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PubMed2025

Development of a multiplex PCR for detection of pathogenic Mycobacterium orygis in cattle tissues harboring tuberculous-like lesions.

Mycobacterium orygis, a recently defined member species of Mycobacterium tubercuolsis complex (MTBC), is emerging as a major threat to zoonotic tuberculosis control, especially in the Asian Subcontinent. The dearth of low-cost diagnostic assay to differentiate M. orygis from other members of the MTBC leads to unavailability of information about the actual burden of this species in human and animal population. In this study, we developed a multiplex PCR for distinguishing M. orygis from other MTBC based on two M. orygis-specific nonsynonymous point mutations in mbtG and fadD23 genes identified by comparative genome analysis. The specificity of the assay shows that a 434 bp IS1081 fragment was amplified from common MTBC species including M. orygis while 240 bp and 181 bp mbtG and fadD23 gene fragments were amplified only from M. orygis. No amplification was observed for nontuberculous Mycobacterium (NTM) and non-Mycobacterial pathogens. The multiplex PCR assay showed a detection limit of 32 pg of M. orygis DNA. Furthermore, a total of 85 tuberculous-like lesions in the different tissues of slaughtered cattle were tested for identification of the M. orygis, and the results showed IS1081, mbtG and fadD23 amplicons in three tissue DNA extracts confirming they contain M. orygis DNA. Also, a single IS1081 amplicon was amplified from one tissue sample signifying presence of DNA of any MTBC species other than M. orygis. An established TaqMan real time PCR assay targeting region of differences (RD) in M. orygis genome was carried out to validate the result of the assay. This showed 100 % accuracy of the in-house developed multiplex PCR.

Mycobacterium orygis↗

PubMed2026

Improving quality control of microbial agri-inputs by confirming strain identity with an easy and low-cost PCR-multiplex: A study case with Azospirillum brasilense.

The first commercial product containing the Azospirillum brasilense elite strains Ab-V5 and Ab-V6 was launched in Brazil in 2009. These strains have demonstrated agronomic efficiency in grasses and in legume co-inoculation, accounting for approximately 43 million doses in 2024. Official identification of these strains is currently performed by rep-PCR, a reliable but time-consuming and laborious method. In this study, a multiplex PCR assay was developed for the simultaneous identification of Ab-V5 and Ab-V6 in a single reaction using strain-specific SNPs. Forward primers were designed so that the terminal nucleotide at the 3' end corresponded to a strain-specific SNP unique to each target strain. To further enhance specificity, artificial mismatches were introduced at the fourth nucleotide from the 3' end of the forward primers. SNPs were identified using Snippy based on genomic alignments between Ab-V5 and Ab-V6 and confirmed by local BLASTn against the genomes of other Azospirillum species. In the multiplex assay, simultaneous and specific amplification of both strains was observed in a single reaction, without non-specific amplification. Primer specificity was also experimentally evaluated against other A. brasilense strains (Ab-V1, Ab-V2, Ab-V4, Ab-V7, Ab-V8, and Sp7T), in silico against bacteria from different genera associated with agricultural inoculants, and in commercial inoculant samples containing Ab-V5 and Ab-V6. The results confirmed the high specificity of the primers for Ab-V5 and Ab-V6 and demonstrated that the assay was capable of identifying the strains in commercial inoculants. This assay facilitates inoculant quality control by enabling strain confirmation using a simple, rapid, and low-cost method.

Azospirillum↗

PubMed2025

Multiplex PCR assay for the rapid detection of Klebsiella pneumoniae pathotypes.

Introduction. Klebsiella pneumoniae (Kp) is a major cause of nosocomial infections, with its evolving pathotypes including multidrug-resistant, hypervirulent (hvKp) and convergent strains posing significant diagnostic and treatment challenges due to combined antimicrobial resistance and virulence.Gap Statement. While there is a pressing requirement for thorough detection of Kp pathotypes, current assays in resource-limited environments are unable to effectively focus on essential carbapenemase and hypervirulence genes with the necessary reliability and precision.Aim. To develop and validate a multiplex PCR (m-PCR) assay capable of simultaneously detecting Kp isolates including those carrying partial or full virulence markers, alongside antimicrobial resistance.Methodology. In this study, an m-PCR assay was designed and optimized for the simultaneous detection of key biomarkers associated with hypervirulent (rmpA, rmpA2, iucA, peg344 and iroB), carbapenem-resistant (bla NDM, bla OXA-48-like and bla KPC) and convergent Kp pathotypes in clinical isolates. The assay was evaluated on clinical isolates and validated against whole-genome sequencing (WGS) data for accuracy, specificity and sensitivity.Results. The developed m-PCR assay exhibited 100% specificity when compared to WGS data, successfully detecting all target genes without cross-amplification in ATCC control strains. The assay demonstrated high sensitivity, efficiently amplifying bacterial genomes from minimal DNA input as low as 1 ng µl-1. Additionally, validation through sequencing confirmed the accuracy of detected amplicons.Conclusion. This m-PCR assay offers a rapid, sensitive and specific diagnostic tool for differentiating Kp pathotypes in clinical settings, aiding in timely intervention and improved infection control measures.

Klebsiella pneumoniae↗

PubMed2026

Molecular diagnostics and integrated management challenges of tobacco streak virus: Current status and future perspectives.

Tobacco streak virus (TSV) is an economically important viral pathogen causing severe yield and quality losses in several agricultural, horticultural and medicinal crops worldwide. Its complex epidemiology involving sap transmission, infected pollen and pollen-feeding thrips, together with symptom similarity to other necrosis-inducing pathogens, frequently results in misdiagnosis and delayed disease management. This review critically evaluates recent advances in TSV diagnostics and integrated disease management strategies. Particular emphasis is placed on the transition from conventional biological and serological assays to advanced molecular diagnostics including reverse transcription polymerase chain reaction (RT-PCR), quantitative real-time PCR, multiplex PCR and emerging isothermal amplification technologies such as recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP). The review also highlights emerging innovations including CRISPR/Cas-based diagnostics in addition, integrated management approaches involving phytosanitation, weed reservoir management, vector ecology-based, host resistance breeding, RNA interference (RNAi) and genome editing technologies are critically analysed. Major challenges including inadequate field validation, limited multiplex capability, poor assay standardization and scarcity of resistant cultivars are discussed. Future objectives to develop quick, field-adaptable and durable TSV detection and management methods are additionally discussed.

CRISPR/Cas diagnostics↗

PubMed2026

Development and evaluation of a multiplex PCR-based dual-platform targeted sequencing framework for precise differentiation of lumpy skin disease virus.

BACKGROUND: Lumpy skin disease virus (LSDV) shares over 96% genomic identity with goatpox and sheeppox viruses, presenting severe diagnostic challenges due to cross-reactivity. METHODS: To address this bottleneck, we established a targeted sequencing framework integrating multiplex PCR with short-read and long-read platforms. By sequentially screening target pathogens, identifying low-homology genes, and designing short and gradient long-fragment primer pools, we evaluated these dual-platform panels using highly homologous poxvirus samples. RESULTS: The short-read panel stably detected target viruses at inputs as low as 5.26 ×101 copies/μL. Under strict alignment criteria, LSDV mapping rates reached 42.91%, suppressing non-target signals to 3.05%. The Nanopore-Targeted Sequencing (NTS) long-amplicon strategy successfully eliminated homologous interference. By applying length-dependent diagnostic thresholds (≥ 100 reads for short amplicons; ≥ 50 reads for long amplicons), precise species-level identification was achieved, maintaining near-zero cross-reads (0-5) in ultra-long regions. Crucially, the field-deployable NTS workflow enabled complete detection in approximately 4 h. CONCLUSION: This complementary strategy seamlessly meets both laboratory demands for high-sensitivity enrichment and frontline requirements for rapid typing, providing a reliable tool for LSDV surveillance, mutation tracking, and outbreak control.

Capripoxvirus differentiation↗

PubMed2025

Distribution and molecular characterization of integron classes from Escherichia coli and Klebsiella pneumoniae isolates in Sulaymaniyah province of Iraq.

UNLABELLED: The environmental pollution from the misuse of antimicrobial drugs is fueling selection pressure in bacteria, thereby exacerbating the threat to global health. In Iraq, the situation is made worse by the poor implementation of the World Health Organization's Global Antimicrobial Resistance and Use Surveillance System (WHO-GLASS). Consequently, this study aimed to increase surveillance of the spread of antimicrobial resistance in Sulaymaniyah, Iraq. A total of 296 Enterobacteriaceae comprising 147 Klebsiella pneumoniae and 149 Escherichia coli were isolated from humans, poultry, and dairy farms. The isolates were screened using multiplex PCR to assess the prevalence of the clinically important integron integrase (intI) classes and antimicrobial resistance genes (ARGs) of commonly used antibiotics. Remarkably, 81.14% of the isolates carried at least 2 ARGs, 10.47% intI1, and 3.72% intI2. No intI3 was detected. A total of 663 ARGs were identified using multiplex PCR in the two Enterobacteriaceae: beta-lactamase genes were 43%, tetracycline resistance genes 25.20%, sulfonamide resistance gene 16.10%, quinolone resistance gene 10.2%, and aminoglycoside resistance genes 5.7%. K. pneumoniae harbored more integrons and ARGs than E. coli, thus posing a higher antimicrobial resistance threat in this province. This study underscores the importance of implementing more stringent WHO-GLASS and antibiotic stewardship to end the multidrug resistance crisis in Iraq. IMPORTANCE: These data are about the prevalence of integrons and resistance genes, helping to fill a significant gap in global surveillance efforts. Results can be used by global health authorities and the World Health Organization to develop national and international antimicrobial resistance (AMR) control strategies. The study is important because integrons are key genetic platforms that capture and disseminate antibiotic resistance genes among bacteria. In addition, Escherichia coli and Klebsiella spp. are among the top causes of hospital- and community-acquired infections, especially urinary tract infections, bloodstream infections, and pneumonia. Therefore, it will be riskier when these bacteria have a high rate of integrons and resistance genes because it impedes treatments during infection. Another importance of this study is that the study was carried out in Iraq. Iraq, like many low- and middle-income countries, faces challenges with unregulated antibiotic use, leading to high rates of AMR.

Escherichia coli↗

PubMed2004

High-throughput method for detecting genomic-deletion polymorphisms.

DNA microarrays have been successfully used with different microorganisms, including Mycobacterium tuberculosis, to detect genomic deletions relative to a reference strain. However, the cost and complexity of the microarray system are obstacles to its widespread use in large-scale studies. In order to evaluate the extent and role of large sequence polymorphisms (LSPs) or insertion-deletion events in bacterial populations, we developed a technique, termed deligotyping, which hybridizes multiplex-PCR products to membrane-bound, highly specific oligonucleotide probes. The approach has the benefits of being low cost and capable of simultaneously interrogating more than 40 bacterial strains for the presence of 43 genomic regions. The deletions represented on the membrane were selected from previous comparative genomic studies and ongoing microarray experiments. Highly specific probes for these deletions were designed and attached to a membrane for hybridization with strain-derived targets. The targets were generated by multiplex PCR, allowing simultaneous amplifications of 43 different genomic loci in a single reaction. To validate our approach, 100 strains that had been analyzed with a high-density microarray were analyzed. The membrane accurately detected the deletions identified by the microarray approach, with a sensitivity of 99.9% and a specificity of 98.0%. The deligotyping technique allows the rapid and reliable screening of large numbers of M. tuberculosis isolates for LSPs. This technique can be used to provide insights into the epidemiology, genomic evolution, and population structure of M. tuberculosis and can be adapted for the study of other organisms.

DNA Probes↗

PubMed2026

Development of a Multiplex Polymerase Chain Reaction Assay for Differentiating Three Lactococcus Species Associated With Piscine Lactococcosis.

Piscine lactococcosis is an important bacterial disease of farmed fish. The causative agents, Lactococcus garvieae, Lactococcus petauri and Lactococcus formosensis, are closely related, which complicates species-level identification. We developed a conventional multiplex PCR assay targeting species-specific genes identified by comparative genomic analysis. Average nucleotide identity reassignment of 441 publicly available genome assemblies identified 111 L. garvieae, 255 L. petauri and 75 L. formosensis genomes. Species-specific primers and a tuf-based Lactococcus common control were evaluated using in silico polymerase chain reaction (PCR) against target genomes and 10,460 off-target assemblies representing 474 taxa in 12 genera. Experimental specificity was assessed using six target strains and 19 non-target fish pathogens. Distinct amplicons of 195, 333 and 500 bp were produced for L. garvieae, L. petauri and L. formosensis, respectively, together with a 132-bp control amplicon. No cross-amplification was observed. All target species were detected in mixed-DNA samples and spiked kidney and spleen homogenates from two fish species. Analytical detection limits were estimated based on microscopic cell counts of bacterial suspensions before DNA extraction and ranged from 0.956 to 8.55 cell equivalents per reaction. This assay represents a rapid, low-cost method for differentiating lactococcosis-causing Lactococcus species using standard PCR and agarose gel electrophoresis.

Lactococcus garvieae↗

PubMed2027

Workflow for Long-Read Amplicon Sequencing of Chikungunya Virus Using Oxford Nanopore Technology.

This protocol provides a comprehensive, step-by-step workflow for whole-genome sequencing of Chikungunya virus (CHIKV) using an amplicon-based strategy optimized for Oxford Nanopore Technologies (ONT) platforms. The procedure includes detailed instructions for sample handling, viral RNA extraction, quality control, cDNA synthesis, multiplex PCR amplification, library preparation, sequencing, and primary bioinformatic processing. The protocol is designed to maximize reproducibility across laboratories and is suitable for genomic surveillance applications, including outbreak investigation and molecular epidemiology, even when working with low-to-moderate viral loads.

Chikungunya virus↗

PubMed2026

VisPan: real-time visualisation of multiplex amplicon-based sequencing panels for rapid syndromic surveillance and pathogen detection.

MOTIVATION: Infectious diseases persist as a major global public health challenge. Diverse factors, including climate change, globalization, deforestation, human-animal interactions, lifestyle choices, and various biological factors, can contribute to their emergence and reemergence. Rapid detection and characterization of (re)emerging pathogens are therefore critical for effective outbreak management and for enhancing our understanding of epidemics by monitoring the transmission, spread, evolution, and genomics of pathogens. In this context, next-generation sequencing technologies (NGS), particularly long-read platforms such as Oxford Nanopore Technologies (ONT), have opened new avenues for real-time pathogen monitoring. However, the bioinformatics bottleneck remains a challenge, emphasizing the need for efficient, accessible, and user-friendly analysis tools. RESULTS: Here, we present a tool adapted from the RAMPART software that enables real-time data visualisation of multiplex PCR syndromic panels combined with Oxford Nanopore sequencing. This real-time analysis enables rapid pathogen detection, from raw data acquisition to taxonomic assignment, within minutes. The interface offers dynamic visual tracking of the sequencing run and amplicon coverage, facilitating immediate insights during diagnostic workflows. Validation experiments confirmed the system's reliability, accurately identifying all pathogens present in complex clinical or environmental samples. This tool provides an integrated, user-friendly solution for genomic pathogen surveillance in field or clinical settings.

Software↗

PubMed2025

Performance evaluation of a commercial multiplex pathogen panel for detection of bacteria in sputum specimens from non-ICU patients with suspected lower respiratory tract infection.

Rapid diagnostic testing can improve pathogen detection and lead to targeted antibiotics. The BioFire FilmArray Pneumonia Panel (BFPP) is a multiplex PCR that has displayed strong concordance with traditional microbiologic techniques. However, most existing literature focuses on deep respiratory specimens, and there is sparse literature on performance in sputum specimens. This retrospective, single-center study included adult patients between 1 September 2022 and 31 August 2024 who had collection of a BFPP with standard of care (SOC) culture from a sputum specimen on a non-intensive care unit (ICU) floor or in the emergency department if admitted to a non-ICU floor. Out of 189 BFPPs performed on 189 sputum specimens, a total of 141 bacterial targets were detected. Between the BFPP and SOC culture, the overall positive percent agreement and negative percent agreement (NPA) were 96.3% and 54.9%, respectively. The positive predictive value (PPV) was 26.3% while the negative predictive value was 98.9%. Patients with greater than 24 h of antibiotic exposure prior to BFPP collection had a lower PPV compared to patients with less than 24 h or no exposure (13.6% vs 29.6% vs 30.4%). The lowest concordance was observed for Haemophilus influenzae (15.4%), Moraxella catarrhalis (18.2%), Streptococcus pneumoniae (19%), and Staphylococcus aureus (22.7%), several of which are fastidious in culture. BFPP showed a high NPA, with all bacterial targets having an NPA greater than 90%, except H. influenzae (82%). Based on these data, a negative BFPP in sputum specimens could help to rule out a bacterial pneumonia, but the benefit of a positive test remains unclear.IMPORTANCEThis study evaluates the BioFire FilmArray Pneumonia Panel (BFPP) by comparing its performance to standard of care cultures exclusively in sputum specimens from non-intensive care unit patients with suspected lower respiratory tract infection. Findings show an overall high positive percent agreement and negative predictive value but a low negative percent agreement and positive predictive value, suggesting that a negative test in sputum specimens could be beneficial when attempting to rule out a bacterial infection, but the benefit of a positive test remains unclear, particularly if common airway colonizing bacteria are detected and at low semi-quantitative thresholds. Clinical symptoms should guide test interpretation in patients with positive BFPP results but negative culture growth.

Humans↗

PubMed2025

Clinical and Epidemiological Insights into a Parainfluenza Virus Type 3 Outbreak in Multiple Myeloma Patients.

Human parainfluenza virus type 3 (HPIV-3) can be responsible for mild to severe respiratory infections and hospital epidemics. We investigated an outbreak in a hematology unit. Respiratory viruses were screened using multiplex PCR. HPIV-3 quantification and whole-genome sequencing were performed on HPIV-3 positive respiratory samples. Clinical characteristics, infection progression, incidence rates of respiratory viruses within the hospital and detection of respiratory viruses were documented, along with the reinforcement of infection prevention and control (IPC) measures implemented. Between November 2022, and January 2023, HPIV-3 was identified in 20 of 113 hematology patients (17.7%), of whom 80% had multiple myeloma. A majority of HPIV-3-positive patients developed pneumonia (60%), and mortality was notably higher (35%) compared to patients who were negative (3%, p < 0.0001). Respiratory HPIV-3 viral loads were similar between patients with and without pneumonia. In parallel, HPIV-3 incidence in the hospital overall was lower than in the hematology unit (p < 0.0001). Air virus screening showed the detection of HPIV-3 in the air in different areas, and whole-genome sequencing confirmed the circulation of a single HPIV-3 strain. Strengthened IPC measures were associated with the containment of the outbreak. HPIV-3 has high epidemic potential in patients with multiple myeloma and causes severe infections. Our findings highlight the need for routine HPIV-3 testing in hematology units.

Humans↗

PubMed2026

Investigation of pmrCAB and mcr associated resistance in colistin-resistant A. baumannii isolates.

BACKGROUND & OBJECTIVES: Colistin is one of the last-resort antibiotics for multidrug-resistant Acinetobacter baumannii. Increasing resistance to colistin limits treatment options, particularly in intensive care units (ICUs). The aim of this study was to compare the expression levels of pmrC, pmrA, and pmrB, among colistin-resistant and colistin-susceptible clinical A. baumannii isolates, to investigate the presence of plasmid-mediated mcr-1-5 genes, and to determine clonal relationships among colistin-resistant isolates. METHODS: A total of 110 A. baumannii isolates recovered from ICU patients in 2020 were included. Colistin minimum inhibitory concentrations were determined using the broth microdilution method. Expression levels of pmrC, pmrA, and pmrB were analyzed by RT-qPCR and compared with the reference strain A. baumannii ATCC 19606. Colistin-resistant isolates (Group 1) were compared with 10 randomly selected colistin-susceptible isolates (Group 2). Detection of mcr-1-5 genes was performed by in-house multiplex PCR. Clonal relationships among resistant isolates were assessed by PFGE. RESULTS: Colistin resistance was detected in 15.45% (17/110) of isolates. The median relative expression levels of pmrC, pmrB, and pmrA in colistin-resistant isolates were 47.84-fold (IQR: 19.29-67.18), 14.72-fold (IQR: 10.13-16.68), and 8.57-fold (IQR: 5.17-12.82), respectively. In colistin-susceptible isolates, the corresponding median expression levels were 5.32-fold (IQR: 3.60-7.97), 3.29-fold (IQR: 0.85-5.95), and 3.31-fold (IQR: 2.58-6.55). Expression levels were significantly higher in colistin-resistant isolates for pmrC (p < 0.001), pmrB (p = 0.002), and pmrA (p = 0.024). None of the resistant isolates carried mcr-1-5 genes. PFGE analysis revealed 12 distinct genotypes among 17 resistant isolates. INTERPRETATION & CONCLUSIONS: Colistin-resistant A. baumannii isolates exhibited significantly higher expression levels of the pmrC, pmrA, and pmrB genes compared to colistin-susceptible isolates. Among the genes evaluated, pmrC showed the largest effect size and the strongest association with the colistin-resistant phenotype. No changes were found in the mcr-1-5 genes among the isolates studied. Further studies, including genomic and functional analyses, are needed to elucidate the underlying mechanisms of these expression changes and their contribution to colistin resistance.

Journal Article↗

PubMed2026

Genomic characterisation of ST233 Pseudomonas aeruginosa co-producing KPC-2 and VIM-2 in Northeastern Brazil during the COVID-19 pandemic: Evidence of independent horizontal acquisition events.

BACKGROUND: Dual-carbapenemase-producing Pseudomonas aeruginosa poses a major therapeutic and epidemiological challenge worldwide, yet systematic data on KPC and VIM co-production in Brazil remain limited. The COVID-19 pandemic intensified antimicrobial use, a period temporally associated with increased carbapenemase detection globally. OBJECTIVES: To characterise the molecular epidemiology and resistance profiles of KPC and VIM co-producing P. aeruginosa isolates from Brazil (2019-2023). METHODS: Between 2019 and 2023, 1489 multidrug-resistant P. aeruginosa isolates were screened by multiplex PCR for carbapenemase-encoding genes. Co-producing isolates underwent pulsed-field gel electrophoresis (PFGE) for clonal profiling, followed by whole-genome sequencing (WGS) for high-resolution phylogenomic analysis. Antimicrobial susceptibility testing and plasmid characterisation using next-generation sequencing platforms were also performed. RESULTS: Forty-two isolates (2.8%) harboured both blaKPC-2 and blaVIM-2, with detection occurring exclusively between 2020 and 2023, temporally coinciding with the COVID-19 pandemic. PFGE identified eight distinct clonal groups, providing evidence for independent horizontal gene transfer (HGT) events, whilst WGS confirmed all isolates as the high-risk ST233 lineage. Chromosomally integrated blaVIM-2 within class 1 integrons predominated; 2 isolates carried dual chromosomal copies. Plasmid-borne blaKPC-2 was identified across heterogeneous replicons (43.3-430.1 kb), suggesting multiple independent acquisition events. All co-producing isolates displayed extensive drug resistance, retaining in vitro susceptibility only to cefiderocol and colistin. CONCLUSIONS: ST233 co-producing KPC and VIM, represents a high-risk resistance phenotype of epidemiological significance. Divergent genomic architectures suggest active horizontal dissemination across diverse genetic backgrounds rather than clonal expansion, highlighting the need for enhanced surveillance and infection control strategies.

Bacterial genomic characterisation↗

PubMed2026

Targeted variant analysis of feline mediastinal lymphoma using MassARRAY and clinical associations.

Lymphoma is the most commonly diagnosed cancer in cats. This study used the Agena MassARRAY to genotype 40 variants across 17 genes in feline mediastinal lymphoma. These variants have previously been identified in tumors, including T- and B-cell lymphomas, acute and chronic lymphocytic leukemias, and mast cell tumors, in humans, dogs, and cats, using various methods. They were selected based on high prevalence reported in prior oncology studies, potential relevance to targeted therapy, and suitability for multiplex PCR amplification. Pleural fluid samples were collected from 76 cats with mediastinal lymphoma, including 69 domestic shorthairs, two Persians, two Siamese, two Wichienmaat, and one Scottish Fold. The most prevalent variants were found in the BCL2, KIT, STAT3, and ZEB1 genes. Specifically, BCL2 c.83275986G > A and c.83275992G > T were present in 71.1% and 57.9%, respectively. In cats with variant-positive in KIT c.163965724C > CT significantly reduced (11 days) compared to wild-type cats (94 days) (p < 0.001). In cats with variant-positive in STAT3 c.42942437C > CA, resulted in shorter median survival compared to wild-type cats (18 days vs. 77 days, p = 0.006). The findings suggest that the variant panel could be useful for the genomic landscape of feline mediastinal lymphoma and warrant further validation.

Animals↗

PubMed2026

A Randomized Clinical Trial to Compare Moxifloxacin Versus Azithromycin for the Treatment of Mycoplasma genitalium: The FARTHEST Study.

BACKGROUND: Mycoplasma genitalium (MG) is increasingly characterized by high rates of macrolide and fluoroquinolone resistance. International guidelines recommend resistance-guided therapy; however, access to genotypic testing is limited, and randomized trial evidence is lacking. We assessed the efficacy of moxifloxacin and azithromycin without resistance assays. METHODS: This monocentric, open-label, superiority, randomized controlled trial enrolled adults with MG infection detected by multiplex PCR, randomized 1:1 to receive moxifloxacin 400 mg daily for 10 days or azithromycin 500 mg daily for 6 days. A test of cure was performed ≥28 days after treatment completion. The primary endpoint was microbiological cure in the intention-to-treat (ITT) and per-protocol (PP) populations. Subgroup analyses assessed symptomatic versus asymptomatic infections, doxycycline exposure, re-treatment, and sexual behavior. RESULTS: Among 358 randomized participants, 87.0% of those treated with moxifloxacin and 61.2% of those treated with azithromycin achieved microbiological cure in the ITT analysis (absolute risk difference 25.8%, 95% CI 16.5, 35.2). The superiority of moxifloxacin was confirmed in the ITT and PP populations. Moxifloxacin remained superior across most subgroups, whereas azithromycin showed comparable efficacy only among heterosexual individuals. Doxycycline coadministration did not improve outcomes. Both regimens were well tolerated, with only one case of discontinuation. CONCLUSIONS: Moxifloxacin demonstrated superior efficacy compared to azithromycin for treating MG infection in the absence of resistance testing. These randomized data support the use of moxifloxacin as a first-line option when resistance assays are unavailable and may inform treatment strategies.

Humans↗

PubMed2026

Prevalence and Genomic Characterization of mcr-Positive Enterobacteriaceae in Retail Meat in Thailand Following the Colistin Ban.

This study aimed to investigate the prevalence and characteristics of mcr-positive Enterobacteriaceae in retail meat in Thailand following the national ban on prophylactic colistin use in food producing animals. A total of 152 meat samples (103 chicken and 49 pork) were collected from supermarkets and open markets between July and September 2023. Samples were screened for mcr-1 to mcr-5 using multiplex PCR. None of the samples from supermarkets tested positive, whereas mcr genes were detected in 15.4% (6/39) and 13.3% (4/30) of chicken and pork samples, respectively, from open markets, with mcr-1 and/or mcr-3 identified. A total of 21 isolates were recovered from PCR-positive samples (11 from chicken and 10 from pork). Escherichia coli was the predominant species (n = 19), followed by Klebsiella pneumoniae (n = 2). All mcr-positive isolates exhibited multidrug resistance. Whole-genome sequencing was performed for 19 non-clonal isolates. One K. pneumoniae strain from a pork sample co-harbored mcr-1 and mcr-8, representing the first report of this combination in the animal sector in Thailand. In addition, virulence-associated genes, including adhesion factors, toxins, and iron acquisition systems, were identified in selected isolates. Core genome SNP-based phylogenetic analysis revealed substantial genomic diversity among the isolates, suggesting relatedness to strains reported prior to the colistin ban. These findings indicate that retail meat from open markets may serve as an important route for the transmission of mcr-positive bacteria in Thailand and highlight the urgent need to incorporate systematic retail meat surveillance into national antimicrobial resistance monitoring programs.

Animals↗

PubMed2026

Near-Whole-Genome Sequencing of Peste Des Petits Ruminants Virus Lineage IV From the Savannah District, Northern Côte d'Ivoire in 2023.

Peste des petits ruminants (PPR) is a highly contagious viral disease affecting sheep and goats, causing substantial economic losses in endemic countries. In the Savannah district of Côte d'Ivoire, knowledge of the genetic diversity and molecular epidemiology of the PPR virus (PPRV) remains limited. This study investigated the genetic diversity and phylogenetic relationships of PPRV circulating in this region using whole-genome sequencing (WGS). A cross-sectional survey was conducted between September and December 2023. Nasal swabs collected from sheep and goats were screened for PPRV ribonucleic acid (RNA) using real-time reverse transcription polymerase chain reaction (RT-qPCR). Samples with low quantification cycle (Cq) values of less than 35 and successful multiplex PCR amplification profiles were selected for sequencing using the Oxford Nanopore MinION platform. Near-complete consensus genomes were generated through reference-based assembly and analysed alongside representative strains from all recognised PPRV lineages. Of the 355 samples analysed, 25 (7.0%) tested positive for PPRV RNA, with positive detections in all three surveyed regions (Poro, Tchologo and Bagoué). The four samples with the lowest Cq values, originating from all three administrative regions, were successfully sequenced, generating genomes that covered 82.0%-86.2% of the reference genome at a depth of ≥ 10 ×. The missing regions were mainly located at the 5' and 3' genomic termini, as well as in limited internal regions associated with amplicon dropout. Phylogenetic analysis revealed that all four sequences belonged to lineage IV and exhibited high nucleotide similarity (98.1%-99.9%). The Ivorian strains clustered with recent lineage IV viruses from West, North and Central Africa, whereas historical Ivorian lineages I and II formed distinct clades. These findings confirm the predominance of lineage IV in northern Côte d'Ivoire and provide baseline genomic data to support molecular epidemiological surveillance in the region.

PPRV↗