Journal of Medical Microbiology

Background The microbial aetiology of early childhood caries (ECC) in sub-Saharan African populations remains poorly characterised, with most studies focusing on conventional cariogenic pathogens like Streptococcus mutans. This study aimed to characterise the salivary microbial profile of children with ECC in urban Kano, northern Nigeria. Methods In this cross-sectional study of 162 children aged 3-5 years in urban Kano, unstimulated saliva samples were collected and analysed using standard bacteriological culture methods. Caries status was assessed using decayed, missing, and filled teeth (dmft) index and International Caries Detection and Assessment System (ICDAS). Microbial isolates were identified through Gram staining, colony morphology, and biochemical tests (catalase, coagulase, oxidase). Results Of 32 microbial isolates obtained, Staphylococcus aureus was the most prevalent (43.8%, n=14), followed by Streptococcus species (28.1%, n=9), Klebsiella species (12.5%, n=4), non-aureus staphylococci (6.3%, n=2), yeast (6.3%, n=2), and Pseudomonas species (3.1%, n=1). Only one isolate demonstrated direct association with dmft-detectable caries. Polymicrobial colonisation occurred in four cases (12.5%), predominantly featuring S. aureus-yeast combinations (n=2). White spot lesions (ICDAS 1-2) were associated with S. aureus and Klebsiella species in two separate cases. Conclusion This study reveals an unexpected predominance of S. aureus in the salivary microbiome of children in northern Nigeria, challenging conventional paradigms of ECC microbiology. The low correlation between microbial isolates and clinical caries suggests complex, multifactorial aetiology. These findings highlight the need for molecular characterisation of oral microbiomes in African populations and reconsideration of caries pathogenesis models in this unique epidemiological context.

BackgroundDeep margin elevation (DME) is a restorative technique that facilitates the placement of restorations in cases of subgingival margins. Although clinically reported, very few data are available on dental practitioners awareness and use of DME. ObjectivesTo evaluate awareness and clinical acceptance toward deep margin elevation (DME) use in subgingival restorative cases among dental practitioners worldwide. MethodologyA cross-sectional questionnaire-based study was conducted among practicing dentists at various dental educational institutions, private dental practices, and a combination of academic and private dental practices across multiple centers globally. The self-administered questionnaire consisted of 20 closed-ended questions to evaluate awareness and clinical acceptance. The data were entered into and analyzed using a Chi-square test and descriptive statistics in the Statistical Package for the Social Sciences (SPSS) software. ResultsOut of 450 invited participants, 349 general dental practitioners completed the survey (77.6%). The purely educational institutions response rate was 79 (23%), the strictly private dental practice response rate was 134 (39%), and the combined academic and private practice response rate was 131 (38%). Sixty-six percent of respondents agreed that predictable adhesive bonding to cervical/root dentin can be achieved in restorations with deep margins. Although a majority of respondents had heard of DME (77%), the majority reported a preference for surgical crown lengthening (75%) when favorable conditions were present. ConclusionThe study highlights moderate DME awareness among the study participants. The findings of this study revealed that the number of dentists who use the technique to restore large subgingival defects in posterior teeth with proximal caries is very small. Thus, it is recommended that dental practitioners introduce this technique in their dental clinics as an alternative to surgical crown lengthening. Although years of experience and a dentists rank may influence clinical decisions, an in-depth factorial analysis with a larger sample size is necessary.

ObjectiveThis study utilizes small-sample periodontitis data to exploratively investigate causal relationships between the oral microbiome and periodontitis in East Asian populations. We aimed to identify specific oral microbial taxa that may drive disease pathogenesis. Given the exploratory nature of the dataset, findings should be interpreted as hypothesis-generating. MethodsWe performed a two-sample Mendelian randomization (MR) analysis using genome-wide association study (GWAS) summary statistics for tongue dorsum and salivary microbiomes alongside periodontitis data in East Asian populations. Primary causal estimates were derived using the inverse-variance weighted (IVW) method, supplemented by MR-Egger, weighted median, weighted mode, and simple mode methods. To ensure robustness, we assessed heterogeneity using Cochrans Q test, evaluated horizontal pleiotropy via the MR-Egger intercept and MR-PRESSO tests, and applied Steiger filtering to rule out reverse causality. ResultsWe identified 60 species-level microbial taxa causally associated with periodontitis, comprising 29 negative and 31 positive associations. These taxa were predominantly enriched within the genera Campylobacter, Pauljensenia, Solobacterium, and Streptococcus. ConclusionThis study provides tentative evidence for causal links between specific species-level oral microbial taxa and periodontitis, highlighting potential targets for prevention and therapeutic intervention.

In the past decade, dengue fever has emerged as a major public health on Reunion Island in the Southwest Indian Ocean. During the 2018-2022 outbreak, an unusual increase in ocular complications was reported in some patients. To investigate a potential viral cause, we analysed 447 blood samples from hospitalized patients with and without ophthalmic symptoms. Genetic sequencing revealed the co-circulation of two strains of dengue virus serotype 1, both genetically linked to strains previously identified in Asia. Notably, all patients with ophthalmic symptoms were infected with viruses from a single cluster within genotype I, which harbored several unique mutations. These findings suggest that the rare ocular complications observed during this outbreak may be associated with specific viral cluster. Further laboratory studies are required to confirm this potential link.

ObjectiveThe rapid availability of phenotypic antimicrobial susceptibility results is crucial for the timely detection of multidrug-resistant Gram-negative organisms and for guiding optimized treatment strategies. Recently, novel methods have been introduced that enable direct antimicrobial susceptibility testing (AST) from positive blood cultures. However, their performance has not yet been systematically compared in head-to-head evaluations. This study aimed to assess the analytical performance of two rapid AST approaches--the agar diffusion-based EUCAST rapid AST (RAST) method and the automated QuickMIC system--using a challenging collection of highly resistant Gram-negative organisms. MethodsA total of 101 Gram-negative bacteria (Escherichia coli, n = 24; Klebsiella pneumoniae, n = 22; Acinetobacter baumannii, n = 30; Pseudomonas aeruginosa, n = 25) were spiked into blood cultures and processed according to the respective AST workflows. Broth microdilution (BMD) was performed from pure cultures as the reference method. Time to result (TTR), categorical agreement (CA), and essential agreement (EA) with BMD were evaluated. Boruta analysis was applied to identify genetic determinants associated with AST errors. ResultsOverall TTR for QuickMIC was 3 h 44 min with a CA of 86.2%, an EA of 92.3 % for Enterobacteriaceae and 97.0 % for non-fermenters. Overall CA of RAST ranged from 90.7%-93.7% across reading time points. Overall, very major discrepancy rates were low (QuickMIC n=0.7%, RAST n=0.1%). Presence of NDM-5 and KPC was most frequently associated with errors for QuickMIC and EUCAST RAST, respectively. ConclusionsBoth rapid AST approaches yielded robust results in this diverse and highly resistant bacterial study population, directly from positive blood cultures, with a short turnaround time. These findings underscore the potential of rapid AST methods to facilitate timely optimization of antimicrobial therapy in bloodstream infections, even in the context of extensively drug-resistant pathogens. ImportanceAccurate antimicrobial susceptibility testing (AST) is essential for stewardship and effective therapy, especially as rising antimicrobial resistance increases the risk of empiric treatment failure. Traditional AST methods are limited by slow turnaround times, creating a need for rapid alternatives. This study evaluated the diagnostic accuracy of two rapid AST methods--EUCAST RAST and QuickMIC--using 101 genetically characterized, carbapenem-resistant Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii tested directly from positive blood cultures. Broth microdilution served as the reference. Both rapid assays provided results within 3.5-6 hours and demonstrated high categorical and essential agreement with few very major discrepancies. Incorrect results were more common in isolates harboring NDM-5 and KPC carbapenemases. Overall, the findings support EUCAST RAST and QuickMIC as reliable tools for challenging resistant pathogens and highlight their potential to enable earlier detection of carbapenem-resistant phenotypes and more timely initiation of appropriate, last-resort antimicrobial therapy.

Aim and ObjectivesThe study aimed to evaluate the effectiveness of titanium inserts for interdental papilla reconstruction, comparing it with the Han and Takei technique using subepithelial connective tissue grafts. The objectives included assessing the black triangle height, papilla height and papilla presence index (PPI) at baseline, 1 month and 3 months postoperatively along with the evaluation of Early Wound Healing Score (EHS) during the first week of post operative healing period. Patients and MethodsThis single-blind randomized clinical trial included systemically healthy individuals aged 18-35 years with Nordland and Tarnows Class I-III papillary loss. A total of 18 participants were randomly assigned to either test group or control group. Clinical parameters were measured pre- and post-operatively at specified intervals. Both groups received standard presurgical care and postoperative follow-up. The surgical protocol for the test group involved titanium insert placement in the interdental bone, while the control group received a connective tissue graft using the Han and Takei method. ResultsBoth groups showed significant intragroup improvements in all parameters from baseline to 1 and 3 months (p<0.05). However, intergroup comparisons showed no significant differences at most time points, except at 3 months for PPI, where the control group showed significantly better results (p=0.04). EHS scores were not significant between the groups. ConclusionTitanium inserts and CTG both demonstrated clinical effectiveness in enhancing interdental papilla dimensions. These findings support the titanium insert as a viable, less invasive alternative, offering clinicians a practical option for esthetic papilla reconstruction.

Microbial keratitis is a sight-threatening corneal infection with varying etiological agents, primarily bacteria and fungi. Assessing and contrasting the virulence factors of microorganisms isolated from a non-contact lens-associated keratitis (NCLAK) and contact lens-associated keratitis (CLAK) is the goal of the current investigation. Samples were collected from over 60 patients and analysed using standard microbiological techniques, including culture, Gram staining, KOH mount, biochemical tests, antimicrobial susceptibility testing, and biofilm assays. The results demonstrated that CLAK isolates were predominantly bacterial, especially Pseudomonas aeruginosa, known for strong biofilm production and high multidrug resistance. In contrast, NCLAK showed a higher incidence of fungal infections, particularly Candida albicans. The results highlight the significance of early diagnosis, tailored and improved awareness regarding contact lens hygiene to prevent complications associated with keratitis.

Statement of problemDental implant failure remains a significant clinical concern, with early loss often attributed to impaired osseointegration. Recent research has considered the role of serum vitamin D in implant integration, yet the precise relationship between vitamin D status and implant loss, particularly when distinguishing early and late failures, is not fully established. PurposeThe objective of this analysis was to evaluate the association between explicit serum vitamin D cut-off values and clinically confirmed dental implant failure, with a particular focus on differentiating early (pre-loading) from late (post-loading) failures. The review also sought to determine whether primary studies used multivariate adjustment for potential confounders. Material and methodsThe draft of this revision was registered in PROSPERO (CRD420251049631, https://www.crd.york.ac.uk/PROSPERO/view/CRD420251049631). A comprehensive literature search was conducted using AI-assisted tools to identify primary research studies, including randomized controlled trials and cohort studies, published in English, Spanish, French, German, or Italian. Eligible studies required explicit vitamin D threshold categorization, clinically verified implant loss, and clear differentiation of early and late failures. Data extraction included study design, vitamin D categorization, analytical methods, and outcomes. ResultsIdentified studies predominantly consisted of retrospective and prospective cohorts examining early implant failures, frequently using cut-offs such as >30 ng/mL, 10-30 ng/mL, and <10 ng/mL for serum vitamin D. Results suggested a higher frequency of early failures in individuals with severe vitamin D deficiency; however, all studies relied on univariate analyses without multivariate adjustment for confounders. Late implant failures were rarely addressed. ConclusionsCurrent evidence indicates a possible association between low serum vitamin D and early dental implant failure, but the lack of robust statistical adjustment prevents definitive conclusions. High-quality studies with rigorous confounder control and explicit early versus late failure analysis are needed.

The rapid diagnosis of Campylobacter infections is important for the management of infectious gastroenteritis. Although stool culture is considered the gold standard, its sensitivity is limited and it requires prolonged incubation times. We performed a prospective multicenter study at nine healthcare facilities in Japan to evaluate a Campylobacter rapid antigen test using stool specimens between March 2024 and August 2025. Patients with suspected infectious gastroenteritis were consecutively enrolled and tested using QuickNavi-Campylobacter and compared with the FilmArray Gastrointestinal Panel as the reference method. Discordant results were further evaluated by culturing and additional PCR assays. In total, 410 patients were included in the final analysis. The positive, negative, and total concordance rates between QuickNavi-Campylobacter and FilmArray Gastrointestinal Panel were 79%, 99%, and 93%, respectively. The positive concordance rate decreased in specimens collected [&ge;] 6 days after the onset of symptoms (50%). QuickNavi-Campylobacter demonstrated relatively good concordance with the FilmArray Gastrointestinal Panel in a real-world multicenter setting. These results suggest that this rapid antigen test may be particularly useful for the early diagnosis of suspected campylobacteriosis.

Influenza A, Influenza B, SARS-CoV-2, Respiratory Syncytial Virus and other respiratory pathogens are an ongoing public health concern. The ability to rapidly identify these viruses early in infection is essential for effective treatment and outbreak control. The AMDI Fast PCR Mini Respiratory Panel (MRP) incorporates sample preparation and real-time RT-PCR for detection of Flu A, Flu B, SARS-CoV-2 and RSV from anterior nasal swab (ANS) specimens in less than 10 minutes at the point of care. We established the analytical performance characteristics of the Fast PCR MRP and determined that the limit of detection (LoD) is 250 copies/mL for Flu A and RSV, and 500 copies/mL for Flu B and SARS-CoV-2. In a reproducibility study at 3 clinical sites, there was at least 98.2% positivity for each target for a weak positive (2x LoD) sample, at least 99.6% positivity for a moderate positive (5x LoD) sample and the negative sample returned a negative result for at least 99.3% of the tests. Fast PCR MRP had 100% analytical reactivity to all strains tested (23 Flu A, 6 Flu B, 8 SARS-CoV-2 and 6 RSV) and at least 98% predicted inclusivity from in silico analysis. There was no cross-reactivity to 40 viruses, bacteria and fungi, nor interference for 15 endogenous and exogenous substances in ANS matrix. The Fast PCR MRP delivers excellent analytical performance comparable to high complexity laboratory assays, at the point of care.

BackgroundOral hygiene is linked with dental caries experience. This systematic review and meta-analysis assessed the associations between oral hygiene status, the frequency of tooth brushing, and the prevalence of dental caries in Nigeria. Tools used for maintaining oral hygiene were also identified. MethodsRegistered with PROSPERO (CRD42022367763), this review searched PubMed, Web of Science, Scopus, African Journals Online, African Index Medicus, and Google Scholar in January 2026. Observational studies and clinical trials reporting baseline caries prevalence were included. There was no language restriction. Studies were excluded if they did not provide information on the sample size, had no study outcome data, or featured duplicate samples, and if they were review articles, systematic reviews and meta-analyses, case reports, case series, in vitro studies, commentaries/letters (editorials, opinion pieces) devoid of primary data. Pooled odds ratios (ORs) were estimated using random-effects models. Subgroup analyses were conducted by dentition type, geopolitical zone, study design, publication year, mean age, and sample size. ResultsTwenty-three cross-sectional studies were included, of which 20 (86.9%) were conducted in Southern Nigeria. After removing an influential outlier, poor oral hygiene was associated with a 38% reduction in caries odds (OR 0.62, 95% CI 0.46-0.84). Brushing at least twice daily was strongly associated with reduced caries (OR 0.01, 95% CI 0.00-0.01). No significant association was found between the type of cleaning device and caries prevalence. Subgroup analyses identified dentition type and publication year as significant moderators. Heterogeneity ranged from low to moderate, and no publication bias was detected for primary associations. The most common cleaning tool was a toothbrush with toothpaste, though chewing sticks, cotton wool, and other traditional tools were also reported. ConclusionTwice-daily tooth brushing is strongly associated with lower caries prevalence in Nigeria. Poor oral hygiene was linked to reduced caries odds in pooled analysis, a finding that may reflect socio-economic and dietary confounding. The type of cleaning tool was not significantly associated with caries risk, highlighting the importance of brushing frequency and technique over tool type. Future research should prioritize Northern Nigeria to address the geographic skewness of the data and improve national representativeness.

Carbapenem-resistant (CR) bacteria have emerged and been spreading beyond healthcare-associated facilities into the environment. It is recognized that toilet bowl water in patient rooms of healthcare-associated facilities can be one of internal reservoirs of CR bacteria. In accordance with this idea, toilet bowl water samples were collected from patient rooms in a tertiary healthcare-associated facility in North Macedonia, and meropenem (MEM)-resistant bacterial isolates were obtained from the toilet bowl water. In this study, because a MEM-resistant C. braakii isolate, that was one of MEM-resistant opportunistic pathogens, was obtained from the toilet water, whole-genome sequencing (WGS) of this isolate was performed to obtain genetic characteristics of the blaNDM-1-positive C. braakii isolate. By the WGS, four contigs were constructed, the longest contig, contig 1 (5,189,681 bp), contained blaCTX-M with some additional antimicrobial-resistance genes (ARGs). Interestingly, blaNDM-1 was detected in contig 2 (177,260 bp) and contig 3 (64,168 bp). Plasmid replicon of contig 2 was IncA/C2 but plasmid replicon of contig 3 was IncN and different from one of contig 2. Genetic structures surrounding blaNDM-1 were different between these two blaNDM-1-positive plasmids implying transfer or insertion of blaNDM-1 had occurred by IS or other mechanism. Further molecular epidemiology will be needed to explain the mechanism that allowed the C. braakii isolate to possess two structurally different blaNDM-1 plasmids.

BackgroundAntibiotic stewardship during stem cell transplantation (SCT) is challenging.. Procalcitonin (PCT) has been employed successfully in critical care patients to safely guide stewardship. However, procalcitonin guided stewardship has not been robustly assessed in SCT recipients. We sought to evaluate the potential utility of PCT to guide antimicrobial de-escalation during engraftment. Methods100 SCT patients were prospectively enrolled in a "strategy trial" and had infectious complications documented. Lab parameters - CBC, BMP, CRP were obtained daily as standard of care (SOC) while PCT was obtained for research purposes. Providers were blinded to PCT results. We compared duration of antimicrobial escalation between actual events (SOC model) and a proposed PCT model. In this hypothetical PCT model, antibiotic de-escalation would occur if CRP remained <100 mg/dl and PCT <0.25 ng/ml after 3 days of escalation. Escalation events were defined as a substitution or addition of an antimicrobial agent after initiation of prophylactic antimicrobials. Results77 patients had escalation events and of these, 33 had bacterial infections. A total of 136 antimicrobial escalations events were identified, and of these only 39(28.7%) were associated with documented infections. The standard of care model had a mean duration (+SD) of 9.08 (+ 6.08) antibiotic days. If the PCT model were employed, the mean duration (+SD) would be 4.44 (+ 6.16) days (p<0.001). The PCT model, however, would have missed 11 infections\ ConclusionProcalcitonin-guided antimicrobial stewardship during autologous stem cell transplantation is feasible however optimization is necessitated for utilization as a tool to guide antibiotic prophylaxis during SCT.

ObjectiveCommunity service-learning (CSL) placements engage with equity-deserving groups. They receive oral health care and students can critically reflect on their experiences. This study aimed to thematically explore the reflections of senior dental students providing oral health services to equity-deserving communities in British Columbia, Canada. MethodsSemi-structured written reflections were collected cross-sectionally from three consecutive graduating cohorts of fourth-year dental students (2022-23, 2023-24, and 2024-25). Reflections were a mandatory component of a community placement course, were approximately 500 words in length, and were prompted as follows: "Describe your personal experience at the assigned community clinic, noting moments of revelation, valuable learning, and/or disappointment for you." An exploratory thematic analysis was conducted using an iterative coding process to identify and interpret categories and themes. ResultsFrom all the three years, 764 reflections were collected (191-625 words each) from 171 students. Of these, 124 reflections were excluded because they consisted solely of descriptions of procedures. Data saturation was reached after in-depth analysis of 205 reflections, yielding four overarching themes, including learning across differences; and pause-breathe-refine. These themes were informed by categories highlighting that detrimental impact of overly controlling mentorship styles and observation-only experiences on students learning. ConclusionTransformative experiences were observed, while students also reflected on less positive practices. Students emphasized the importance of CSL placements for their education, professional growth, and understanding of underserved populations, while also highlighting implementation challenges. Future research should examine the long-term impact of CSL activities once these challenges are addressed.

Over-the-counter (OTC) lateral flow tests for respiratory viruses have newly emerged since the beginning of the coronavirus disease 2019 (COVID-19) pandemic and are increasingly available to consumers. While all marketed tests have met standards for Emergency Use Authorization (EUA), De Novo classification or 510(k) clearance, limited data are available to inform consumers of their relative performance. We performed a head-to-head benchtop analytical assessment of OTC tests available for purchase from retailers in the United States in the fall of 2025. Contrived specimen dilution panels were prepared from propagated viral stocks of influenza A H1N1pdm09, influenza A H3N2, influenza B (Victoria lineage), and SARS-CoV-2 JN.1 (Omicron variant) in clinical matrix and applied to the swab provided with each kit. Tests were subsequently performed according to the manufacturers instructions for use and results were interpreted by two readers blinded to the starting test material. Lower limits at which 3 of 3 replicates of test material were detected were within a 4-fold dilution for all four viruses and all eight OTC tests evaluated. We found that at low viral concentrations many OTC tests were interpreted as negative at the start of the stated interpretation window but converted to a positive result by the end of the interpretation window. We conclude that eight OTC tests that are currently readily available to consumers perform similarly in a contrived specimen analytical study, but we recommend that users of OTC lateral flow tests allow for the full incubation time before concluding that a test is truly negative.

This study describes the establishment of the National Dental Center Singapore Oral Disease Registry (NDCS-ODR), a large-scale, electronic health records registry designed to capture real-world data on oral diseases. The NDCS-ODR was developed to standardize and integrate oral health data within Singapore Health Services, the countrys largest healthcare cluster. Its development, governance, and data architecture are described, with an overview of individuals with oral diseases recorded in the registry. Data collection from 2013 to June 2025 has been completed. As of June 2025, the NDCS-ODR comprises 229,249 unique patients, with a mean (SD) age of 49.1 (19.5) years and an approximately equal sex distribution. Most were of Chinese ethnicity (77.6%), and Singapore citizens (92.5%). Clinical variables indicated substantial disease and treatment burden, with a mean of 7.9 (7.7) missing teeth, 4.8 (6.2) restored surfaces, and 2.8 (3.4) restored teeth per patient. Among 108,517 recorded periodontal diagnoses, Stage III periodontitis (2018 EFP/AAP Classification) and severe chronic periodontitis (1999 Classification of Periodontal Diseases and Conditions) were most common. The NDCS-ODR represents Singapores first large-scale, real-world oral disease registry embedded within a national specialty center, demonstrating the feasibility of leveraging electronic health record data for research and service evaluation.

Clostridioides difficile (C. diff) is a leading cause of hospital-acquired infections with severity ranging from mild diarrhea to fulminant colitis and death. Current antigen tests lack adequate sensitivity and DNA-based nucleic acid amplification tests (DNA-NAATs) exhibit limited specificity for active infection, leading to either underdiagnosis or inappropriate treatment of colonized individuals. Unlike DNA, mRNA is expressed only by metabolically active bacteria and is rapidly hydrolyzed, providing natural advantages to distinguish active infection from colonization. In this study, we developed and evaluated a novel multiplexed reverse-transcriptase PCR assay (RNA-NAAT) targeting C. diff-specific sequences. No cross-reactivity was observed with other gastrointestinal pathogens, commensal enteric flora, or closely-related Clostridioides or Clostridium species. Toxin gene RNA expression was detected only in samples spiked with metabolically active C. diff at a limit of detection 30-to-50-fold less than current diagnostic methods. Sequential clinical samples (n=260) were collected in proprietary RNA-preservative solution from patients receiving standard of care C. diff diagnostic testing. All samples were evaluated with RNA-NAAT, Alere GDH/toxin EIA, Solana DNA-NAAT, and both forward- and reverse-2-Step Algorithms (2-SA). Samples yielding valid results on all platforms (n=239) with discordant test results (n=14) were adjudicated via toxigenic culture and blinded chart review by infectious disease physicians. RNA-NAAT outperformed all comparator test strategies, simultaneously exhibiting higher sensitivity and specificity, including a higher specificity for active infection than the specific toxin EIA (99.5% vs 98.2%) and a higher sensitivity for organism identification than the sensitive DNA-NAAT (100% vs 88.2%), with significantly reduced false positive test results (1 vs 7). One Sentence SummaryA novel RNA diagnostic distinguishes clinically-relevant C. difficile infection from toxigenic carrier states, improving sensitivity and specificity.

Mycobacterium tuberculosis (MTB) disease is a major global health threat with most tuberculosis (TB) cases occurring in low-and middle-income countries (LMIC) with limited healthcare infrastructure. Near-point-of-care testing which can be deployed at peripheral clinical settings is needed to start treatment earlier and thereby improve treatment outcomes. Here we report the development and preliminary characterization of an MTB detection assay that utilizes tongue swab or sputum specimens for The DASH(R) Rapid PCR System which employs cartridge-based automated sequence specific capture sample prep combined with dual target qPCR multicopy MTB insertion sequences IS6110 and IS1081 amplification and detection. MTB is resistant to conventional bacterial lysis techniques; therefore, we evaluated two pre-cartridge lysing techniques, mechanical lysis and sonication, and selected sonication for all subsequent studies. The DASH MTB assay demonstrated a limit of detection of 2.5 MTB cells/swab with no detection of 10 non-tuberculosis Mycobacterium strains. Clinical testing of 100 (49 positive and 51 negative) de-identified blinded sputa from South African symptomatic clinic attendees yielded an overall test sensitivity of 96% (100% for smear positive samples and 88% for smear negative samples) and specificity of 88% when compared to sputum culture. In a separate study of 110 tongue swab specimens (70 positive and 40 negative) from South African symptomatic clinic attendees, the sensitivity was 93% and the specificity was 100%. We further demonstrated that the test is compatible with peripheral LMIC settings via external battery operation and cartridge stability at 45{degrees}C for up to one year. ImportanceTuberculosis (TB) is the single most deadly infectious disease with 1.23 million deaths in 2024. Near-point-of-care testing which can be deployed at peripheral settings that lack laboratory infrastructure to deliver prompt and accurate diagnosis is needed to start treatment earlier and thereby improve treatment outcomes. In this study, we have developed an automated test to detect Mycobacterium tuberculosis (MTB), the cause of TB, from sputum and tongue swab specimens. Its high sensitivity and specificity, rapid time to result, and compatibility with environments that lack air conditioning and consistent electricity make this assay suitable for diverse clinical settings.

BackgroundAntimicrobial resistance (AMR) represents one of the most serious threats to global public health, with a disproportionate burden borne by low- and middle-income countries. Rural healthcare settings in India remain under-represented in molecular AMR surveillance despite high antimicrobial use and limited diagnostic capacity. ObjectivesTo describe longitudinal phenotypic and molecular AMR patterns in clinical bacterial isolates from a rural district hospital in Gujarat, India, and to assess the feasibility of clinician-led molecular surveillance in a resource-limited setting. MethodsA retrospective observational study was conducted on 242 non-duplicate discarded clinical bacterial isolates collected between January 2021 and August 2025 at Banas Civil Hospital, Palanpur, Gujarat. Antimicrobial susceptibility testing was performed according to Clinical and Laboratory Standards Institute guidelines (CLSI M100, 31st edition) using Kirby-Bauer disk diffusion and broth microdilution methods. Molecular detection of blaCTX-M, blaNDM, blaOXA-48, mecA, and vanA resistance genes was carried out using polymerase chain reaction, with confirmation by Sanger sequencing. Data were analysed using R software (version 4.3.2). ResultsThe most common pathogens were Escherichia coli (38%), Klebsiella pneumoniae (26%), and Pseudomonas aeruginosa (17%). Resistance to third-generation cephalosporins was high in E. coli (64%) and K. pneumoniae (59%). Carbapenem resistance peaked at 17% in 2024. Molecular analysis identified blaCTX-M in 42% of Gram-negative isolates, while blaNDM and blaOXA-48 were detected in 11% and 7%, respectively. Genotype-phenotype concordance exceeded 80%. Ethical ApprovalEthical approval for the study was obtained from the Institutional Ethics Committee of Banas Medical College and Research Institute, Palanpur, Gujarat, India. No patient identifiers were accessed, and no patient interviews, direct contact, or additional specimen collection were performed for research purposes. ConclusionThis study demonstrates a substantial burden of ESBL- and carbapenemase-mediated antimicrobial resistance in rural Gujarat. Clinician-led molecular surveillance using discarded clinical samples is feasible and essential for guiding empirical therapy and strengthening antimicrobial stewardship in resource-limited settings

BackgroundTalaromycosis, caused by the fungus Talaromyces marneffei, is a leading cause of HIV-associated death in Southeast Asia. Current culture-based diagnosis only identifies late-stage infection, limiting understanding of disease burden and disease spectrum. We evaluated the clinical performance of anti-Mp1p IgM and IgG enzyme immunoassays (EIA) for talaromycosis diagnosis. MethodsThis diagnostic study included 423 adults with advanced HIV disease and culture-confirmed talaromycosis as cases, and 206 non-talaromycosis individuals with and without HIV who have never traveled to Asia as controls. Anti-Mp1p IgM and IgG antibodies were measured using conventional double-sandwich EIA. Diagnostic performance was assessed using the healthy control group and the HIV control group separately. Assay cut-offs were based on both the Youden index generated from the receiver operating characteristic curves, and separately using a pre-defined specificity of 95%. ResultsAt the pre-defined 95% specificity, IgM had low to moderate accuracy of 62.3% and 87.9%, and a low sensitivity of 8.3% and 21.3%, when evaluated with healthy and HIV controls, respectively. IgG had similarly low accuracy of 52.2% and 78.4%, and a low sensitivity of 21.5% and 30.5%, when evaluated using healthy and HIV controls, respectively. Both IgM and IgG assays performed better in HIV controls than healthy controls. ConclusionsThe anti-Mp1p IgM and IgG EIAs show low utility for the diagnosis of acute talaromycosis. However, at the high specificity cut-off of 95%, the assays have utility in the detection of T. marneffei exposure at both individual and population levels, and. provides a foundation for future sero-epidemiological studies. IMPORTANCETalaromycosis, caused by the dimorphic fungus Talaromycosis marneffei endemic in Southeast Asia, southern China, and northeastern India, is an invasive fungal infection that causes over 25,000 cases and 6,000 deaths annually but remains neglected in the global health community. Current diagnosis requiring culture-based testing is too slow, often resulting in patient death before treatment can begin. This study presents the first large-scale clinical evaluation of antibody tests for talaromycosis. While the antibody tests showed limited sensitivity for diagnosing acute disease, the high specificity makes them useful in determining prior exposure to T. marneffei, providing a new tool for public health investigation of disease prevalence at a population level, and for clinicians to identify individuals at risk for disease reactivation who may benefit from prevention strategies. The research provides important groundwork for improving disease control efforts in regions where this neglected infection is endemic.