The Journal of Infectious Diseases

BackgroundMycoplasma genitalium (MG) is an emerging sexually transmitted infection (STI) associated with pelvic inflammatory disease and tubal factor infertility. Its relationship with impaired fecundity remains unclear and is rarely examined in the context of co-seropositivity with other STIs. MethodsWe conducted a secondary analysis of the Effects of Aspirin in Gestation and Reproduction (EAGeR) trial, a prospective preconception cohort of women with proven fecundity and prior pregnancy loss. MG serostatus was determined using Western blot-based IgG assays on 1146 stored serum specimens. Chlamydia trachomatis (CT) and other STIs were also measured. Associations between MG seropositivity and measures of impaired fecundity were assessed. Pregnancy loss and live birth were modeled using inverse-probability weighted quasi-Poisson and unweighted log-binomial models to calculate relative risks (RR). Fecundability-odds-ratio (FOR) was estimated using a discrete Cox proportional hazards model accounting for left truncation and right censoring. Propensity score (PS) weighted versions of these models assessed risks associated with CT co-seropositivity. Analyses were adjusted for demographic and reproductive history variables. ResultsOverall, 17.1% (n=210) of participants were MG seropositive, with 27.6% (n=58) co-seropositive with CT. Compared to STI-seronegative women, MG seropositivity was not associated with any outcome, although trends were observed for reduced fecundability (FORadj: 0.87, 95% CI 0.70-1.08) and live birth (RRadj: 0.94, 95% CI 0.79-1.11). Co-seropositivity with CT was associated with lower likelihood of live birth [Relative Risk (RR)PS-weighted: 0.82, 95% CI: 0.70-0.96]. Sensitivity analyses supported the robustness of these findings. ConclusionsBeing co-seropositive for MG and CT preconception may impair fecundity.

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.

BackgroundNK cells are major innate and adaptive responders to malaria, with multiple roles in protection. Function of NK cells is heterogeneous, underpinned by expression of a diversity of receptors. One driver of NK cell heterogeneity is latent CMV infection, which drives the expansion of memory-like NK cells. We have recently reported that latent CMV infection can negatively impact the adaptive immune response to malaria, but whether CMV-mediated changes to the NK cell compartment also impact innate responses to malaria is unknown. MethodsWe investigated the impact of latent CMV infection on NK cell response to the malaria parasite Plasmodium falciparum in vitro, and in CMV seronegative and seropositive individuals during controlled human malaria infection. We analysed NK cell activation, cytotoxicity and NK cell receptor expression. Additionally, we investigated the impact of CMV serostatus on cytokine production in response to TLR stimulation in the myeloid cell compartment. The impact of CMV and NK cell responses on parasite control and malaria symptoms was investigated. ResultsNK cells from CMV seropositive individuals had reduced responsiveness to P. falciparum parasites in vitro and had reduced activation during controlled human infection. Reduced activation was not restricted to NK subsets modulated by CMV but occurred across the entire NK cell compartment. Consistent with global NK cell attenuation, IL-12 production from myeloid cells, a response that supports NK cell activation on exposure to P. falciparum parasites, was lower in CMV infected individuals. Linking NK cell activation to clinical outcomes, NK cells expressing perforin were associated with parasite control in CMV seronegative individuals. ConclusionCMV infection modulates NK cell responses during malaria by disruption of IL-12, leading to reduced parasite control.

BackgroundCambodia is nearing Plasmodium falciparum elimination, but targeted interventions are needed to prevent resurgence. Identity-by-descent (IBD)-based methods offer insight into parasite relatedness and transmission dynamics. MethodsWe estimated pairwise genetic relatedness between 107 P. falciparum isolates collected from military personnel and farmers in Cambodia between 2014 and 2016. All isolates underwent whole genome sequencing. Relatedness was defined as the proportion of the genome shared IBD between isolates. We evaluated associations between relatedness and epidemiologic factors, including occupation, location, time, age, and sex. Infomap community detection was used to identify clusters of highly related parasites and their association with covariates. ResultsMean IBD sharing was high (40%), with greater relatedness among isolates from Oddar Meanchey than from Kratie province (p<2.2e-16). Within Oddar Meanchey, isolates from individuals with different occupations shared 7.2% less of their genome IBD than farmer pairs (probability of direction (pd)=99.9), after adjusting for age. Highly related clusters (>90% IBD) were associated with occupation, K13 mutation (C580Y vs. R539T), and PfCRT mutation (p= 0.0025, 0.0001, and 0.0001, respectively). Isolates from mixed-occupation pairs had lower odds of high relatedness (>90% IBD) than pairs from farmers (pd=0.958). ConclusionsHigh overall IBD sharing may reflect a combination of recent elimination efforts and expansion of drug-resistant lineages. Substantial differences in relatedness between provinces underscore the role of geography in shaping relatedness patterns. Lower relatedness among mixed-occupation pairs compared to farmer pairs suggests potential occupation-specific transmission sources and supports tailoring interventions to local micro-epidemiologic factors.

BackgroundTuberculosis (TB) is a leading cause of morbidity and mortality among children living with HIV (CLHIV). Poor diagnostic performance is a significant contributor. Serological assays that determine levels of Mycobacterium tuberculosis reactive antibodies inconsistently detect TB. However, antigen-specific antibody Fc receptor engagement and effector functions are promising biomarkers of TB disease. MethodsThis study evaluated serum from a well-characterized cohort of Kenyan CLHIV via two orthogonal approaches: 1) longitudinally following over the course of TB treatment and 2) assessing a cross-section with and without clinical TB disease. For each individual sample, 13 antibody functional properties against 8 Mtb and 4 non-Mtb microbial antigens were measured and analyzed via univariate and multivariate machine-learning approaches. FindingsFcR/CD89 immune complex formation with antibodies reactive to four Mtb antigens including ESAT-6 & CFP-10, Fc{psi}RI/CD64 associated with one Mtb antigen, and HIV gp120 IgA1 levels decreased during the intensive and continuation/consolidation phases of TB therapy. This antibody signature also highlighted treatment non-responsiveness and distinguished children with from those without TB disease with predictive capacity similar to Xpert. InterpretationAn Mtb and HIV reactive peripheral blood antibody functional signature of FcR/CD89, Fc{psi}RI/CD64, and IgA1 has the potential to complement current clinical tools and those in development to diagnose pulmonary TB disease in CLHIV. FundingThis work is supported by UT Southwestern Disease Oriented Scholars Award (LLL), NIAID 5R01AI158858 (LLL), Burroughs-Wellcome Fund UTSW Training Resident Doctors as Innovators in Science (YJK), NICHD K12HD000850, NIAID K23AI143479 and R21AI192086 (LMC), NICHD R01 HD023412 (GJS), NIAID 75N93019C00071 (CW).

BackgroundExisting ex vivo mucosal explant models for HIV assess viral replication but do not fully capture the ability of tissue to support productive infection and transfer to target cells, an important indicator of tissue susceptibility. Additional tools to investigate relative mucosal susceptibility to HIV are necessary to better understand risk factors, such as substance use, or to evaluate therapeutics. ResultsWe developed an ex vivo gut mucosal explant assay incorporating a CCR5/CXCR4-expressing GFP-reporter cell co-culture to quantify both viral replication and transfer. Susceptibility was quantified using a composite metric based on time to infection endpoint across viral doses. Modulators of susceptibility, including anti-CD3 stimulation, tenofovir, and methamphetamine, were evaluated. The assay reliably detected differences in tissue susceptibility under various experimental conditions. We detected enhanced susceptibility following anti-CD3 stimulation, while tenofovir pretreatment reduced susceptibility in a dose-dependent manner. Methamphetamine exposure resulted in a modest but significant increase in gut tissue susceptibility to HIV. ConclusionsThis novel ex vivo explant susceptibility assay advances HIV mucosal transmission research by measuring both viral production and transfer. It can detect subtle changes in susceptibility, providing a versatile platform for evaluating prevention therapeutics, host and microbial factors, and substance use effects.

BackgroundMalaria transmission in southwestern Uganda is low, but persists despite ongoing control efforts. Identifying whether infections are locally sustained or imported by travelers is critical for guiding interventions. We integrated epidemiologic surveillance with parasite genomics to characterize imported malaria episodes at three health facilities in southwestern Uganda. MethodsBetween January 2023 and June 2024, we enrolled microscopy-confirmed malaria cases at three health facilities, Maziba and Muko (very low transmission) and Kamwezi (low-to-moderate transmission), administered travel history questionnaires, and collected dried blood spots for genotyping. Plasmodium falciparum infections were genotyped using MAD4HatTeR, a highly sensitive multiplex amplicon sequencing panel targeting 165 diversity markers and 38 drug resistance loci. Complexity of infection and pairwise relatedness were estimated using MOIRE and Dcifer, respectively. Plasmotrack, a Bayesian transmission network framework, was used to infer network structure, transmission directionality, reproduction numbers, and importation rates. ResultsAmongst malaria cases, recent overnight travel was common in Maziba (87%) and Muko (96%) but infrequent in Kamwezi (12%). Most travel in cases from Maziba and Muko was from high-transmission regions in northern and eastern Uganda. Parasites in Maziba and Muko cases exhibited higher within-host diversity and lower within-site relatedness compared to those in Kamwezi cases. Transmission network inference identified most infections in Maziba and Muko as imported, with the majority of inferred secondary transmission linked to recent travelers. In contrast, Kamwezi showed multiple highly related clusters, indicating sustained local transmission. Validated and candidate markers of artemisinin partial resistance (K13 P441L and R561H) were more prevalent in Kamwezi. ConclusionMalaria in Maziba and Muko was driven largely by importation from other parts of Uganda, while local transmission played a larger role in Kamwezi . Tailored interventions addressing travel-associated risks and local transmission, supported by travel histories and parasite genetic data will be valuable to advance malaria elimination in this region.

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)

Placental malaria may negatively impact acquisition of immunity to malaria in infants. To study the effect of maternal antibodies on early-life infections, a birth cohort of infants were enrolled in a prospective observational study in Busia, Uganda. We measured P. falciparum-specific antibody responses to 19 P. falciparum antigens and tetanus toxoid in 183 infants using a Luminex multiplex bead array. We also assessed the frequency and phenotype of peripheral T follicular helper cells and B cells using flow cytometry. Placental malaria and lower gestational age adversely impacted antibody transfer overall. Maternal antibodies present in cord blood were not associated with protection in the first year of life, and neither was maternal malaria status. Infants generated antibodies against P. falciparum antigens in response to infection, but these were also not associated with protection, only exposure. Interestingly, infections in the first six months of life correlated with decreased levels of antibodies to P. falciparum antigen at one year of age. Early-life infections were not clearly associated with circulating follicular T cell or B cell phenotypic composition. These data suggest the infant antibody response has little, if any, impact on preventing clinical malaria or patent parasitemia in the first year of life.

BackgroundTuberculosis (TB) and HIV co-infection cause profound immune dysregulation. Understanding how these infections alter immune cell distribution across systemic and tissue compartments is critical for improving therapeutic and vaccine strategies. MethodsFlow cytometry was used to quantify CD4 and CD8 T cells, B cells, and tissue-resident memory (TRM) T and B cells in peripheral blood mononuclear cells (PBMCs), lung tissue, bronchoalveolar lavage (BAL), spleen, and lung-draining hilar lymph nodes (HLN) from individuals with pulmonary TB (PTB), disseminated TB (Diss TB), HIV only, or both TB and HIV infections. ResultsCD4 T cell frequencies were significantly reduced in multiple compartments of HIV infected subjects, irrespective of TB status, indicating systemic immune suppression. CD8 T-cell frequencies were elevated in the blood of HIV-infected individuals, suggesting a compensatory response to CD4 T-cell loss. B-cell frequencies were reduced in PBMCs and lung tissue of TB subjects, regardless of HIV status. Notably, CD4 TRM T cells were specifically depleted in lung tissue of HIV/TB co-infected individuals, whereas TRM B cells were selectively depleted in TB subjects, independent of HIV infection. ConclusionTB and HIV drive distinct and compartment-specific TRM cell loss in infected tissues. HIV primarily targets CD4 TRM T cells, while TB specifically depletes TRM B cells, highlighting separate mechanisms of tissue-resident immune disruption. These findings emphasize the importance of tissue-specific immune analyses and provide new insights for targeted vaccine and immunotherapy strategies.

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.

BackgroundWe assessed associations between antibody concentrations within 7 days of symptom onset and testing positive for influenza virus infection among outpatients enrolled in a test-negative study. MethodsFrom November 2018[boxh]May 2019, study sites in five states obtained serum and respiratory specimens from outpatients aged [&ge;]18 years presenting with acute respiratory illness. Respiratory specimens were tested for influenza virus, and viral clades were identified by genomic sequencing. We measured influenza antibody titers against vaccine and circulating viruses by hemagglutination inhibition (HI), microneutralization (MN) and neuraminidase inhibition (NAI) assays. Percent of patients with HI, MN and NAI titers [&ge;]10 and [&ge;]40 were compared among patients with and without influenza-associated illness, and reduction in odds of confirmed influenza at increasing HI, MN and NAI antibody titers was estimated using logistic regression adjusting for influenza vaccination status and time since beginning of influenza season. ResultsAmong 175 patients with confirmed influenza virus infection, including 112 with influenza A(H1N1)pdm09 and 63 with A(H3N2) (44 clade 3C.3a), and 130 test-negative control patients, higher antibody titers against influenza hemagglutinin or neuraminidase proteins at enrollment were associated with lower odds of influenza virus infection. HI and MN antibody titers against circulating viruses were more strongly associated with protection than titers against vaccine reference viruses. Odds of A(H1N1)pdm09 infection were 44% and 54% lower for each two-fold increase in A(H1N1)pdm09 HI or NAI titer, respectively. Odds of A(H3N2) infection were 46% and 30% lower, respectively, for each two-fold increase in MN or NAI titer against circulating A(H3N2) virus clade. NAI titers were independently associated with lower odds of influenza A(H1N1)pdm09 and A(H3N2) after controlling for HI titer. ConclusionHigher influenza antibody titers against circulating viruses were associated with lower likelihood of influenza virus infection among adult patients with acute respiratory illness. SUMMARYFrom November 2018[boxh]May 2019, we assessed the association between antibody concentrations during acute illness and laboratory-confirmed influenza among adult patients enrolled in a test-negative study in five US states. We found that higher influenza antibody titers were associated with lower likelihood of symptomatic influenza virus infection.

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.

BackgroundPlasmodium vivax is the predominant cause of malaria in South Asia. P. vivax cases have fallen over the past decade, but cross-border transmission remains a major challenge to elimination. Genetic data can generate valuable insights into transmission; however, until now, only low-resolution data have been available from Nepal, Bhutan and Bangladesh. We piloted high-resolution genotyping using a new microhaplotype (multiallelic) assay to monitor P. vivax transmission across borders. MethodsGenotyping was conducted using the 93-microhaplotype vivaxGEN panel on P. vivax parasites collected from patients enrolled in clinical trials in Bangladesh, Bhutan, and Nepal between 2013 and 2023. These data were compared with open-access microhaplotype and genomic data derived from Afghanistan, India, and Pakistan between 2014 and 2024. Polyclonality and relatedness (identity by descent (IBD)) were determined within and between countries. ResultsHigh-quality genotyping data were generated for Nepal (n = 19), Bhutan (n = 27), and Bangladesh (n = 35); comparative data were sourced from Afghanistan (n = 159), India (n = 24), and Pakistan (n = 213). Overall, 29.6% (47/159) of isolates from Afghanistan and 20.2% (43/213) from Pakistan had polyclonal infections, whereas all parasites from Bhutan, Nepal and Bangladesh were monoclonal, suggesting lower superinfection. Country-wide IBD analyses revealed three genetic clusters partitioning Bangladesh and Bhutan (partial) from the remaining countries. There were two sub-populations in Bhutan, which separated local and cross-border imported cases. ConclusionsOur results highlight the use of regional high-resolution genetic data to enhance monitoring of transmission intensity and cross-border importations.

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.

BackgroundCombination antiretroviral therapy (cART) reduces morbidity among children living with HIV (CHIV) but does not restore measles immunity. Many CHIV now initiate cART before measles vaccination but the impact on measles-specific immunity is not understood. This study compared measles antibodies between CHIV initiating cART before and after 9 months of age in Zambia. MethodsThis retrospective study was nested within an open cohort study of CHIV and community-based malaria studies in Macha, Zambia conducted from 2007-2020. Samples were tested for measles IgG antibodies from CHIV defined by age at cART initiation (aged 0-8, 9-23, and 24-59 months) and age-matched community-based controls. A cross-sectional analysis compared antibody concentrations between CHIV groups after 6-12 months of cART and aged >12 months and controls. A longitudinal analysis evaluated antibody trajectories from 9 months of age through 48 months of cART and during the 2010-11 measles outbreak. ResultsIn the cross-sectional analysis, 31.9% of 301 CHIV were seropositive after 6-12 of cART, with no significant difference by group (0-8: 30.8%; 9-23: 37.0%; 24-59 months: 26.6%). Within each CHIV group, the proportion seropositive was significantly lower than age-matched controls. Among 242 CHIV included in the longitudinal analysis, 46-56% had an antibody response, with an estimated time to seroreversion of 1.8-3.2 years. Among CHIV followed in 2010-11, 50% seroconverted or boosted; the estimated time to seroreversion was 2.1 years. ConclusionsLow levels of measles antibodies were found among CHIV, even when cART was started before 9 months of age, supporting a recommendation that CHIV, including infants, would benefit from revaccination.

Gout is driven by an interleukin-1{beta}-mediated intense innate immune reaction to monosodium urate (MSU) crystals (MSUc). In cell culture models of inflammatory gout there is a synergistic effect of phagocytosis of MSUc and TLR2 and TLR4 activation by agonists such as free fatty acid and lipopolysaccharide (LPS) in NLRP3-inflammasome activation and IL-1{beta} secretion. A substantial number of gout patients do not report a dietary trigger, and observational studies associate airborne particulate matter with incident gout and flares. Airborne particulate matter contains LPS and airborne-derived particulate matter stimulates IL-1{beta} secretion in cell culture. We hypothesized that air-borne particulate matter could co-stimulate, with MSUc, IL-1{beta} secretion and inflammation. We tested the hypothesis using MSUc with extracted airborne PM4 in human cells (the THP-1 monocyte cell line, primary human monocytes and PBMCs) or carbon black particles with ozone (CB+O3) in a murine foot-pad injection model of gout. There was strong NLRP3-inflammasome-dependent co-stimulation of IL-1{beta} secretion in THP-1 cells with PM4+MSUc and a moderate additive effect in primary human PBMCs. However, there was no added effect on IL-1{beta} secretion of PM4 in isolated primary human monocytes. Inhalation of CB+O3 persistently exacerbated MSUc-induced murine paw inflammation, with an increase of alveolar/lavage macrophages that contained CB+O3 particles and increased lavage expression of IL-1{beta}. In conclusion, airborne-derived PM4 particulate matter enhanced MSUc-induced IL-1{beta} secretion in THP-1 cells and PBMCs. Combined with exacerbation of MSUc-induced inflammation by fine particulate matter in in vivo experiments, these data provide evidence that exposure to fine particulate matter may play a role in the etiology of gout.

BackgroundPost-tuberculosis (TB) lung disease (PTLD) affects approximately 50% of persons with pulmonary TB. We recently discovered whole blood transcriptional signatures associated with PTLD. We examined whether a minimum gene signature predicts PTLD as a clinically useful biomarker. MethodsWe prospectively enrolled 301 treatment naive adults with newly diagnosed pulmonary TB (PTB) (cohort A). We collected whole blood at 0 and 6-month visits, isolated RNA, and measured a modified MTB Host Response (HR) signature (mHR) based on expression of DUSP3, GBP5, and TMBIM6. We recorded spirometry at 6 (n=216) and 12 months (n=210) after treatment initiation and examined the association of the mHR score with PTLD and Mtb aerosolization. We recruited household contacts of cohort A to compare mHR score with non-PTB participants (cohort B). FindingsmHR was associated with TB (p=4.15e-66) when compared to HHCs, treatment response (p=1.07e-53), and characteristics including CD4 count (p=0.003), bacillary load (p=3.02e-05), lung cavities (p=1.59e-04), and lung quadrants involved (p=3.87e-06). The mHR score was not associated with Mtb aerosolization. In total, 105 (50%) participants had PTLD at 12 months including 61 with restriction, 26 with obstruction, and 18 with mixed obstruction and restriction. Baseline mHR was associated with obstructive PTLD at both 6 (p=0.003) and 12 months (p=0.012) in bivariate and multivariate analyses. The mHR score was not associated with restrictive lung disease. InterpretationBaseline mHR was associated with obstructive PTLD at 6 and 12 months and may have applications in targeting treatment and prognostication.

BackgroundTesting blood samples with multiplex bead assays can assess elimination and identify residual foci of transmission for multiple pathogens simultaneously. In Ecuador, onchocerciasis and yaws are presumed eliminated, and the status of trachoma is unknown. We assessed their elimination status by measuring IgG antibodies in children 6-24 months. MethodsIn a birth cohort of 404 children measured between 2021-2024 in Esmeraldas province, Ecuador, we tested dried blood spots at ages 6, 9, 12, 18, and 24 months using a multiplex IgG assay that included antigens to Onchocerca volvulus (Ov16), Treponema pallidum (rp17, tmpa), and Chlamydia trachomatis (Pgp3, Ct694). We estimated seroprevalence and incident seroconversion rates across an urban-rural gradient. ResultsOf 404 children enrolled, 370 contributed 1,606 samples. Seroprevalence was near zero for onchocerciasis (0.4%, 95% CI: 0.2% to 0.8%) and yaws (0.2%, 95% CI: 0.0% to 0.5%). Conversely, C. trachomatis Pgp3 seroprevalence increased along the urban-rural gradient from Esmeraldas city (3.4%, 95% CI: 1.8% to 5.8%) to remote, river-accessible rural villages (22.4%, 95% CI: 16.0% to 30.1%), and incident seroconversion was common in rural villages (16.1 per 100 child-years, 95% CI: 11.3 to 21.9). ConclusionsSerologic surveillance found no evidence of O. volvulus or T. pallidum transmission, consistent with elimination. High Pgp3 seroconversion rates suggest ongoing C. trachomatis transmission in rural villages. Results highlight the value of integrated serologic surveillance and motivate in-depth trachoma surveillance in Esmeraldas. Lay SummaryAntibodies measured in blood provide a sensitive measure of past infection. At the population-level, antibody responses are widely used to monitor pathogen elimination. Antibodies measured in blood samples from children along an urban-rural gradient in Ecuador showed no evidence of exposure to the pathogens that cause onchocerciasis or yaws, confirming their continued elimination in the region. However, antibody responses to chlamydia were common among children in rural villages during their first two years, motivating additional surveillance to assess trachoma endemicity in the area as its endemicity status is currently unknown. Trachoma is caused by ocular chlamydia infection, and these results indicate a need for additional surveillance to assess ocular infections and clinical signs of trachoma in this population.