The research, published in PLOS Global Public Health, followed 636 children under fifteen years of age who were being investigated for suspected pulmonary tuberculosis between January 2018 and January 2024. Each child was followed for twelve months after enrollment. The team, led by Juaneta Luiz and Heather J. Zar along with colleagues from South African and American institutions, tested induced sputum samples with both liquid culture, the traditional diagnostic gold standard, and Xpert MTB/RIF Ultra, a rapid molecular assay that detects genetic material from Mycobacterium tuberculosis and also flags mutations associated with rifampicin resistance.

Inducing sputum in a child is a carefully choreographed procedure. After a nebulized dose of hypertonic saline loosens secretions in the lungs, a health worker uses gentle suction through the nose or mouth to collect the loosened material. The technique is considered safe and is widely recommended, but questions have persisted about how well rapid molecular tests perform on these small, often bacteria-sparse samples, particularly in the very groups where tuberculosis hits hardest. Previous studies frequently excluded infants, malnourished children, and children with HIV, leaving clinicians with limited evidence to guide them.

The study population reflected exactly those high-risk profiles. Among the 636 enrolled children, the median age was just 27.4 months, and 163 children, or 25.6 percent, were infants under one year. A total of 223 children, 37.8 percent, were underweight for their age, and 168 children, 26.5 percent, were living with HIV. Using the consensus definitions established by the United States National Institutes of Health, the researchers classified 173 children, or 27.2 percent, as having confirmed tuberculosis, 384 children, or 60.4 percent, as having unconfirmed tuberculosis, and 79 children, or 12.4 percent, as having unlikely tuberculosis. The large unconfirmed category underscores a familiar frustration: most children with suspected tuberculosis never receive microbiological proof of their disease.

Among the 557 children ultimately classified with tuberculosis, the induced sputum samples proved surprisingly productive. Overall, 145 children, or 26 percent, had their tuberculosis confirmed microbiologically on induced sputum. Xpert MTB/RIF Ultra accounted for the bulk of these confirmations, detecting the bacterium in 137 of the 557 children, or 24.6 percent, while liquid culture confirmed 88 children, or 15.8 percent. Notably, 46 percent of the Ultra-positive results fell into the assay’s lowest positive band, the so-called trace positive category, a finding that mirrors the low bacterial burden typical of childhood tuberculosis and highlights the assay’s enhanced sensitivity compared with its predecessor.

One of the most striking findings was the degree to which the molecular test outperformed culture in this setting. Of the 145 children with microbiologically confirmed tuberculosis, 57, or 39.3 percent, tested positive on Ultra but negative on culture. These culture-negative, Ultra-positive cases contributed a full 10.2 percent to the overall diagnostic yield. Because culture can miss infections when bacteria are scarce or when samples are tiny, the molecular assay’s ability to amplify even faint genetic signals makes it a particularly powerful tool for pediatric diagnosis. The researchers calculated that Ultra achieved an overall sensitivity of 90.9 percent and a specificity of 89.6 percent against culture, with confidence intervals indicating consistently high accuracy.

Performance varied somewhat across subgroups, and the patterns are informative. The test performed better in children over five years of age and in children with severe wasting, defined by a weight-for-height Z-score below minus three. The latter finding may seem counterintuitive, but severely malnourished children in this cohort may have had more advanced disease with higher bacterial loads, or their clinical presentation prompted more thorough sampling. Crucially, accuracy remained high across all the high-risk groups examined, including infants and children living with HIV, populations in whom diagnostic data have historically been thinnest. This suggests that induced sputum paired with Ultra can serve as a reliable first-line diagnostic across the pediatric spectrum in high-burden settings.

The stakes of getting this right are measured in lives. Among the 557 children treated for pulmonary tuberculosis, 27 died, a mortality rate of 4.9 percent. When the researchers adjusted for other factors, the risk of death was highest among children with stunting, who faced an adjusted relative risk of 7.3 compared with their better-nourished peers. Children living with HIV had an adjusted relative risk of 3.5, and infants faced an adjusted relative risk of 3.3. These figures lay bare the lethal intersection of tuberculosis with malnutrition, HIV, and extreme youth, and they reinforce why rapid, accurate diagnosis in these groups is not merely a technical goal but a matter of survival.

The implications for clinical practice are substantial. Xpert MTB/RIF Ultra returns results within about two hours, whereas liquid culture can take weeks to yield a definitive answer, if it yields one at all. In the interim, children may deteriorate, treatment may be delayed, and families may face repeated clinic visits. A rapid test that confirms nearly a quarter of all pediatric tuberculosis cases on a single induced sputum specimen, with high specificity that limits false positives and unnecessary treatment, could reshape diagnostic algorithms in the countries that bear the greatest tuberculosis burden. The test’s built-in rifampicin resistance detection adds a further layer of value, since drug-resistant tuberculosis requires entirely different treatment regimens that must be started promptly.

The study also carries lessons for global health policy. The World Health Organization has recommended Xpert assays as the initial diagnostic test for children with signs and symptoms of tuberculosis, but evidence from high HIV and malnutrition settings has been limited. By prospectively enrolling more than six hundred children over six years and following them for a full year, this study provides some of the strongest real-world evidence to date that the recommended approach works where it matters most. The authors conclude that Ultra on induced sputum was feasible, with high yield and diagnostic accuracy for high-risk children. As countries work toward eliminating tuberculosis as a public health problem, closing the persistent diagnostic gap in children, especially the youngest and most vulnerable, remains a critical frontier, and this study shows that the tools to close it are already within reach.

Subject of Research: Diagnostic accuracy of Xpert MTB/RIF Ultra on induced sputum for childhood pulmonary tuberculosis in a setting with high HIV and malnutrition burden

Article Title: Rapid diagnosis of pulmonary tuberculosis on induced sputum in children in a high burden setting for HIV and malnutrition

Article References: Luiz, J., Workman, L. J., Baard, C. B., Hlombe, Y., Prins, M., Makubalo, N., Jaganath, D., Cattamanchi, A., Nicol, M. P., & Zar, H. J. (2026). Rapid diagnosis of pulmonary tuberculosis on induced sputum in children in a high burden setting for HIV and malnutrition. PLOS Global Public Health, 6(10), e0007271. https://doi.org/10.1371/journal.pgph.0007271

Image Credits: AI Generated

DOI: 10.1371/journal.pgph.0007271

Keywords: tuberculosis, children, Xpert MTB/RIF Ultra, induced sputum, diagnosis, HIV, malnutrition, South Africa, pediatrics, molecular diagnostics, PLOS Global Public Health, public health

Post COVID-19 condition is characterized by persistent and highly heterogeneous symptoms that most prominently affect the cardiopulmonary system, including breathlessness, fatigue, exercise intolerance, and dysautonomia. Despite years of research, the biological mechanisms driving these symptoms remain poorly understood. Previous work has documented immune dysregulation and epigenetic alterations in peripheral blood following acute infection, but the lung itself, the primary site of infection and immune activation, has remained largely unexplored at the epigenetic level in this patient group.

The research team, led by Frida Nikesjö, Kristofer Hedman, and Maria Lerm of Linköping University, recruited 13 patients who had experienced persistent symptoms affecting daily life for more than 12 weeks after COVID-19 in 2020 and 2021. Participants were referred to the Department of Clinical Physiology at Linköping University Hospital, where they provided blood and induced sputum samples at study inclusion and again one year later. Induced sputum has been validated as a reliable and biologically informative source of pulmonary immune cells for methylome analysis, allowing the researchers to isolate neutrophil-enriched and macrophage-enriched cell fractions from the lung environment.

At each visit, patients also completed validated questionnaires covering fatigue, quality of life, and dyspnea, and underwent objective physiological testing including symptom-limited cardiopulmonary exercise testing, dynamic spirometry, and single-breath carbon monoxide diffusion capacity measurement. To reduce dimensionality in this small cohort, the team combined questionnaire scores and physiological outcomes into a single symptom-physiology composite variable, categorizing each measure from normal to severely abnormal using clinical reference limits.

Genome-wide DNA methylation profiling was performed using the Illumina Infinium Methylation EPIC 850K array, with rigorous preprocessing that removed failed and cross-reactive probes, sex chromosome sites, and probes containing common single nucleotide polymorphisms. Cell type proportions were estimated using the EpiDISH reference-based deconvolution method, and longitudinal differential methylation was assessed within individuals using the limma framework, with patient identity as a blocking factor and estimated cell proportions, sex, age, body mass index, and smoking status included as covariates. Differentially methylated CpG sites required both an absolute mean methylation difference greater than 0.15 and a false discovery rate-adjusted p-value below 0.05.

The most striking finding was a sharp divergence between compartments. No significant longitudinal DNA methylation changes were detected in peripheral blood mononuclear cells, yet pronounced changes emerged in both lung-derived fractions. In the neutrophil-enriched fraction, the researchers identified 446 differentially methylated CpG sites between the two time points, 70 hypermethylated and 376 hypomethylated, mapping to 274 unique genes. Pathway enrichment analysis across 334 KEGG pathways revealed 43 significantly enriched processes, with Wnt signalling and circadian entrainment among the most prominent. Notably, 87 of the affected genes are known to interact with SARS-CoV-2 proteins according to the BioGRID database.

In the macrophage-enriched fraction, 34 differentially methylated CpG sites were identified, evenly split between hyper- and hypomethylation. Although these changes did not map significantly to any KEGG pathways, an overlap analysis revealed a single gene, KIAA0930, that was differentially methylated in both lung cell fractions. This gene has been implicated in hypoxia adaptation and lung cancer development, and protein interaction analyses place it at a network hub connected to the 14-3-3 protein family, part of the Reactome pathway describing how SARS-CoV-2 targets host intracellular and regulatory pathways. BioGRID further identifies a direct interaction between KIAA0930 and ORF3A, a viral accessory protein known to modulate immune responses and cellular stress pathways.

Crucially, the epigenetic changes were not merely molecular noise. Seventy differentially methylated CpG sites, mapping to 54 genes, showed changes in methylation that correlated with changes in the symptom-physiology composite variable over the year of follow-up. Seven participants improved symptomatically, three remained stable, and three worsened, and their methylation trajectories tracked these clinical courses. Protein interaction and Gene Ontology analyses of the correlated genes revealed significant enrichment in biological pathways related to cardiac function, including a module connected to the KEGG pathway for viral myocarditis, a finding that resonates with the cardiopulmonary symptoms, such as exercise intolerance and dysautonomia, that dominate the clinical picture of post COVID-19 condition.

The authors are careful to frame the study as exploratory and hypothesis-generating. The cohort of 13 patients is small, control samples from pre-pandemic healthy subjects were used only for contextual reference rather than direct statistical comparison, and the lung cell isolation approach yielded enriched rather than fully homogeneous fractions. Neutrophil proportions were higher at the first time point, and residual confounding from comorbidities or treatments cannot be excluded. Without longitudinally matched controls, age-related or time-dependent methylation changes unrelated to post COVID-19 condition cannot be fully ruled out, and no causal relationship between the epigenetic alterations and symptoms can be inferred from these data.

Nevertheless, the findings carry significant implications. The tissue-specific nature of the rewiring suggests that peripheral blood, the workhorse of most long COVID biomarker studies, may be an incomplete window into the disease process, and that neutrophil biology in particular may persist in an altered state well beyond acute infection, consistent with reports of immature and activated neutrophils, neutrophil extracellular trap formation, and epigenetic reprogramming of bone marrow progenitors after COVID-19. The enrichment of pathways involving AMPK signalling, circadian entrainment, and dopaminergic synapses also aligns intriguingly with recent clinical observations, including evidence that the AMPK activator metformin reduces the incidence of post COVID-19 condition. Larger longitudinal studies with matched controls and mechanistic validation will be needed to determine whether these pulmonary epigenetic signatures are drivers of persistent illness, scars of prior infection, or downstream echoes of physiological stress, but this first longitudinal map of the lung methylome in long COVID provides a clear and testable starting point.

Subject of Research: Longitudinal DNA methylation changes in lung immune cells of patients with post COVID-19 condition

Article Title: Longitudinal epigenetic rewiring in lung immune cells in patients with post COVID-19 condition

Article References: Nikesjö, F., Smiljanić, J., Sayyab, S., Martínez-Enguita, D., Gustafsson, M., Rosvall, M., Hedman, K., & Lerm, M. (2026). Longitudinal epigenetic rewiring in lung immune cells in patients with post COVID-19 condition. Epigenetics Communications, 6(1), Article 5. https://doi.org/10.1186/s43682-026-00046-6

Image Credits: AI Generated

DOI: 10.1186/s43682-026-00046-6

Keywords: post COVID-19 condition, long COVID, DNA methylation, epigenetics, lung immune cells, neutrophils, macrophages, induced sputum, SARS-CoV-2, cardiopulmonary symptoms, KIAA0930, viral myocarditis