimate adherence to prophylactic antiviral medication for a full influenza season, to the compare efficacy of antiviral prophylaxis to that of the seasonal influenza vaccine and to identify exposures that increase risk of acute respiratory illnesses (ARI) in healthy adults., Methods: Participants were randomized 1:2 to receive the 2008-2009 influenza vaccine or daily prophylaxis with 10 mg of zanamivir during the season. Web-based questionnaires collected information on demographics, symptoms, exposures, medication use and side effects., Results: Sixty-four healthy adults were recruited in November 2008. Three of 40 active participants discontinued zanamivir due to side effects; the remaining 37 took >85% of scheduled doses for a median of 121 days. Symptomatic, laboratory-confirmed influenza was detected in one person randomized to zanamivir (2·5%) and 2/20 (10%) who received the vaccine (P = 0·25). Forty-seven participants reported 109 episodes of ARI. Factors associated with an ARI were exposure to a spouse (OR 7·2), child (OR 2·4) or patient (OR 2·0) with symptoms of an ARI in the previous 7 days., Conclusions: Breakthrough influenza infection occurred in both vaccinated participants and those receiving antiviral prophylaxis. Most adults were willing and able to comply with season-long prophylaxis. Report of recent exposure to family members and patients with an ARI increased the risk of developing an ARI in healthy adults., (© 2015 The Authors. Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd.)

Background: Little is known about risk factors for severe outcomes in patients infected with H5N1 and no systematic review has been conducted. Understanding risk factors is an important step for prioritizing prophylaxis or treatment in the event of a pandemic., Objectives: To systematically evaluate risk factors for severe outcomes in patients with avian influenza H5N1 infection., Data Sources: MEDLINE, EMBASE, CINAHL, GlobalHealth, and CENTRAL through March 2011., Eligibility Criteria for Selecting Studies: Observational studies of any design published in English, French, Spanish, German or Korean that reported on risk factor-outcome combinations of interest in participants with confirmed H5N1 infections. Outcomes considered included death, ventilator support, hospital and ICU admission, pneumonia, and composite outcomes., Study Appraisal: Risk of bias was assessed using the Newcastle-Ottawa scale (NOS)., Results: We identified 20 studies reporting on 999 patients infected with H5N1. The majority of studies (n = 14, 70%) were at intermediate risk of bias, i.e. 4-6 points on the NOS. Females were at increased risk of death (OR 1.75, 95% CI 1.27-2.44), while young age, in particular <5 years of age (OR 0.44, 95% CI 0.25-0.79 for death), was protective. Data on traditional risk factors was scarce and requires further studies. Another major limitation in the published literature was lack of adjustment for confounders., Interpretation: Females were at increased risk for complications following H5N1 infection while young age protected against severe outcomes. Research on traditional risk factors was limited and is required.

Objective: To evaluate risk factors for severe outcomes in patients with seasonal and pandemic influenza., Design: Systematic review., Study Selection: Observational studies reporting on risk factor-outcome combinations of interest in participants with influenza. Outcomes included death, ventilator support, admission to hospital, admission to an intensive care unit, pneumonia, and composite outcomes., Data Sources: Medline, Embase, CINAHL, Global Health, and the Cochrane Central Register of Controlled Trials to March 2011., Risk of Bias Assessment: Newcastle-Ottawa scale to assess the risk of bias. GRADE framework to evaluate the quality of evidence., Results: 63 537 articles were identified of which 234 with a total of 610 782 participants met the inclusion criteria. The evidence supporting risk factors for severe outcomes of influenza ranged from being limited to absent. This was particularly relevant for the relative lack of data for non-2009 H1N1 pandemics and for seasonal influenza studies. Limitations in the published literature included lack of power and lack of adjustment for confounders was widespread: adjusted risk estimates were provided for only 5% of risk factor-outcome comparisons in 39 of 260 (15%) studies. The level of evidence was low for "any risk factor" (odds ratio for mortality 2.77, 95% confidence interval 1.90 to 4.05 for pandemic influenza and 2.04, 1.74 to 2.39 for seasonal influenza), obesity (2.74, 1.56 to 4.80 and 30.1, 1.74 to 2.39), cardiovascular diseases (2.92, 1.76 to 4.86 and 1.97, 1.06 to 3.67), and neuromuscular disease (2.68, 1.91 to 3.75 and 3.21, 1.84 to 5.58). The level of evidence was very low for all other risk factors. Some well accepted risk factors such as pregnancy and belonging to an ethnic minority group could not be identified as risk factors. In contrast, women who were less than four weeks post partum had a significantly increased risk of death from pandemic influenza (4.43, 1.24 to 15.81)., Conclusion: The level of evidence to support risk factors for influenza related complications is low and some well accepted risk factors, including pregnancy and ethnicity, could not be confirmed as risks. Rigorous and adequately powered studies are needed.

Background: Although the presence of tumour infiltrating lymphocytes (TIL) is a constant feature in melanomas, their immunophenotypic characterisation is still incomplete. We hypothesise that the transition from normal skin to benign naevi (BN) to melanocytic dysplastic naevi (MDN) to radial growth phase cutaneous malignant melanoma (RGP-CMM) to vertical growth phase cutaneous malignant melanoma (VGP-CMM) is associated with alterations in TIL. This study attempted to test this hypothesis and to characterise TIL in the melanocytic skin lesions., Methods: In total, 74 lesions (12 BN, 12 MDN, 13 RGP-CMM, 26 VGP-CMM, and 11 metastatic melanomas) were examined using immunoperoxidase staining methods and antibodies targeting leukocyte common antigen (LCA+), T (CD3+) and B (CD20+) lymphocytes, and resting cytotoxic T cells (TIA-1+)., Results: Histologically, the transitions from normal skin to BN to MDN to RGP-CMM to VGP-CMM was associated with a gradual increase in the numbers of TIL (total, parenchymal, stromal, perivascular, and epidermal TIL, as well as TIL at the base of the lesions). The numbers of TIL were higher at the stroma than at the parenchyma. Similarly, immunostaining revealed that these transitions were associated with a gradual increase in the staining values (staining intensity, percentage of positive cells, and immunoreactivity score) for LCA+, CD20+, CD3+, and TIA-1+cells. The number of CD3+ cells was higher than that of CD20+ cells. All these differences between the normal skin and the lesional ones reached statistical significance (p<0.01). The majority of CD3+ cells were TIA-1+ T cells with cytotoxic potential. Compared with primary melanomas, there was a decrease in TIL in metastatic melanomas., Conclusions: The gradual increase in TIL during melanoma tumorigenesis may reflect increased antigenicity of the tumour cells. Although both humoral and cell mediated immunity are involved in melanomagenesis, the latter seems to have the major role. The immune profile of MDN suggests their intermediacy between BN and CMM.

Immunizations early in life, when the host is most susceptible to infection, allow protective immunological memory to develop. Decreasing the dose of Cas-Br-E murine leukemia virus when priming neonatal mice results in adult-like, Type 1 protective responses, but the resulting memory cell populations are smaller than after adult priming. After secondary challenge, virus-specific CD8+ memory cell populations expand twice as much in neonate-primed mice as in adult-primed mice. We found that when equivalent numbers of virus-specific cells were transferred into virus-susceptible mice, protection from disease was similar whether donor, immune mice were primed as neonates or adults, and IL-4 did not alter in vivo virus-specific CD8+ memory cell effector function. Hence, neonate-primed CD8+ cells develop into memory cells that rival adult-primed cells in proliferation and effector function.