The Journal of Infectious Diseases

BackgroundAfter the global deployment of pneumococcal conjugate vaccines (PCVs), serotype 12F has become the predominant serotype responsible for invasive pneumococcal disease (IPD) worldwide. As PCVs that include serotype 12F are gradually introduced, we aim to characterise the global population structure and genetic diversity of the 12F capsule locus using whole-genome sequencing. Capsule variants with vaccine evasion potential were further investigated by functional experiments. MethodsA global collection of pneumococcal serotype 12F genomes (n=806) from 37 countries across six continents were included in this study. To characterise the serotype 12F population, Global Pneumococcal Sequence Cluster (GPSC), in silico serotype, and antimicrobial resistance profile were inferred from whole-genome data for each isolate. The capsule biosynthesis (cps) locus was analysed for gene content variations that could alter polysaccharide capsule production or structure, thereby influencing recognition by vaccine-induced antibodies. These isolates were further investigated by assessing their capsule production using immunofluorescence assays and its susceptibility to vaccine-elicited antibody killing by opsonophagocytosis assays. FindingsThe global increase in serotype 12F was driven by both distinct pneumococcal lineages across different continents, and a globally-disseminated and multidrug-resistant lineage GPSC26. We identified six capsule variants in nine isolates that had disruptive mutations in cps genes including wze, wcil, wciJ and fnlA. Most (6/9) of the disruptive mutations were a result of strand-slippage mutations. A convergent strand-slippage mutation disrupting the glycosyltransferase gene wciJ was identified in four isolates from distinct lineages and countries. Despite the truncation, three of four isolates with available Quellung typing results still identified them as 12F, indicating the production of the capsule. We then created a genetically engineered lab strain with wciJ knockout and complemented with wciJ containing the strand-slipppage mutation. The knockout strain did not produce any capsule. In contrast, the lab strain with wciJ containing the strand-slippage mutation produced a mixed population of encapsulated and non-encapsulated pneumococci, even within the same chain of pneumococcal cells. This observation indicated encapsulated subpopulation possesses a functional WciJ and rapidly reversible strand-slippage mutation during replication. Opsonophagocytosis assays indicated that the clinical 12F strain with strand-slippage mutation in wciJ exhibited reduced susceptibility to vaccine-elicited serum killing, compared to a genetically closely related 12F clinical strain with an intact wciJ. However, substantial inter-individual antisera variation limits definitive interpretation. InterpretationOur work revealed the global rise of serotype 12F pneumococci has been driven by both regional-specific lineages, and a globally-disseminated and multidrug-resistant lineage GPSC26. We demonstrated that strand-slippage mutation is one of the major drivers of serotype 12F capsule variants and represents a novel mechanism enabling reversible on-off switching of capsule production. The ability to switch off capsule expression in a subpopulation may enable evasion of antibody-mediated killing but increase susceptibility to innate immune clearance. FundingBill & Melinda Gates Foundation, Wellcome Sanger Institute, and the US Centers for Disease Control and Prevention.

Background: Partial resistance to artemisinins (ART-R) has emerged in East Africa, associated with mutations in the Plasmodium falciparum kelch13 gene. It is currently unclear whether ART-R has implications for gametocyte production or for onward transmission to mosquitoes. Methods: In a cohort of uncomplicated malaria patients attending Kalongo Hospital in northern Uganda, we quantified carriage of PfKelch13 mutant parasites by conventional sequencing and droplet digital PCR (ddPCR) for the C469Y and A675V mutations. Prevalence and density of gametocytes and ring-stage parasites were assessed by microscopy and quantitative reverse-transcriptase PCR (qRT-PCR). Lumefantrine concentrations, indicative of prior malaria treatment, were determined by ultra-high performance liquid chromatography-tandem mass spectrometry. Transmission potential of wild-type and PfKelch13 mutant parasites was assessed by mosquito feeding assays and complemented with molecular characterization of parasites in wild-caught mosquitoes from household resting catches. Findings: We enrolled 235 patients with symptomatic P. falciparum infection; PfKelch13 C469Y or A675V mutations were detected in 35.8% (78/218) of infections by sequencing and 59.1% (136/230) by ddPCR. Gametocyte carriage was 24.0% (56/233) by microscopy and 56.6% (133/235) by qRT-PCR and not associated with the abundance of PfKelch13 mutant parasites by ddPCR (p=0.603). Among a total of 227 mosquito feeds with patient whole blood, 1.4% (120/8745) of mosquitoes became infected. Mosquito infection rates were positively associated with gametocyte density ({beta} = 0.39, 95% CI = 0.23-0.59, p < 0.001) without an observed interaction with the abundance of PfKelch13 mutant parasites (p = 0.452). PfKelch13 C469Y or A675V mutations were detected in 40.1% (21/52) of malaria-infected bloodmeals of field-caught mosquitoes and in 28.0% (7/25) of sporozoite-positive mosquitoes. Interpretation: We conclude that pfkelch13 mutations are very common in patients in northern Uganda with uncomplicated malaria, mostly in multiclonal infections. We observed no evidence that ART-R affected gametocyte production or transmission to mosquitoes. Funding: Dutch Research Council (NWO)

Tuberculosis (TB) is a significant cause of morbidity and mortality in children and adolescents, causing 172,000 deaths in 2024 in children and adolescents worldwide. Diagnostic challenges are pronounced in pediatrics, in which collecting respiratory specimens is challenging and TB is often paucibacillary, leading to delayed diagnosis and increased mortality. We describe the protocol and methodology of the Pediatric TB Diagnostic (PDTBDx) cohort, a study with the primary aim of evaluating non-sputum-based TB diagnostics for diagnosis and treatment response in children. This is a prospective observational cohort study of >400 children recruited from inpatient and outpatient clinical sites in Nairobi, Kenya. Children <15 years presenting to study clinical sites with TB symptoms will be considered for enrollment as symptomatic participants. Enrolled participants will undergo rigorous clinical assessment and longitudinal follow-up to ensure appropriate diagnostic classification by NIH consensus statement guidelines for pediatric TB. Baseline evaluation includes symptom assessment, chest x-ray, HIV testing, respiratory TB culture and GeneXpert Ultra, and urine LAM. Subsequent visits occur at week 2, months 1, 2, 4, 6,12 and 24. Blood and urine specimens will be collected at baseline and at follow-up visits for storage for evaluation of novel diagnostic assays, including exosome-based and CRISPR-based TB biomarkers. This large, prospective cohort of pediatric participants with and without TB follows a consistent and rigorous protocol for diagnosing childhood TB, in concordance with internationally recognized guidelines. Assays evaluated in PDTBDx will guide improved diagnostic strategies for pediatric TB.

Klebsiella pneumoniae (KP) isolates belonging to multi-locus sequence type 258 (ST258) are a frequent cause of hospital-associated outbreaks and display extensive multidrug resistance. The KP ST258 lineage consists of two genetically distinct clades, called Clade 1 and Clade 2. These two clades are genetically related to one another, but are historically distinguished by having different capsular polysaccharide types. While bacteria belonging to both clades are isolated from clinical infections, Clade 2 is isolated more frequently compared to Clade 1. To investigate drivers of this difference in clade prevalence, we collected 172 clinical KP ST258 isolates from patients at a single medical center. Clinical review showed that patients infected with Clade 2 isolates were more acutely ill than Clade 1-infected patients, despite having fewer comorbidities. We also found that Clade 2 isolates were more resistant to killing by human serum, despite binding more complement protein C3 than Clade 1 isolates. Additionally, mice infected with a Clade 2 isolate had increased bacterial dissemination from the lungs to the liver and spleen than mice infected with a Clade 1 isolate, and this dissemination required an intact capsule locus. Increased dissemination in mice was not due to differential serum killing, as mouse serum was unable to kill isolates of either clade, but dissemination was associated with decreased macrophage uptake of the Clade 2 isolate. Taken together, these data suggest that KP ST258 Clade 2 is more virulent than Clade 1, though the specific mechanisms at play appear to differ between mice and humans.

Background: Prolonged SARS-CoV-2 infection in immunocompromised individuals may accelerate virus evolution within the host, raising concerns about the virus evading immunity, developing resistance, and forming novel variants of concern. However, the determinants and public health implications of within-host viral evolution in this population remain incompletely understood. Methods: We performed longitudinal analyses of SARS-CoV-2 genomes from 91 patients with COVID-19 who were classified as being severely or moderately immunocompromised. Using serial measurements of viral RNA loads and infectious titers, we modeled the shedding dynamics of the virus and stratified the infected cases by upper respiratory virus shedding duration to assess associations with within-host evolutionary dynamics. Results: Shedding modeling identified two profiles of shedding duration: intermediate and long. The long shedding profile (shedding lasting >21 days) was found in 14.8% of moderately immunocompromised cases and 72.1% of severely immunocompromised cases. Frequent single-nucleotide variants accumulated specifically in severely immunocompromised individuals with the long shedding phenotype, correlating positively with shedding duration. By contrast, mutations remained limited in moderately immunocompromised individuals with the long shedding phenotype and in severely immunocompromised individuals with the intermediate shedding phenotype. We identified mutations in the spike receptor-binding domain associated with monoclonal antibody resistance; however, we found no fitness-enhancing mutations for inter-host transmission, and antiviral drug resistance mutations were rare. Instead, mutations were introduced frequently and randomly across the entire viral genome. Conclusions: Prolonged upper respiratory virus shedding exceeding 21 days combined with severe immunocompromise is a risk factor of the accumulation of within-host SARS-CoV-2 mutations. Although no variants of concern emerged, the introduction of genome-wide random mutations suggests that the risk for novel variant generation cannot be excluded. These findings highlight the need for intensive antiviral strategies to limit shedding duration to less than 21 days in severely immunocompromised patients, and for immunological investigations to elucidate the host factors underlying residual shedding control in those who achieve clearance within this threshold.

Introduction: Intermittent preventive treatment in pregnancy (IPTp) with sulfadoxine-pyrimethamine (SP) has become less effective at preventing malaria due to rising parasite resistance. IPTp with dihydroartemisinin-piperaquine (DP) alone or in combination with SP (DP+SP) dramatically lowers the risk of malaria in pregnancy compared to SP but is associated with lower birthweight and early life wasting. We estimated the effect of IPTp-DP, DP+SP, and SP on infant growth outcomes and assessed possible treatment mechanisms through a causal mediation analysis. Methods: We used infant follow-up data (N=761) from a trial (NCT04336189) that randomized pregnant women to receive monthly IPTp-DP, SP, or DP+SP. We compared weight-for-length (WLZ) and length-for-age (LAZ) z-scores between treatment arms. We assessed possible mediation through pregnancy, birth, and infancy factors using interventional indirect effect models. Results: Compared to IPTp-SP, IPTp-DP+SP decreased mean WLZ by 0.18 [95% confidence interval (CI) -0.03, 0.39] between 1-3 months and 0.28 (95% CI 0.07, 0.49) between 4-6 months, with the largest differences among primigravidae. Lower risk of active placental malaria in IPTp-DP+SP helped reduce differences in mean WLZ vs IPTp-SP (+0.06, 95% CI 0.02, 0.10). The IPTp-DP+SP arm had up to 0.28 lower mean LAZ between 7-13 months compared to IPTp-DP, particularly among children who were wasted between 0-6 months; low birthweight had a persistent, mediating effect on linear growth. Conclusion: Adverse birth outcomes contributed to early growth faltering among children born to mothers receiving IPTp-DP+SP vs IPTp-SP, but the prevention of placental malaria partially counteracted the negative effects of IPTp-DP+SP on ponderal growth.

Background: Host genetics alone explains limited susceptibility to tuberculosis (TB), particularly in people with HIV (PWH). Protein quantitative trait loci (pQTLs), genetic variants that regulate plasma protein levels, may bridge genetic and immunological mechanisms underlying TB progression. Methods: We conducted cis-pQTL mapping in 60 PWH who progressed to active TB and 194 matched controls from the Swiss HIV Cohort Study. Plasma proteomes were quantified via high-resolution mass spectrometry (dia-PASEF), and genotype-protein associations were analyzed separately in TB and control groups. Results: TB progressors harbored 26 cis-pQTLs linked to 12 proteins uniquely enriched in immune pathways (antigen presentation, complement activation, phagocytosis, and T-cell regulation). Controls showed 107 cis-pQTLs linked to 14 targets. Gene Ontology enrichment revealed 46 immune biological processes in TB versus only 1 in controls, with HLA-C, C4B, and CHIT1 as key TB-specific proteins. Conclusions: Integrating proteomics with genomics suggests differential regulation of immune proteins associated with TB progression in PWH. hese genetically anchored protein candidates support follow-up studies and future biomarker evaluation for TB risk prediction.

The upper respiratory tract is a primary niche for Streptococcus pyogenes colonisation and disease. Lower respiratory tract infection (pneumonia) is the most common invasive S. pyogenes syndrome. Studies have not previously examined how epithelial cells, from the airway to the alveolus, respond to S. pyogenes infection. Here, we established a scalable human in vitro model by differentiating induced pluripotent stem cells (iPSCs) into mature pseudostratified airway epithelium or alveolar type 2 epithelial cells, cultured at air-liquid interface and infected with S. pyogenes (M1UK and M75 strains). Both strains attached to iPSC-derived lung epithelial cells, with significantly greater adherence to the airway epithelium by M75 compared to M1UK. Moreover, invasion by both S. pyogenes strains of alveolar epithelial cells was greater than for the airway epithelium. Dynamic S. pyogenes gene expression changes were evident between 6 and 24 hours after infection, which was influenced by the infected cell type; however, virulence genes were not significantly altered. While infection of the airway epithelium induced rapid and dynamic inflammatory signalling, the alveolar epithelium demonstrated augmented cell death and mounted a transcriptional pro-inflammatory and proliferative response that was uncoupled from cytokine secretion. The airway epithelium model exhibited consistently higher baseline type I interferon (IFN) signalling than the alveolar epithelium. Invasion by S. pyogenes and inflammation was significantly reduced in IFN-{beta}-treated alveolar epithelial cells. In summary, we have established the first model of S. pyogenes infection in physiologically relevant airway and alveolar epithelial cells. Our findings suggest that host responses to infection are influenced by lung compartment, the S. pyogenes strain type, and infection timepoint, highlighting context-specific pathways that could be leveraged therapeutically.

Background: Binary interpretation of Mycobacterium tuberculosis (Mtb) interferon gamma release assay (IGRA) results discards information about recency of exposure and disease risk. We analysed quantitative IGRA responses to Mtb in a community--based survey to investigate associations with response magnitude and inform understanding of transmission dynamics. Methods: We included QuantiFERON--TB Gold Plus (QFT--Plus) results from 2,895 participants (10--40 years old) in Blantyre, Malawi. Bayesian regression models assessed the probability of a positive response ([&ge;]0.35 IU/mL), response magnitude, and associated factors. We also investigated associations with a TB2-TB1 differential >0.6 IU/mL (proposed to reflect recent transmission), and how hypothetical alternative IGRA positivity thresholds affected inference about age-- and sex--specific transmission. Results: 17.4% (503/2,895) of participants had positive TB1 or TB2 responses at the QFT--Plus positivity threshold (0.35 IU/mL). The distributions of TB1 and TB2 responses, among participants with positive QFT--Plus, were similar across age and sex. A TB2-TB1 differential >0.6 IU/mL occurred in 3.8% (109/2,895) of participants and was not associated with age or sex. However, participants with HIV had reduced odds of TB2-TB1>0.6 IU/mL (adjusted odds ratio 0.37 [0.14--0.93]). At higher hypothetical positivity thresholds, the mean predicted Mtb immunoreactivity prevalence among males exceeded that in females at an earlier age: at 19 years, predicted immunoreactivity prevalence ratios were 0.90 (0.83--0.99) and 1.02 (0.89--1.15) at 0.1 IU/mL and 0.5 IU/mL thresholds, respectively. Conclusions: Quantitative IGRA responses offer information about age-- and sex--specific immunoreactivity and transmission risks that dichotomisation obscures. In high-burden settings, quantitative IGRA responses may clarify Mtb transmission patterns and guide targeted public health strategies.

Rwanda is a malaria endemic country and a focal point for emerging Plasmodium falciparum artemisinin partial resistance (ART-R). While Demographic and Health Surveys (DHS) provide both national and province-level representative data, malaria testing in Rwandan DHS (RDHS) studies has been limited to a subset of adult women and children under 5 years using RDT and/or microscopy. Recent work using ultra-sensitive quantitative real time PCR on residual dried blood spots (DBS) from the 2014-15 RDHS revealed a significantly higher P. falciparum prevalence than detected by standard DHS diagnostics. Building on this study, we analyzed 7,127 adult DBS samples collected for HIV testing in the 2019-20 RDHS to generate updated prevalence measures. We found a national P. falciparum infection prevalence of 7.7% (95%CI [6.8%, 8.7%]), with predominantly low-density infections (median parasitemia: 7.3 parasites/uL). We assessed covariates of P. falciparum malaria infection, identifying male sex, lower household wealth, lower educational achievement, and residence at lower elevation as significant predictors. Notably, national P. falciparum prevalence decreased 53% relative to the parallel 2014-15 RDHS study, despite reports of increasing ART-R-associated mutations in Rwanda. These findings demonstrate the utility of ultra-sensitive molecular surveillance, and suggest that national malaria control efforts have substantially reduced malaria burden in Rwanda even amid rising antimalarial parasite prevalence. Subsequent studies on this data set will provide measures of minor Plasmodium species prevalence, as well as temporospatial analysis of antimalarial resistance markers in P. falciparum positive samples.

Background Five-biomarker-defined hypervirulent Klebsiella pneumoniae (hvKp) causes invasive infections, but its burden in bloodstream infections versus classical K. pneumoniae (cKp) is unclear. Methods This retrospective cohort study at a tertiary hospital in Japan included K. pneumoniae bloodstream infection episodes from January 2022-December 2024. hvKp was defined by the presence of all 5 genotypic biomarkers (rmpA, rmpA2, iucA, iroB, and peg-344). The primary outcome was abscess complications, and secondary outcomes were length of stay and antibiotic duration. Whole-genome sequencing was performed for 164 isolates. Results Among the 207 episodes, 28 (14%) were of hvKp. Abscess complication occurred in 17 (61%) hvKp versus 23 (13%) cKp episodes (adjusted odds ratio 10.7; 95% CI, 4.36-26.2). Median length of stay in hvKp versus cKp was 28 versus 14 days (adjusted ratio 1.60; 95% CI, 1.18-2.16) and median antibiotic duration was 43 versus 14 days (adjusted ratio 2.13; 95% CI, 1.64-2.77). These associations were attenuated after adjusting for abscess-related complications. No significant difference in 30-day mortality was observed, although the study was underpowered. Multidrug resistance was less frequent in hvKp strains than in cKp strains (11% vs. 30%; P = .040). Among the sequenced hvKp episodes, abscess rates varied across lineages, from 9 of 10 in ST23 to 1 of 4 in ST412. Conclusions Five biomarker-defined hvKp strains delineated a bloodstream infection subgroup with frequent abscess complications and prolonged care. hvKp and cKp present distinct clinical challenges; diagnostic tools distinguishing these subgroups may aid abscess evaluation and source control.

BackgroundThe Toto Bora trial tested whether a course of azithromycin reduced rates of re-hospitalization or death in the 6 months following hospitalization among Kenyan children. We hypothesized that azithromycin would reduce enteric bacteria and increase carriage of macrolide resistance in the subsequent 3 months. MethodsKenyan children (1-59 months) hospitalized and subsequently discharged for non-traumatic conditions provided fecal samples before and 3 months after randomization to a 5-day course of azithromycin or placebo. Quantitative PCR identified enteropathogens and AMR-conferring genes in fecal samples. Generalized estimating equations assessed the impact of the randomization arm on pathogen and resistance gene detection, accounting for baseline presence and site. ResultsAmong 1,393 baseline stools, 12.4% had at least one bacterial enteropathogen, 94.7% had at least one macrolide-resistance gene, and 92.6% had at least one beta-lactamase-resistance gene identified. At month 3, children randomized to azithromycin had a 6.1% higher likelihood of carrying a macrolide resistance gene compared to placebo (adjusted prevalence ratio [aPR], 1.06; 95% CI, 1.04-1.08; P<0.001). Specifically, azithromycin randomization was associated with a higher relative prevalence of erm(B) (aPR, 1.09 [95% CI, 1.04-1.15]; P=0.001), erm(C) (aPR, 1.23 [95% CI, 1.14-1.31]; P<0.001), msr(A) (aPR, 1.14 [95% CI, 1.04-1.25]; P=0.007), and msr(D) (aPR, 1.07 [95% CI, 1.03-1.11]; P=0.001). There was no difference in overall bacterial pathogen prevalence (18.9% vs 17.3%) between randomization arms, but a slightly lower proportion of children had Shigella after randomization in the azithromycin arm (3% vs. 5%, aPR, 0.79 [95% CI, 0.62, 1.01]; P=0.063). InterpretationAzithromycin at hospital discharge was associated with higher carriage of macrolide-resistance-conferring genes in the post-discharge period compared with placebo, without significant declines in enteric pathogen carriage other than modest changes to Shigella. The potential benefits and risks of empiric azithromycin need to be considered, as children are increasingly exposed to this broad-spectrum antibiotic.

Background: Although the hemagglutination inhibition (HAI) titer remains the gold standard correlate of protection against influenza, it does not fully capture the broader antibody responses that contribute to immunity. Methods: We analyzed immune responses in paired pre-infection and convalescent sera from 306 RT-PCR-confirmed A/H3N2 infections from two household studies (2014-18) in Managua, Nicaragua. Antibody responses were measured by HAI and enzyme-linked immunosorbent assays (ELISAs) against full-length hemagglutinin (HA), the HA stalk, and neuraminidase (NA). Participants were classified as HAI responders ([&ge;]4-fold HAI rise), alternate responders (no HAI rise but [&ge;]4-fold boost in [&ge;]1 ELISA), or no-response individuals (no [&ge;]4-fold rise in any assay). We compared demographic, clinical, and pre-infection antibody characteristics across these groups. We also analyzed predictors of an NA response. Results: Overall, 77% of participants had HAI seroconversion or a 4-fold rise. Among the 23% HAI non-responders, 62% had alternate antibody responses. No-response individuals had the highest pre-infection HAI and full-length HA titers (p < 0.0001), the lowest viral loads, and the fewest fever or influenza like illness (ILI) symptoms (p < 0.01). An NA response was more common among symptomatic individuals (p = 0.0483) and those with low or high baseline NA titers. Conclusions: High baseline HAI titers can limit detectable 4-fold rises and are associated with milder illness. Evaluating additional immune responses may capture a more complete picture of the host response to infection, thereby improving surveillance and informing vaccine development. Keywords: Influenza A/H3N2; Hemagglutination inhibition (HAI); Neuraminidase antibodies; symptomatic vs asymptomatic infection; correlates of protection.

Numerous factors may influence the optimal rollout of new gonococcal antibiotics. We compared eight rollout strategies using a gonorrhea transmission model and ranked strategies by the number of gonococcal infections and clinically useful antibiotic lifespan. Rankings were most sensitive to the starting ceftriaxone resistance prevalence and screening frequency.

BackgroundMalnutrition compromises mucosal immunity, especially in the respiratory tract, increasing susceptibility to pathogens like Streptococcus pneumoniae. This study assessed whether nasal administration of Lacticaseibacillus rhamnosus CRL1505 or its peptidoglycan could promote the recovery of nasopharynx-associated lymphoid tissue (NALT) structure and functionality, thereby enhancing resistance to S. pneumoniae infection in protein-malnourished mice. MethodsMale Swiss albino mice were fed to a protein-free diet to induce malnutrition, followed by nutritional repletion with or without nasal supplementation of CRL1505 or its peptidoglycan. Resistance to S. pneumoniae infection, NALT architecture, immune cell composition in NALT and regional lymph nodes, and nasal cytokine production were evaluated. ResultsProtein deficiency caused marked NALT atrophy, immune cell depletion, and heightened susceptibility to S. pneumoniae. Nutritional repletion alone partially reversed these effects. In contrast, nasal supplementation with CRL1505 or its postbiotic fully restored NALT structure and cellularity, normalized lymphoid and myeloid populations, and reduced pathogen burden. Both treatments increased B and T lymphocytes, immature B cells, dendritic cells, and macrophages. The postbiotic also enhanced MHCII expression and balanced neutrophil-like Gr-1 cells. Notably, immune enhancement was evident even before infection, indicating a mucosal priming effect. Cytokine levels in nasal fluids remained largely unchanged. ConclusionsNasal delivery of L. rhamnosus CRL1505 or its postbiotic effectively reestablished NALT integrity and mucosal immunity in malnourished mice, providing significant protection against respiratory pathogens. These findings support the development of nasal immunobiotic formulations as non-invasive interventions to bolster respiratory defenses in immunocompromised hosts.

BackgroundIn the course of testing mAb-based therapies to eradicate the persistent reservoir of HIV infection, we investigated the efficacy and mode of killing of HIV-infected cells by two categories of cytotoxic immunoconjugates (CICs) targeted by the same mAb, an immunotoxin (IT) and antibody-drug conjugate (ADC). MethodsWe performed metabolic and transcriptional analyses of treatment effects on the persistently-infected cell line H9/NL4-3. Cells were treated with CICs consisting of the anti-gp41 mAb 7B2 conjugated to either deglycosylated ricin A chain (dgA) or to the highly cytotoxic anthracycline derivative PNU-159682. At intervals up to 24 hr, intracellular metabolites were quantified by 1H nuclear magnetic resonance spectroscopy, and the transcriptome analyzed by RNA-Seq. ResultsSix hr post treatment, 7B2-dgA elicited both metabolic and transcriptional alterations, whereas 7B2-PNU treated cells did not differ from untreated cells. 7B2-dgA treated cells exhibited elevated intracellular levels of many amino acids, and activation of gene pathways for apoptosis, intracellular signaling, and immune activation. By 24 hr, both 7B2-dgA and 7B2-PNU treated cells differed markedly from untreated. Many of the changes observed following 7B2-PNU treatment at 24 hr were similar to those observed at 6hr following 7B2-dgA, likely indicating processes involved in cell death, but a number of alterations were unique to either IT or ADC treated cells. ConclusionsAn IT and ADC showed both similarities and differences in their cytotoxic effects. These results raise the question of whether the mode of cell killing could be a determinant of clinical efficacy. Although these studies were aimed at targeting the persistent reservoir of HIV infection, they have relevance for the design of CICs to treat cancer and other conditions. SUMMARYThe use of cytotoxic immunoconjugates, wherein an antibody is attached to a cellular poison, is effective in the treatment of cancer and other conditions. We seek to extend these results to treating HIV and other chronic viral infections. We analyzed the molecular mechanisms of cell killing when the same antibody was attached to different toxic structures. We report that each immunoconjugate induced both common and distinct patterns of killing. Such differences may have clinical relevance.

RationaleBronchopulmonary dysplasia (BPD), the lung disease associated with premature birth, is a significant health problem, often with long-term respiratory consequences. Recent research has highlighted the potential role of the lung and gut microbiome in the development and progression of BPD, yet it is unclear what aspects of the microbiome may contribute to BPD susceptibility. ObjectivesTo comprehensively characterize the lung and gut microbiomes of preterm infants and identify shared microbial taxa that are associated with BPD development. MethodsTracheal aspirate and stool samples were collected from 39 premature infants over the first month of life. To assess the taxonomic microbial composition of the lung and gut, samples were analyzed using shotgun metagenomic sequencing. BPD classification was determined using the National Institute of Child Health and Human Development severity-based definition at 36 weeks postmenstrual age. Measurements and Main ResultsMicrobial communities of the lung and gut were significantly different between infants who went on to develop BPD and those who did not, with an enrichment of skin-associated microbial genera such as Staphylococcus, Corynebacterium, and Cutibacterium in infants who developed BPD. Specifically, Staphylococcus epidermidis was enriched in premature infants who developed BPD and was the most prominent species shared between lung and gut communities. Temporal changes in gut microbial communities co-occurred with feeding practices and antibiotic exposure, suggesting an influence of external factors on microbiome composition. ConclusionsOur findings provide evidence that certain microbial colonization patterns among premature infants are closely associated with the pathogenesis and progression of BPD.

ObjectiveStudies of nutritional status and host responses during severe and critical illness have focused predominantly on obesity; in contrast, the relationship between undernutrition, host responses, and clinical outcomes in adults hospitalized with severe infection remains poorly defined. We sought to determine whether severe undernutrition is associated with distinct host responses and clinical outcomes in adults hospitalized with severe infection. DesignProspective cohort study. SettingTwo public referral hospitals in Uganda. PatientsNon-pregnant adults ([&ge;]18 yr) hospitalized with severe, undifferentiated infection. InterventionsNone. Measurements and Main ResultsWe analyzed clinical data and serum Olink proteomic data from 432 participants (median age, 45 yr [IQR, 31-57 yr]; 44% male). Overall, 213 participants (49%) met prespecified criteria for undernutrition, including 52 (12%) with severe undernutrition. Clinically, severe undernutrition was associated with HIV coinfection, microbiologically diagnosed tuberculosis, greater physiological instability, and higher mortality. After adjustment for age, sex, illness duration, study site, and HIV, malaria, and tuberculosis coinfection, severe undernutrition was associated with higher expression of proteins involved in pro-inflammatory immune signaling, endothelial and vascular remodeling, hypoxia and oxidative stress responses, and extracellular matrix remodeling, together with lower expression of proteins linked to growth signaling, anticoagulant regulation, and lipid homeostasis. ConclusionsSevere undernutrition is associated with a distinct high-risk clinical phenotype and biologic signature in adults hospitalized with severe infection. These findings suggest that undernutrition may potentiate key domains of sepsis pathobiology, with implications for strengthening nutritional support and informing host-directed treatment strategies in low- and middle-income countries where malnutrition is common. Key PointsO_ST_ABSQuestionC_ST_ABSHow does undernutrition influence immune, metabolic, and endothelial responses to severe infection in adults? FindingsIn this multicenter cohort study of 432 adults hospitalized with severe infection in Uganda, severe undernutrition was associated with greater physiologic instability, higher mortality, and a distinct proteomic host-response profile. Adults with severe undernutrition exhibited a proteomic signature characterized by pro-inflammatory immune signaling, endothelial and extracellular matrix remodeling, and hypoxia and oxidative stress responses, together with lower expression of proteins involved in growth signaling, anticoagulant regulation, and lipid homeostasis. MeaningSevere undernutrition is associated with a distinct high-risk clinical and biologic phenotype during severe infection, with implications for nutritional support, risk stratification, and host-directed therapeutic strategies, particularly in low- and middle-income countries.

The intermittent preventive treatment with sulfadoxine-pyrimethamine (IPTp-SP) remains the main strategy to prevent malaria in pregnancy. However, continued drug pressure may also contribute to the emergence of resistant parasites and impact the gametocyte carriage and subsequent infectiousness. Pregnant women are thought to be a potential reservoir for malaria transmission due to the increased carriage of gametocytes following long-lasting infections. We used molecular methods to examine 100 Plasmodium falciparum (P. falciparum) isolates collected from Mozambican women at delivery in 2014-15, to determine SP resistance polymorphisms in P. falciparum dihydrofolate reductase (pfdhfr) and dihydropteroate synthetase (pfdhps) genes as well as the presence of gametocytes by RT-qPCR. Overall, 54% and 7% of parasites harbored quintuple and sextuple pfdhfr/pfdhps mutant haplotypes, respectively. Gametocytes were detected in 34% of isolates. Gametocyte carriage was significantly associated with quintuple mutant infections (AOR = 7.5, p = 0.001), which accounted for 80% of infections with detectable gametocytes. Results indicate the relevance of ongoing surveillance of SP resistance in Mozambique to guide future evaluation of alternative IPTp approaches as resistance levels evolve and to anticipate potential implications for parasite transmission and maternal-fetal health.

BackgroundLung ischemia-reperfusion (IR) injury drives early morbidity after lung transplantation and cardiothoracic surgery, yet targeted preventive therapies are lacking. The gut-lung axis and microbiota-derived tryptophan metabolites, including indole-3-propionate (IPA), may regulate pulmonary immunity and inflammation. We investigated whether a tryptophan-rich (Trp-Rich) diet attenuates sterile lung IR injury by increasing microbiota-derived indole metabolites and reprogramming alveolar macrophage (AM) inflammatory responses. MethodsC57BL/6 mice received isocaloric tryptophan-standard (Trp-Std; 0.18%) or Trp-Rich (0.60%) diets for 14 days, then underwent unilateral left lung IR (60 min ischemia followed by 60 min reperfusion). Oxygen saturation, lung cytokines, and aryl hydrocarbon receptor (AhR) signaling readouts (Cyp1a1/Cyp1b1) were evaluated. Gut microbiota was profiled by 16S rRNA sequencing, and targeted metabolomics quantified tryptophan metabolites in feces, portal vein (PV) plasma, and lung tissue. To further assess inflammatory priming in vivo, mice were additionally challenged with intratracheal lipopolysaccharide (LPS). Mechanistic studies compared IPA with related indole metabolites in MH-S cells and primary human AMs, including ex vivo nutritional IR, LPS stimulation, and AhR stimulation and blockade using synthetic agonists and antagonists. ResultsTrp-Rich feeding improved post-IR oxygenation, reduced lung IL-1{beta}, and increased pulmonary Cyp1a1/Cyp1b1 gene expression. Trp-Rich diet remodeled the gut microbiota, including enrichment of Bifidobacterium and Lactobacillus, and increased IPA levels across feces, PV plasma, and lung tissue, with lower kynurenine/IPA ratios across matrices. In the LPS intratracheal challenge, Trp-Rich feeding reduced IL-6 levels in lung tissue and systemic plasma. Primary murine AMs isolated from Trp-Rich mice also showed reduced IL-1{beta} and IL-6 release in an ex vivo nutritional IR model. Among tested indole metabolites, IPA showed the strongest dose-dependent suppression of LPS-induced cytokines and chemokines in MH-S cells and primary human AMs, remained active in the ex vivo nutritional IR model, and its anti-inflammatory effect was abrogated by AhR blockade and enhanced by co-treatment with other indole metabolites. ConclusionsA Trp-Rich diet attenuated sterile lung IR injury, coinciding with gut microbiota remodeling, increased systemic and pulmonary IPA, reduced inflammatory priming, and reprogrammed AM responses. These data support diet- or microbiome-directed strategies targeting IPA-AhR signaling to mitigate perioperative lung IR injury. Caption for graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=190 SRC="FIGDIR/small/714281v1_ufig1.gif" ALT="Figure 1"> View larger version (67K): org.highwire.dtl.DTLVardef@1b06a9corg.highwire.dtl.DTLVardef@1273f33org.highwire.dtl.DTLVardef@1a63a2borg.highwire.dtl.DTLVardef@350e1c_HPS_FORMAT_FIGEXP M_FIG A tryptophan-rich diet remodels the gut microbiota and indole metabolite profiles, including IPA, enhances alveolar macrophage AhR signaling, and attenuates sterile lung ischemia-reperfusion injury. C_FIG