搜索结果：Diagnostic microbiology and infectious disease

Among the Indian population, hepatitis B virus (HBV) is one of the major burdens and the hepatitis C virus (HCV) chronically infects around 1% Indian population. CRISPR-based detection platforms have shown to be a novel low-cost technology with high sensitivity and specificity. In the presence of target nucleic acids, Cas13a molecule is activated to trans-cleave the fluorophore quencher (FQ)-labeled ssRNA reporter, and illuminate detectable fluorescent signals. Leptotrichia wadei (Lwa) cas13a was expressed and purified. Recombinase Polymerase Amplification (RPA) was implemented to produce T7 RNA polymerase appended amplicons of conserved regions of HBV and HCV at 37°C and 42°C respectively. Corresponding crRNAs have been designed against amplified regions and produced using In-Vitro Transcription (IVT). With T7 RNA polymerase, the RPA-amplified HBV and HCV templates are transcribed into ssRNAs, which are further used in detection assay containing expressed Cas protein, crRNA, and fluorescent probes. This detection was performed in the microplate reader in kinetic format. LwaCas13a was expressed and was purified using the strep tag. Conserved regions among Indian HBV and HCV genotypes are selected as targets of detection. RPA and Nested-RPA was performed using primers against conjunct region of HBV polymerase and surface antigen and RNA-dependent RNA polymerase (RdRp) region of HCV. The detection assay was performed from 45 HBV and 30 HCV human samples, out of which it could differentiate positive and healthy samples. This approach can be a better alternative to be used in rural India and at the same time with high sensitivity, for a rapid detection of HBV and HCV, which could be used as a novel a low-cost diagnostics platform for identification of HBV DNA and HCV RNA.

Entamoeba histolytica is a protozoan parasite that causes amebic colitis, a leading cause of severe diarrheal disease worldwide, and amebic liver abscess, the most common extraintestinal manifestation of infection. The disease burden is highest in resource-limited settings but remains clinically important in travelers and men who have sex with men in non-endemic regions. Although most infections are asymptomatic, severe and fulminant disease is associated with high mortality, particularly among individuals exposed to corticosteroids or other forms of immunosuppression. The increasing use of molecular diagnostic tools has improved understanding of the epidemiology of E. histolytica and enabled distinction from morphologically identical but nonpathogenic Entamoeba species; however, these diagnostics remain underutilized in many endemic settings due to cost and infrastructure limitations. Treatment options remain limited, with nitroimidazoles constituting the only drug class available for symptomatic invasive disease, leaving few alternatives for patients who cannot tolerate therapy or in the event of emerging resistance. Despite advances in understanding parasite pathogenesis and the application of high-throughput technologies, no licensed vaccine exists, and progress toward vaccine development has been minimal. These persistent gaps highlight the need to reprioritize amebiasis as a neglected tropical disease and to accelerate investment in diagnostics, therapeutics, and preventive strategies.

Ureteroscopy (URS) is a widely performed procedure for urinary stone disease, yet infectious complications, including sepsis, remain a concern despite negative preoperative urine cultures. Intraoperative urine dipstick testing offers a rapid point-of-care assessment, but its diagnostic performance for predicting culture positivity and severe infection at different sampling sites during URS remains unclear. In this retrospective, single-center study, 238 patients undergoing URS were included. Paired urine dipstick and culture samples were obtained from bladder urine, intraoperative pelvic urine, and post-laser pelvic urine. Dipstick positivity was defined using two criteria: leukocytes and/or nitrites (OR definition) and leukocytes and nitrites (AND definition). Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) with 95% confidence intervals were calculated for predicting culture positivity. Receiver operating characteristic (ROC) curve analysis was performed, including a composite dipstick score (0-2). Severe postoperative infection was assessed as a secondary exploratory outcome. Culture positivity ranged from 18.4% (bladder) to 22.9% (intraoperative pelvic urine). Using the OR definition, pooled sensitivity was 80.5% and specificity 47.6%, whereas the AND definition yielded lower sensitivity (43.8%) and higher specificity (86.4%). Diagnostic performance varied by sampling site, with intraoperative pelvic urine demonstrating the most balanced metrics. ROC analysis showed moderate discriminative performance, with the highest AUC observed for pelvic urine sampled prior to laser fragmentation (AUC 0.723, 95% CI 0.646-0.801). For severe infection (4.2%), dipstick testing demonstrated low sensitivity (30%), and this analysis was considered exploratory. Intraoperative urine dipstick testing demonstrates moderate diagnostic performance for predicting culture positivity during URS, with results influenced by sampling site and positivity definition. Pre-laser pelvic sampling provides the most informative results. A negative intraoperative dipstick, particularly from pre-laser pelvic urine, may help identify patients at lower likelihood of culture positivity; however, the test remains insufficient to guide antimicrobial or postoperative decision-making in isolation. Further prospective studies are required to validate its role in perioperative risk stratification.

Chronic pulmonary aspergillosis (CPA) is a frequent complication of pulmonary tuberculosis (PTB), particularly in high-burden settings where access to reliable serological diagnostics remains limited. We evaluated the diagnostic performance of two immunochromatographic technology (ICT) lateral flow assays (LFAs) and an ELISA for CPA screening among patients with active or previously treated PTB. In this two-year prospective multicentre diagnostic evaluation, serum from adults with prior or active PTB was tested using the Era Biology Aspergillus IgG ICT LFA, LDBio Aspergillus IgG/IgM ICT LFA, and Bordier Aspergillus fumigatus IgG ELISA. CPA diagnosis was established using a consensus composite reference standard incorporating clinical, immunological, radiological, and microbiological criteria. The Bordier ELISA was used as part of the immunological component of the consensus CPA diagnosis, with a cutoff optical density of ≥1.0. Diagnostic accuracy, agreement statistics, receiver operating characteristic analysis, and latent class analysis (LCA) were performed. Among 340 participants, 24 (7.06%) had CPA. Proportion of participants with positive antibody tests among all tested individuals were 6.76% for LDBio ICT LFA, 20.0% for Era Biology ICT LFA, and 11.47% for Bordier ELISA. Against consensus CPA diagnosis, Bordier ELISA showed 87.50% sensitivity and 94.30% specificity, LDBio ICT LFA 58.33% sensitivity and 97.15% specificity, and Era Biology LFA 66.67% sensitivity and 83.54% specificity. LCA estimated CPA prevalence at 7.72%. LCA-derived sensitivities and specificities were 86.58% and 99.92% for LDBio ICT LFA, 83.39% and 85.31% for Era Biology LFA, and 79.10% and 94.19% for Bordier ELISA. The Bordier ELISA showed high sensitivity and specificity, while the LDBio ICT LFA demonstrated very high specificity with strong LCA-derived performance. These findings support the use of ELISA for laboratory diagnosis and ICT as a point-of-care screening tool for CPA in resource-limited settings. Era Biology Aspergillus IgG LFA demonstrated moderate sensitivity and acceptable diagnostic performance, indicating its potential utility as a supplementary screening assay for CPA in settings where rapid, point-of-care testing is required.

Septic arthritis (SA) is a destructive disease requiring urgent diagnosis and treatment. The low sensitivity of conventional culture methods necessitates rapid molecular diagnostic approaches. This study aimed to evaluate the diagnostic performance of the multiplex PCR-based BioFire® Joint Infection Panel (BJIP) compared with BACTEC liquid culture and solid media culture methods in a country with high rates of antibiotic use. Synovial fluid samples from 86 patients diagnosed with SA were retrospectively analyzed. Diagnostic performance was compared using sensitivity, specificity, positive and negative predictive values, Cohen's kappa agreement coefficient, and McNemar's test. The mean patient age was 59.9 ± 17.1 years, and 59.3% were male. The BJIP detected pathogens in 12 patients (14.0%), BACTEC in 9 (10.5%), and solid media in only 3 (3.5%). The detection proportion of BJIP was higher than that of solid media (p = 0.004). No statistically significant difference was found between BJIP and BACTEC detection rates (p = 0.549). Inter-method agreement analysis demonstrated moderate concordance between BJIP and BACTEC (κ = 0.41, p = 0.002) and between BACTEC and solid media (κ = 0.472, p = 0.001), while agreement between BJIP and solid media was fair-to-moderate (κ = 0.37, p = 0.002). The BJIP demonstrated significantly superior detection proportion compared to solid media culture and comparable performance to BACTEC. Given the high rate of prior antibiotic use (69.8%) in our study population, PCR-based methods are particularly recommended, as they provide rapid results and enable detection of pathogens in culture-negative cases.

Co-infection with both hepatitis B virus (HBV) and hepatitis D virus (HDV) can aggravate the severity of the end-stage liver disease and accelerate its progression. However, no combined diagnostic method for HDV and HBV nucleic acids exists. In this study, we have developed a highly sensitive and specific dual detection method for HDV RNA and HBV DNA using the CRISPR-Cas system. We established a dual detection method combining CRISPR-Cas12a with recombinase polymerase amplification (RAA) for HBV, and CRISPR-Cas13a with RT-RAA for HDV. Validation was performed using specimens from 70 co-infected patients. RAA primers and crRNAs were designed and optimized to establish a dual fluorescence detection method (DF) and lateral flow strip-based dual detection (DL) within the same CRISPR-Cas13a/Cas12a system for HDV RNA and HBV DNA. The system demonstrated 100% specificity, and both DF and DL methods exhibited a sensitivity of 10 copies/μL for synthetic positive plasmids and samples. Peak fluorescence detection was achieved with T7 RNA polymerase, while the best detection efficiency was at ssRNA: ssDNA ratio of 1:1.5. In the validation of plasma samples from 70 co-infected clinical patients, the positive concordance rates for RT-RAA-CRISPR-Cas13a/Cas12a DF and DL were 85.7% (60/70) and 82.9% (58/70), respectively. We developed a CRISPR-Cas13a/Cas12a-based dual assay for sensitive, specific, and accurate detection of HDV RNA and HBV DNA, offering an effective tool for the early detection, treatment, and monitoring of HDV and HBV infections.

Mycoplasma pneumoniae (MP) is a key cause of community-acquired pneumonia (CAP) in children. Accurate laboratory diagnosis is essential for appropriate therapy and prevention of complications. However, variable clinical presentation and uncertainty regarding optimal diagnostic tests contribute to underdiagnosis in India. To evaluate and compare the diagnostic performance of culture, serology, and polymerase chain reaction (PCR) for early diagnosis of MP pneumonia in children, assess pulmonary and extrapulmonary manifestations, and determine the prevalence of macrolide-resistant strains among confirmed cases. Between April and November 2021, 81 children aged 6 months to 15 years with clinical features of CAP were enrolled. Throat and nasopharyngeal swabs were cultured using pleuropneumonia-like organism (PPLO) media and tested by PCR targeting the P1-adhesin gene. Serum samples were analyzed for specific IgM and IgG antibodies. PCR-positive samples were further evaluated for macrolide resistance using 23S rRNA gene targets. MP infection was detected in 30/81 (37.04%) children by culture and/or serology and/or PCR. Positivity by culture was observed in 3/81 (3.70%) cases, serology in 17/81 (20.98%), and PCR in 15/81 (18.5%). Higher serological positivity likely reflected delayed presentation after symptom onset and reduced PCR sensitivity associated with upper respiratory tract specimens. Both serology and PCR were positive in 9 (11.11%) cases. All culture-positives were also positive by serology and PCR. Macrolide resistance was detected in 60% of PCR-positive samples. The high proportion of macrolide resistance highlights the need for combined ELISA-based serology and PCR, including resistance detection, for timely diagnosis and optimal therapeutic management.

Cystic echinococcosis (CE), caused by Echinococcus granulosus sensu lato (E. granulosus s.l.), is a major zoonotic disease causing substantial health and economic losses, particularly in pastoral communities. WHO and WOAH recommend combining regular dog deworming with sheep vaccination (EG95) for control. However, genotype data are important for vaccination planning, as EG95 mainly targets G1/G3 in sheep and evidence of protection against other genotypes is limited. In Mongolia, molecular data from livestock, especially sheep, remain scarce despite the large sheep population. We therefore aimed to determine the genotype distribution of E. granulosus s.l. among livestock, particularly sheep, across Mongolia's endemic provinces. From August to December 2024, we conducted an abattoir-based cross-sectional survey in four provinces (Umnugobi, Bayankhongor, Dundgobi, and Tuv). Sheep, goats, and camels were examined for hydatid cysts. DNA was extracted, the COX1 gene was amplified by PCR and sequenced using the Sanger method, and published diagnostic COX1 sites were used to refine G6/G7 assignments; phylogenetic and haplotype analyses were performed with regional reference data. Cysts were detected in 2.5% (115/4,578) of animals, and 0.31% (14/4,578) were molecularly confirmed as E. granulosus s.l. Among them, 93% (13/14; 10 sheep, 2 goats, 1 camel) were Echinococcus canadensis (G6/G7) and 7% (1/14; 1 sheep) were Echinococcus granulosus s.s. (G1/G3). Five COX1 haplotypes were identified: four E. canadensis haplotypes (H1-H4) and one E. granulosus s.s. haplotype (H5). Haplotypes H1-H3 were consistent with G6, whereas H4 was consistent with G7a. These haplotypes grouped with reference isolates from Mongolia and neighbouring countries, as well as from other endemic regions included in the network. This study strengthens evidence that E. granulosus s.l. infections in Mongolian livestock are predominantly caused by Echinococcus canadensis G6/G7 and shows, for the first time, that this taxon predominated among successfully genotyped isolates from sheep in the surveyed areas. These findings support genotype-informed surveillance for vaccination planning, particularly in mixed-genotype settings, while underscoring the importance of dog deworming and safe offal disposal. Our findings further clarify CE transmission patterns in Mongolia and the wider region, highlight the transboundary nature of CE, and support the need for coordinated cross-border surveillance and control.

Rapid and accurate diagnosis of infectious diseases in aquaculture is essential for preventing major economic and ecological losses. Traditional culture-based methods focus on isolation of individual pathogens, and often are burdened with extended processing times, particularly during investigations of polymicrobial infections. Application of Oxford Nanopore Technologies (ONT) sequencing offers a rapid, culture-independent workflow for the identification of bacterial and fungal pathogens directly from fish tissues. Swab and organ samples from four cases (1: Salmo spp.; 2: Cyprinus carpio; 3: Salvelinus fontinalis; 4: Heniochus acuminatus) were analyzed using ONT long-read sequencing for metagenomic screening and bioinformatic classification. The results revealed case-, species-, and tissue-specific microbial profiles, with external tissues showing higher microbial diversity and internal organs enriched in pathogenic taxa. Dominant pathogens included Streptococcus iniae, Aeromonas hydrophila, Pseudomonas spp., and Saprolegnia parasitica, alongside opportunistic zoonotic bacteria such as Escherichia coli and Acinetobacter baumannii. We demonstrate the potential for diagnostic application of ONT sequencing in investigations and detection of multi-pathogen infections, including assessments of microbial community structure changes during disease outbreaks in aquatic species. The presented workflow enables rapid, cost-effective, and comprehensive pathogen profiling, supporting early disease surveillance and improved management in aquatic veterinary practice.

Reliable biomarkers are needed to support the early diagnosis of UTI. This study investigated the diagnostic value of urinary Neutrophil Gelatinase-Associated Lipocalin (uNGAL) and conventional inflammatory markers in the diagnosis and differentiation of UTI types. In this prospective study (2024-2025), 259 adults with positive urine cultures were classified as having asymptomatic bacteriuria, uncomplicated UTI, complicated UTI, recurrent UTI, or catheter-associated UTI according to the 2025 IDSA Complicated UTIs Guideline. uNGAL was measured by ELISA. uNGAL levels were numerically higher in UTI patients than controls (p=0.458). uNGAL levels did not show significant discrimination between controls and UTI subtypes (p=0.643). uNGAL levels were slightly higher in patients with recurrent UTI than in those without recurrent UTI and in patients with catheter-associated UTI than in non-catheterized patients (p=0.657 and p=0.058, respectively). Although NGAL could not distinguish complicated from uncomplicated UTI (p=0.272), CRP and WBC levels were significantly higher in complicated UTI (median 21 vs. 6 mg/L, p< 0.001; median 9800 vs. 7300 /mm&#xb3;, p< 0.001, respectively). ROC analysis identified optimal cut-off values of 10.5 mg/L for CRP and 7850/mm&#xb3; for WBC; with sensitivities of 65% and 76% and specificities of 75% and 60%, respectively. Multivariate analysis showed that each 10 mg/L increase in CRP and each 1000/mm&#xb3; increase in WBC was associated with a 1.2 fold higher likelihood of complicated UTI. uNGAL does not provide diagnostic value beyond conventional inflammatory markers and should not be used as a standalone biomarker in adult UTI. CRP and WBC remain more reliable markers for predicting complicated UTI.

Malaria remains endemic in Indonesia, with high transmission rates observed in the Papua region. Routine surveillance data are essential to inform service delivery and optimize case management. This cross-sectional study examined malaria cases in Sorong City in 2024 reported through Indonesia's national surveillance system to describe malaria burden across healthcare facilities, diagnostic and treatment practices, and predictors of hospital attendance and hospitalization. All laboratory-confirmed malaria cases were analyzed, including demographics, malaria diagnosis, disease severity, treatment received, and hospitalization data. Descriptive statistics and logistic regression were used to identify determinants of hospital attendance and inpatient admission. Among 3,953 malaria cases, most patients were &#x2265;15 years old (65.9%), males (59.4%), Papuan (54.2%), students (36.6%), and living in highly endemic areas (78%). Community health centers reported most cases (61.7%). Microscopy was the primary diagnostic tool (70.1%). Plasmodium vivax was the predominant species (65.5%), and nearly all infections were uncomplicated (99.9%). Notably, nearly 21% of patients received non-standard antimalarial regimens. Predictors of hospital attendance include older age (aOR: 1.02; 95% CI: 1.00-1.02; p&#x2009;=&#x2009;0.001), non-Papuans ethnicity (aOR: 1.77; 95%CI: 1.51-2.07; p&#x2009;=&#x2009;0.001), being housewife (aOR: 1.57; 95%CI: 1.02-2.42; p&#x2009;=&#x2009;0.041) or student (aOR: 1.56; 95%CI: 1.05-2.31; p&#x2009;=&#x2009;0.029), and living in moderate- (aOR: 2.01; 95%CI: 1.69-2.40; p&#x2009;=&#x2009;0.001) or low- (aOR: 4.57; 95%CI: 2.01-10.39; p&#x2009;=&#x2009;0.001) endemicity areas. Children <15 years were more likely to be hospitalized (aOR 1.9; 95% CI 1.18-3.06; p&#x2009;=&#x2009;0.009). Malaria remains a substantial public health burden, dominated by P. vivax, with community health centers serving as primary care providers. Older individuals were more likely to attend hospital, while younger children had a higher likelihood of hospitalization once diagnosed. Strengthening community-based services, promoting early treatment-seeking among children, and ensuring consistent adherence to national treatment guidelines are critical to reducing severe disease and hospital burden.

Cryptococcal antigen (CrAg) lateral flow assays (LFA) are widely used for the rapid diagnosis of cryptococcal meningitis because of their excellent sensitivity and specificity. We report a rare case of culture- and sequencing-confirmed Cryptococcus gattii VGI meningitis in an immunocompetent 43-year-old man with persistently negative IMMY CrAg LFA and semi-quantitative CrAg SQ results in both cerebrospinal fluid (CSF) and serum. The patient presented with fever, chronic progressive headache, diplopia, and papilledema. CSF analysis demonstrated lymphocytic pleocytosis, hypoglycorrhachia, elevated protein, and budding yeast cells on direct microscopy and India ink preparation. BioFire FilmArray meningitis/encephalitis panel detected C. neoformans/gattii species complex, while fungal culture and sequencing confirmed C. gattii VGI. False-negative CrAg LFA results persisted despite serial sample dilutions up to 1:2560. This case highlights a rare diagnostic pitfall of CrAg testing and underscores the importance of integrating microscopy, culture, and molecular methods when clinical suspicion remains high despite negative antigen assays.

Acute mastoiditis is an infectious complication of acute otitis media, potentially leading to life-threatening extracranial and intracranial sequelae. In recent years, there has been a noticeable shift in the epidemiology of causative pathogens, with an increasing incidence of less common bacteria. This article presents the case of a 1.5-year-old child diagnosed with acute mastoiditis and additional complications, who failed to respond to empirical antibiotic therapy and had persistently negative cultures. Only through the use of polymerase chain reaction (PCR) testing was the causative organism, Fusobacterium necrophorum, identified. We discuss the epidemiological and clinical features of F. necrophorum, the diagnostic challenges due to its difficult cultivation, treatment approaches, distinctive imaging findings, and the role of advanced imaging in patient follow-up. This case highlights the importance of recognizing this pathogen in culture-negative mastoiditis, the need for early consideration of PCR testing, tailored antibiotic and surgical management based on disease severity, and the necessity of radiological follow-ups.

Accurate species identification within the genus Actinotignum is difficult because 16S rRNA gene sequencing and conventional phenotypic methods lack sufficient discriminatory power. Practical molecular markers that enable reliable species-level identification are needed for clinical and research laboratories. Whole-genome-based phylogenetic analyses were performed to clarify species boundaries among Actinotignum strains. Sixteen housekeeping genes were evaluated, and their phylogenetic relationships were compared with whole-genome results. gyrA and gyrB were selected as candidate markers, and complete or PCR-amplified gene sequences were analyzed to determine similarity thresholds. Species-specific PCR assays targeting these genes were developed and assessed for specificity and analytical sensitivity. Whole-genome analyses clearly distinguished Actinotignum species. Among the housekeeping genes examined, only gyrA and gyrB reproduced the whole-genome phylogeny. Identity thresholds of &#x2265;97.6% for gyrA and &#x2265;98.4% for gyrB enabled species-level discrimination. The species-specific PCR assays amplified only the corresponding species and detected genomic DNA down to 100-10 pg. Sequence analysis of gyrA and gyrB provides a simple and reliable alternative to whole-genome analysis for identifying Actinotignum species. The developed PCR assays offer rapid, accurate, and cost-effective species identification, supporting improved detection of Actinotignum infections and facilitating future epidemiological studies.

This study evaluated the diagnostic performance of metagenomic next-generation sequencing (mNGS) in identifying the etiological agents of thoracolumbar spine infections and examined its clinical relevance in facilitating timely diagnosis and therapeutic decision-making. A total of 54 patients with suspected thoracolumbar spinal infection admitted to the Department of Spinal Orthopedics between June 1, 2022, and January 15, 2026, were enrolled. Tissue specimens from all patients underwent microbial culture, histopathological examination, and metagenomic next-generation sequencing (mNGS). Based on established clinical diagnostic criteria, patients were classified into an infection group (n = 49) and a non-infection group (n = 5). The pathogen detection rate, and diagnostic sensitivity of mNGS and conventional culture were compared using the paired &#x3c7;&#xb2; test. Among the 54 patients with suspected thoracolumbar spine infection, the male-to-female ratio was 2:1. The overall positive detection rate of mNGS was 75.9% (41/54), which was significantly higher than that of microbial culture at 57.4% (31/54) (&#x3c7;&#xb2; = 4.500, p < 0.05). When clinical diagnosis served as the reference standard, mNGS demonstrated greater sensitivity for diagnosing thoracolumbar spinal infections compared to microbial culture (83.7% vs. 63.3%), and this difference reached statistical significance (&#x3c7;&#xb2; = 4.500, p < 0.05). mNGS shows a high pathogen detection rate and superior sensitivity for diagnosing thoracolumbar spinal infection, providing valuable support for clinical diagnosis and guiding therapeutic management in suspected cases.

Highly pathogenic avian influenza A (H5N1) remains one of the most significant zoonotic threats of the 21st century due to its wide host range, high case-fatality rates in humans, and ability to cause devastating losses in poultry and wildlife. Since its first detection in humans in 1997, H5N1 has diversified into multiple genetic clades, with clade 2.3.4.4b now driving a true global panzootic affecting birds, mammals, and livestock across continents. This review synthesizes current knowledge on the virology, genomic evolution, transmission dynamics, and ecological spread of H5N1, with particular emphasis on its recent incursions into dairy cattle, companion animals, and marine mammals. Mechanistic insights into viral proteins, immune dysregulation, and mammalian adaptation highlight the virus's expanding zoonotic potential. Surveillance data demonstrate repeated spillovers at the human-animal-environment interface, although sustained human-to-human transmission has not yet been observed. Preparedness requires a One Health approach that integrates surveillance, early detection, and coordinated responses across veterinary, medical, and environmental sectors. Advances in next-generation vaccines, diagnostics, and therapeutics offer promising countermeasures, but equitable access, ethical oversight of high-risk research, and socio-economic resilience remain critical challenges. Lessons from past pandemics underscore the urgent need for proactive global action to mitigate the pandemic risk posed by H5N1.

To improve clinical decision-making about Carbapenem-resistant Gram-negative bacteria (CR-GNB) infections and halt the spread of resistant microbes, quicker and less expensive diagnostic techniques are required. Thus, the purpose of this study was to thoroughly evaluate the diagnostic efficiency (sensitivity, specificity, and concordance) of direct-from-specimen multiplex lateral flow immunoassay (LFIA) across diverse raw clinical specimens and pathogen types from critically sick patients. A total of 300 non-duplicate samples were tested to detect CR-GNB. Five major Carbapenemase genes were detected directly from the specimen using carbapenem-resistant K.N.I.V.O. detection K-Set and from culture using culture-enhanced multiplex PCR. Turnaround time (TAT) of each method was calculated. The direct LFIA revealed 100% specificity for NDM, KPC, and IMP enzymes in all tested clinical matrices (blood, urine, and respiratory samples). The study demonstrated 100% sensitivity and specificity with perfect categorical agreement (&#x3ba; = 1.000) for the blaKPC in the Klebsiella pneumoniae and for blaOXA-48 and blaIMP in the Acinetobacter baumannii; however, sensitivity of blaVIM was significantly diminished across all isolates and samples. TAT decreased significantly (p < 0.001) from 30 to 70 h to about 50 min. The tested direct LFIA facilitates the prompt enhancement of lifesaving tailored antibiotic treatment for severe illnesses.

The SwabSeq COVID-19 diagnostic assay [7] enables scalable SARS-CoV-2 detection using next-generation sequencing (NGS) of barcoded samples without RNA extraction. To adapt this approach for routine clinical use, we developed a fully automated workflow on the Opentrons FLEX liquid-handling platform. Automated pre-PCR setup processed 96 samples in approximately 15 min with minimal hands-on time, while post-PCR thermocycling, pooling, and solid-phase reversible immobilization (SPRI) bead cleanup were completed autonomously in about 2.5 h. The automated workflow produced results highly concordant with the manual method, showing complete agreement among negative samples, very high agreement among positive samples, and no discordant calls. This work demonstrates that a complex NGS-based diagnostic can be translated from a pandemic-scale pipeline into a practical, routine laboratory workflow using an accessible, open-source automation platform.

Accurate detection and monitoring of antimicrobial resistance (AMR) in Helicobacter pylori mainly rely on phenotypic methods and culture, which can sometimes fail when bacterial load is low or after recent treatment. We investigated whether gastric biopsies classified as H. pylori-negative by standard diagnostic techniques still contain detectable bacterial DNA, including regions linked to AMR, and assessed whether selected DNA fragments can mediate allelic exchange in vitro. Gastric biopsies from 46 dyspeptic patients in the Democratic Republic of the Congo (including 23 phenotypically positive and 23 phenotypically negative individuals) were analyzed using long-read amplicon sequencing of seven resistance-associated loci, selective whole-genome amplification (sWGA) followed by long-read sequencing of H. pylori-enriched reads, and a proof-of-concept natural transformation assay. Phenotypically negative biopsies exhibited significantly lower sequencing depth across multiple loci (including 23S rRNA, gyrA, gyrB, and pbp1A; p = 0.003-0.014), indicating a reduced H. pylori DNA burden. However, AMR-associated mutations linked to various antibiotic classes were found in both groups. sWGA enabled recovery of fragmentary H. pylori sequence data from phenotypically negative samples, including reads that map to resistance- and virulence-associated genes. In vitro, 23S rRNA A2143G amplicons from both phenotypically positive and negative biopsies produced clarithromycin-resistant transformants in strain 26695. These findings indicate that phenotypically negative gastric biopsies might contain low-abundance and fragmentary H. pylori DNA. Although certain DNA fragments can mediate allelic exchange under controlled in vitro conditions, these results do not confirm bacterial viability, active infection, or clinically relevant in vivo resistance transfer. Therefore, they should be interpreted with caution in molecular AMR surveillance and detection contexts.