Molecular Assays For Detection Research Articles

Mismatch repair (MMR) and microsatellite instability (MSI) phenotypes across solid tumors: A comprehensive cBioPortal study on prevalence and prognostic impact.

Mismatch repair deficiency (MMR-d) and microsatellite instability (MSI) are prognostic and predictive biomarkers in oncology. Current testing for MMR/MSI relies on immunohistochemistry (IHC) for MMR proteins and molecular assays for MSI detection. This combined diagnostic strategy, however, lacks tumor specificity and does not account for gene variants. This study provides an in-depth analysis of MMR mutations frequency, spectrum, and distribution in solid tumors. Data from 23,893 patients across 11 tumor types, using 66 publicly available studies, were analyzed. MMR-mutated (MMR-m) status was defined by alterations in MLH1, PMS2, MSH2, and/or MSH6; MSI was assessed by MSIsensor. Cases with indeterminate labelling were excluded. Survival was analyzed using the Kaplan-Meier method. Among 19,353 tumors, 949 MMR variants were identified, comprising 432 pathogenic and 517 variants of unknown significance (VUS), as defined by OncoKB. MSH6 mutations were the most frequent (n = 279, 29.4 %), followed by MSH2 (n = 198, 20.9 %), MLH1 (n = 187, 19.7 %), and PMS2 (n = 161, 16.9 %). MMR-m cases were more frequent in endometrial (EC, 20.5 %), colorectal (CRC, 8.2 %), bladder (BLCA, 8.7 %), and gastroesophageal cancers (GEC, 5.4 %). Pathogenic mutations were more common than non-pathogenic in EC, CRC, and GEC (p < 0.001, p = 0.01, p = 0.32, respectively). MMR-m status was not associated with MSI in 247 (48.9 %) cases, including 67 (13.2 %) with pathogenic mutations. The highest concordance between MMR-m and MSI was observed in CRC (65.7 %), EC (91.2 %), and GEC (69.6 %), while the lowest in pancreatic (0.2 %) and lung cancers (0.1 %). MMR-m GECs showed improved overall survival compared to MMR-wt (p = 0.009), a relationship not observed in other tumor types. This study demonstrates that the MMR spectrum is extremely hetoerogeneous in solid tumors, highliting the need for comprehensive and tumor-specific testing strategies.

P-2078. Performance of Anyplex and Xpert for the Detection of Mycobacterium tuberculosis complex in Respiratory and Non-Respiratory Samples

Abstract Background Tuberculosis diagnosis requires rapid and sensitive diagnostic methods. The aim was to evaluate the performance of Anyplex MTB/NTM and Xpert MTB/RIF assays for the detection of Mycobacterium tuberculosis complex (MTC) in respiratory and non-respiratory samples from patients suspected of having tuberculosis in a third-level hospital in Mexico. Methods Samples (n=436) from January 2020 to December 2023 in a hospital from Monterrey, Mexico underwent paired microbiological (MGIT or Lowenstein-Jensen culture) and molecular assays (Anyplex™ MTB/NTM Real-time Detection or Xpert MTB/RIF™) for mycobacterial detection. Results Of the 279 (63.8%) respiratory samples (bronchoalveolar lavage 71.3%, sputum 20.7% and tracheal aspirate 7.8%), positive culture and detected PCR were found in 26/201 samples by Anyplex and in 20/78 samples by Xpert whereas negative culture and detected PCR were found in 32 and 7 samples, respectively. Anyplex showed 65% sensitivity, 80.1% specificity, 26.5% positive predictive value (PPV), and 90.2% negative predictive value (NPV) whereas Xpert showed 100% sensitivity, 87.9% specificity, 74% PPV and 100% NPV. Of the 157 (36.1%) non-respiratory samples (cerebrospinal fluid 37.3%, soft tissues 21.5%, pleural 22.1%, lymph nodes 11.3%, and synovial 7.5%), positive culture and detected PCR were found in 14/118 samples by Anyplex and in 4/39 by Xpert whereas negative culture and detected PCR were found in 17 and 4 samples, respectively. Anyplex showed 73.6% sensitivity, 82.8% specificity, 45.1% PPV, 94.2% NPV whereas Xpert showed 66.6% sensitivity, 87.8% specificity, 50% PPV and 93.5% NPV. Conclusion Xpert assays showed higher sensitivity than Anyplex for the detection of MTC in respiratory samples and showed higher specificity compared to Anyplex in both respiratory and non- respiratory samples. Xpert showed higher PPV for respiratory samples, indicating its superiority disregarding true positive cases. Disclosures All Authors: No reported disclosures

The Combinational Effect of Enhanced Infection Control Measures and Targeted Clinical Metagenomics Surveillance on the Burden of Endemic Carbapenem and Other β-Lactam Resistance Among Severely Ill Pediatric Patients.

Background: Antimicrobial resistance (AMR) is recognized as one of the most important global public health threats. There is an urgent need to reduce the spread of these multidrug-resistant bacteria (MDR-B), particularly in extremely vulnerable patients. The aim of this study was to investigate whether targeted gene amplification performed directly on clinical samples can be used simultaneously with a bundle of enhanced infection control measures in a Pediatric Intensive Care Unit (PICU) endemic to MDR-B. Methods: This study had three phases: (1) the baseline phase was performed prior to intervention when first screening and sample collection were performed; (2) the intervention phase was performed when various enhanced infection control measures (EICM) were applied; and (3) the maintenance phase occurred when EICMs were combined with the implementation of targeted molecular surveillance. The presence of four carbapenemase genes, blaKPC, blaOXA-48-like, blaVIM, and blaNDM, as well as the β-lactamase genes blaTEM and blaSHV, was evaluated by PCR after DNA isolation directly from stool samples. The results were compared to culture-based phenotypic analysis. Results and Conclusions: The implementation of EICM appeared to reduce the resistance burden in this sample endemic to an MDR-B clinical setting. The direct implementation of a targeted and customized rapid molecular detection assay to clinical samples seems to be an effective clinical tool for the evaluation of EICM measures.

Alternative hosts of banana bunchy top virus in the Philippines and the first evidence of seed transmission of BBTV.

Banana bunchy top disease is caused by banana bunchy top virus (BBTV). BBTV is transmitted locally by aphids (Pentalonia spp.), but the long-distance spread is through the movement of infected planting materials. This study investigated potential alternative hosts of BBTV in ornamental Musa and related species in the Zingiberales in the Philippines. Artificial inoculation of BBTV, molecular detection and transmission assay were used to evaluate 15 plant test species. The potential for seed transmission of BBTV through Canna indica seeds was also investigated. Seed samples were validated and quantified for BBTV presence using molecular tools, and then grown for transmission assay. Typical symptoms of BBTV in bananas, including dark green streak on the midrib and petiole and rosetting were observed on inoculated Musa coccinea (banana blossom), M. velutina (velutina), M laterita. (bronze banana) and Canna indica (Bandera Espanola). PCR assays confirmed BBTV infection in these symptomatic test plants, as well as in Curcuma longa (turmeric) which exhibited large chlorotic blotches on the leaf. BBTV was detected from both seeds and germinated seedlings of artificially inoculated and field-collected C. indica samples. This study identified M. laterita as a new host of BBTV. The susceptibility to BBTV of M. coccinea, M. velutina, C. indica, and C. longa was also confirmed. The study also provided the first evidence of seed transmission of BBTV. C indica is an ornamental plant popularly used for landscaping in the Philippines and seeds were shown to be an efficient mode of transmission of the virus with rates up to 34%. The discovery of natural infection in ornamental plants and seeds poses a risk to the banana industry and responsible propagation and appropriate quarantine protocols must be implemented.

Validation of a molecular multiplex assay for the simultaneous detection and differentiation of bluetongue virus and epizootic haemorrhagic disease virus in biological samples

Bluetongue virus (BTV) and epizootic haemorrhagic disease virus (EHDV) are Culicoides-transmitted viruses, circulating in multiple serotypes, that cause two relevant WOAH-listed diseases of ruminants. Following its first identification in Tunisia in 2021, a novel EHDV strain belonging to serotype 8 has been detected in cattle showing BTV-like symptoms in Italy and Spain in 2022, and soon after in Portugal and France. These are European regions with recurrent circulations of different BTV serotypes. Hence, in this study we describe the validation of a TaqMan RT-qPCR panBTV/panEHDV assay, based on well-established primers and probes sets, able to simultaneously detect and distinguish between BTV and EHDV. The implemented assay, characterized by high sensitivity, specificity and good reproducibility, can be successfully applied for the rapid and affordable diagnosis needed in the current epidemiological situation and represents a powerful tool to be employed in surveillance and control strategies with a significant reduction of costs.

Specific detection of duck adeno-associated virus using a TaqMan-based real-time PCR assay.

Duck adeno-associated Virus (DAAV) is a novel pathogen that was recently discovered in ducks. To establish a molecular detection assay for DAAV for further epidemiological investigation and pathogenic mechanism. Here, we designed specific primers and probes according to the sequence characteristics of the newly discovered DAAV and then established a TaqMan real-time PCR method (TaqMan-qPCR) for the detection of DAAV. Our data showed that the established TaqMan-qPCR for detecting DAAV had high sensitivity, with the lowest detection limit of 29.1 copies/μL. No cross reaction was found with duck circovirus (DuCV), H9N2 subtype avian influenza virus (AIV), avian Tembusu virus (ATmV). duck hepatitis A virus 1 and 3 (DHAV-1 and DHAV-3), duck adenovirus A (DAdV-A), duck adenovirus 3 (DAdV-3), or duck enteritis virus (DEV). The repeatability was excellent, with the coefficients of variation of repeated intragroup and intergroup tests ranging from 0.12-0.21% and 0.62-1.42%, respectively. Seventy-eight clinical samples collected from diseased or deceased ducklings were tested. The results showed that the DAAV positive rate was 21.79%, and a triple infection (DAAV+MDPV+GPV) was found. These data provide technical support for further molecular epidemiological surveillance and pathogenic mechanism studies of DAAV infection.

Validation of the Level 2 Modification for the Neogen® Molecular Detection Assay 2 - Salmonella Enteritidis/Salmonella Typhimurium Method for Detection of the Monophasic Variant Salmonella enterica 1,4,[5],12:-:1,2.

The Neogen® Molecular Detection Assay 2-Salmonella Enteritidis/Salmonella Typhimurium (MDA2-SE/ST) method, Performance Tested Method SM (PTM) 122302, is a nucleic acid amplification test for specific detection of Salmonella enterica ser. Enteritidis (SE) and Salmonella enterica ser. Typhimurium (ST), including the ST monophasic variant Salmonella enterica 1,4,[5],12:i:-, in select poultry samples. Results for SE and ST are generated separately. The objective of the study was to validate the MDA2-SE/ST method for detection of an additional monophasic variant of ST, S. enterica 1,4,[5],12:-:1,2. A single strain of S. enterica 1,4,[5],12:-:1,2 was tested as part of a seven-strain inclusivity/exclusivity test panel. Strains were tested after growth in two enrichment media used in the method, buffered peptone water (BPW) and buffered peptone water-ISO formulation (BPW-ISO), and at two incubation temperatures, 35 ± 2 °C and 41.5 ± 1 °C. S. enterica 1,4,[5],12:-:1,2 produced positive results in the ST assay and negative results in the SE assay in both enrichment media and at both incubation temperatures. Results for the other six test strains were as expected under all conditions. The MDA2-SE/ST method can detect the monophasic variant S. enterica 1,4,[5],12:-:1,2 as well as S. enterica ser. Typhimurium and the monophasic variant S. enterica 1,4,[5],12:i:-. The MDA2-SE/ST method maintains inclusiveness for S. enterica ser. Typhimurium despite flagellar antigen variation. The data were reviewed by the AOAC PTM Program and approval was granted for modification of the MDA2-SE/ST method, PTM 122302.

Validation of the Neogen® Molecular Detection Assay 2 - Salmonella Enteritidis/Salmonella Typhimurium Method for Specific Detection of Salmonella enterica Ser. Enteritidis and Salmonella enterica Ser. Typhimurium in Chicken Carcass Rinse and Raw Ground Chicken AOAC Performance Tested MethodSM 122302.

The Neogen® Molecular Detection Assay 2-Salmonella Enteritidis/Salmonella Typhimurium (MDA2-SE/ST) method is a rapid, nucleic acid amplification-based test for specific detection of Salmonella enterica ser. Enteritidis (SE) and Salmonella enterica ser. Typhimurium (ST), including the ST monophasic variant Salmonella enterica 1,4 , [5] , 12: i:-, in select poultry samples. Results for SE and ST are generated separately. The objective of the study was to validate the MDA2-SE/ST method for detection of SE and ST in chicken carcass rinse and raw ground chicken (325 g) for Performance Tested Methods SM (PTM) certification. The study consisted of inclusivity/exclusivity testing and independent laboratory testing of chicken carcass rinse and raw ground chicken using inoculated matrixes. Data were analyzed using a paired probability of detection model to determine if differences in the number of positive results obtained with the MDA2-SE/ST and reference methods were significant. In inclusivity testing, all 50 SE strains produced positive results in the SE assay and negative results in the ST assay. All 53 ST strains (including the monophasic variant) produced positive results in the ST assay and negative results in the SE assay. All 35 exclusivity strains produced negative results in both assays. The exclusivity panel included multiple non-SE group D1 Salmonella serovars, multiple non-ST group B serovars, Salmonella spp. from other somatic groups, and other Enterobacteriaceae. In matrix testing, results for the MDA2-SE/ST and reference methods were in complete agreement for both matrixes. Results of the validation study showed that the MDA2-SE/ST method is an accurate, specific method for detection of SE and ST in select poultry matrixes. The data were reviewed by the AOAC PTM Program and approval was granted for certification of the Molecular Detection Assay 2-Salmonella Enteritidis/Salmonella Typhimurium method as PTM 122302.

Level 2 Modification (Matrix Extension) Study of the Neogen® Molecular Detection Assay 2 - Salmonella Enteritidis/Salmonella Typhimurium Method: Detection of Salmonella enterica ser. Enteritidis and Salmonella enterica ser. Typhimurium in Cooked Breaded Chicken and Raw Ground Chicken (25 g).

The Neogen® Molecular Detection Assay 2-Salmonella Enteritidis/Salmonella Typhimurium (MDA2-SE/ST) method is a nucleic acid amplification test for specific detection of Salmonella enterica ser. Enteritidis (SE) and Salmonella enterica ser. Typhimurium (ST), including the ST monophasic variant Salmonella enterica 1,4,[5],12:i:-, in select poultry samples. Results for SE and ST are generated separately. The method was previously validated for testing of chicken carcass rinse and raw ground chicken (325 g). The objective of the study was to validate the MDA2-SE/ST method for detection of SE and ST in cooked breaded chicken and raw ground chicken (25 g) as a Level 2 modification for Performance Tested Methods SM certification. Inclusivity/exclusivity testing and independent laboratory testing of cooked breaded chicken and raw ground chicken (25 g) were conducted. In inclusivity testing with BPW-ISO broth at 41.5 °C, all 50 SE strains produced positive results in the SE assay and negative results in the ST assay. All 53 ST strains (including the monophasic variant) produced positive results in the ST assay and negative results in the SE assay. All 35 exclusivity strains produced negative results in both assays. The exclusivity panel included multiple non-SE group D1 Salmonella serovars, multiple non-ST group B serovars, Salmonella spp. from other somatic groups, and other Enterobacteriaceae. In matrix testing, there were no significant differences in the number of positive results obtained with the MDA2-SE/ST and reference methods by probability of detection analysis at the 95% confidence level. Results of the matrix extension study showed that the MDA2-SE/ST method is an accurate method for detection of SE and ST in cooked breaded chicken and raw ground chicken (25 g). Validation of the MDA2-SE/ST method for two additional matrixes provides users with additional capability for specific detection of SE and ST in poultry products.

Development and preliminary assessment of the iFIND TBR: all-in- one molecular diagnostic assay for rapid detection of Mycobacterium tuberculosis and rifampicin resistance.

Early and accurate diagnosis of tuberculosis (TB) is crucial for initiating timely treatment and preventing new infections. In this study, we introduced the iFIND TBR assay, an automated all-in-one tuberculosis detection approach that simultaneously detect Mycobacterium tuberculosis (MTB) and rifampicin (RIF) resistance. The limits of detection (LOD), sensitivity, specificity, and RIF-R rpoB mutation detection of the iFIND TBR were tested on Mycobacterium tuberculosis DNA or sputum samples spiked with known numbers of M.tuberculosis H37Rv. Frozen clinical samples from patients suspected of having TB were also tested. The LOD of the iFIND TBR for MTB detection were 13.34 CFU/ml (95% CI, 11.71-16.47), and for RIF resistance was 109.79CFU/mL (95% CI, 95-138.19). The iFIND TBR assay accurately distinguish MTB strains from non-tuberculous mycobacteria (NTM) without any cross reactivity. Testing on 157 clinical sputum samples, compared with the bacteriologically TB standard, the overall sensitivity and specificity of the iFIND TBR was 100% (95%CI, 94.64, 100) and 85.29% (95% CI, 74.61, 92.72), respectively. When assessing RIF susceptibility, the iFIND TBR achieved a sensitivity of 98.15% (95% CI, 90.11-99.95) and a specificity of 85.71% (95% CI, 67.33-95.97), compared with phenotypic drug susceptibility testing. Discordant RIF susceptibility results were more frequently observed in samples exhibiting heteroresistance. These findings demonstrate that iFIND TBR assay performs well in detecting TB and RIF resistance, and shows promise as a point-of-care tool in resource-limited areas.

Multigeographic clinical assessment of a molecular diagnostic assay for detection of key codons to predict decreased susceptibility or resistance to cephalosporins in Neisseria gonorrhoeae.

Cephalosporin resistance in Neisseria gonorrhoeae has severely compromised the efficacy of World Health Organization (WHO)-recommended therapies. This study aimed to methodologically evaluate the optimized Six-CodonPlus assay, and additionally conducted a multicenter evaluation to assess its clinical application, especially for predicting antimicrobial resistance (AMR). For methodological evaluation, 397 sequence-known N. gonorrhoeae isolates were evaluated for specificity, 17 nongonococcal isolates were assessed for cross-reactivity, 159 uncultured urogenital swabs and urine samples were evaluated for sensitivity at the clinical level. For multicenter evaluation, 773 isolates with confirmed phenotypic data and 718 clinical urogenital swabs collected from four geographical cities were, respectively, utilized for the evaluation of AMR-prediction strategies and the clinical application of the assay. The assay accurately identified specific single-nucleotide polymorphisms in resistance-associated genes, the detection limits dropped to 10 copies/reaction for individual targets. The specificity reached 100% and no cross-reactivity occurred with double-target confirmation. The assay could be directly applied to clinical samples containing over 20 copies/reaction. Multicenter evaluation formulated two optimal strategies for decreased susceptibility prediction in specific scenarios, and one tactic for prediction of resistance and identification of FC428-like strains. High sensitivity of 86.84% (95% CI, 71.11-95.05) and specificity of 99.59% (95% CI, 98.71-99.89) for resistance prediction were demonstrated for ceftriaxone (CRO). Regarding N. gonorrhoeae identification among multicenter swabs, specificity reached 97.53% (95% CI, 95.49-98.69), and sensitivity reached 93.77% (95% CI, 90.04-96.22). The Six-CodonPlus assay exhibited excellent detection performance and formulated optimal AMR-related prediction strategy with regional adaptability, providing critical information for population screening and clinical treatment.

Multiplexed Assay for Small-Molecule Quantification via Photo-cross-linking of Structure Switching Aptamers.

There is an unmet need for molecular detection assays that enable the multiplexed quantification of small-molecule analytes. We present xPlex, an assay that combines aptamer switches with ultraviolet-cross-linkable complementary strands to record target-binding events. When the aptamer's small-molecule target is present, the cross-linkable strand is displaced, enabling PCR amplification and detection of the relevant aptamer. In the absence of that target, the aptamer is readily cross-linked to the strand, preventing amplification from happening. The resulting aptamer-specific amplicons can be detected and quantified in a multiplexed fashion using high-throughput sequencing. We demonstrate quantitative performance for a pair of small-molecule analytes, dopamine and glucose, and show that this assay retains good specificity with mixtures of the two molecules at various concentrations. We further show that xPlex can effectively evaluate the specificity of cross-reactive aptamers to a range of different small-molecule analytes. We believe that the xPlex assay format could offer a useful strategy for achieving multiplexed analysis of small-molecule targets in a variety of scenarios.

Molecular detection and phylogenetic tree assay of Pseudomonus aeruginosa isolated from otitis cases of cats and humans, Iraq

The aim of this study was toward isolation and identification of P. aeruginosa from cats with otitis externa in Basra city Using culture media, Biochemical tests and Polymerase chain reaction. Pseudomonas aeruginosa was isolated from 46 isolates that involved 32 cases out of 75 of feline otitis externa (21.3%) and in 14 (9.3%) from 75 human samples .the clinical signs of otitis externa were head shaking, scratching, excessive ear wax, malodor, pain during palpation, alopecia and pus and/or blood. Culture was done on Pseudomonas chrome agar revealed positive samples with production of greenish-blue pigmentation by the bacteria .polymerase chain reaction revealed that isolates were Pseudomonas aeruginosa using specific primers for P. aeruginosa identification 16S rRNA and the result was positive for the 15 isolates. a phylogenetic tree analysis of P. aeruginosa was performed using the 16S rRNA gene sequence analysis which illustrated the phylogenetic relationship between P. aeruginosa strains isolated from cats and other related genera. In conclusion, this study confirmed the higher prevalence of P. aeruginosa in otitis externa infection of cats when compared to human. The using of molecular assay and 16S rRNA gene contributed effectively in detection of P. aeruginosa. Therefore, this study suggests the application of molecular techniques in identification of different microorganisms. Additionally, moreover studies are necessary to detect the prevalence of P. aeruginosa in different infections in animals as well as human.

Evaluation of cobas® Liat® Cdiff, STANDARD™ M10 C. difficile and Xpert® C. difficile BT assays for rapid detection of toxigenic Clostridioides difficile

We evaluated the analytical performance of three commercial molecular assays for rapid detection of Clostridioides difficile toxin B in stool samples. The results were compared with results from the BD MAX™ Cdiff assay. We analyzed forty negative and thirty-two positive stool samples with three rapid assays: Roche cobas® Liat® Cdiff, SD Biosensor STANDARD™ M10 C. difficile and Cepheid Xpert® C. difficile BT. The assays demonstrated a sensitivity of 96.9 %, 96.9 % and 100.0 % and a specificity of 100 %, 97.5 % and 97.5 %, respectively. There is limited data available on the analytical performance of the newly introduced STANDARD™ M10 C. difficile assay. In this study, all three rapid assays demonstrated similarly high analytical performance and can be used for detection of toxigenic C. difficile.

A comprehensive review of influenza B virus, its biological and clinical aspects.

Influenza B virus (IBV) stands as a paradox, often overshadowed by its more notorious counterpart, influenza A virus (IAV). Yet, it remains a captivating and elusive subject of scientific inquiry. Influenza B is important because it causes seasonal flu outbreaks that can lead to severe respiratory illnesses, including bronchitis, pneumonia, and exacerbations of chronic conditions like asthma. Limitations in the influenza B virus's epidemiological, immunological, and etiological evolution must be addressed promptly. This comprehensive review covers evolutionary epidemiology and pathogenesis, host-virus interactions, viral isolation and propagation, advanced molecular detection assays, vaccine composition and no animal reservoir for influenza B virus. Complex viral etiology begins with intranasal transmission of influenza B virus with the release of a segmented RNA genome that attacks host cell machinery for transcription and translation within the nucleus and the release of viral progeny. Influenza B virus prevalence in domesticated and wild canines, sea mammals, and birds is frequent, yet there is no zoonosis. The periodic circulation of influenza B virus indicates a 1-3-year cycle for monophyletic strain replacement within the Victoria strain due to frequent antigenic drift in the HA near the receptor-binding site (RBS), while the antigenic stability of Yamagata viruses portrays a more conservative evolutionary pattern. Additionally, this article outlines contemporary antiviral strategies, including pharmacological interventions and vaccination efforts. This article serves as a resource for researchers, healthcare professionals, and anyone interested in the mysterious nature of the influenza B virus. It provides valuable insights and knowledge essential for comprehending and effectively countering this viral foe, which continues to pose a significant public health threat.

Managing fruit rot diseases of Vaccinium corymbosum.

Blueberry is an important perennial fruit crop with expanding consumption and production worldwide. Consumer demand for blueberries has grown due to the desirable flavor and numerous health benefits, and fresh market production in the U.S. has risen in turn. U.S. imports have also increased to satisfy year-round consumer demand for fresh blueberries. Pre- and post-harvest fruit diseases such as anthracnose (caused by Colletotrichum spp.) and botrytis fruit rot (caused by Botrytis spp.) have a significant impact on fruit quality and consumer acceptance. These are also among the most difficult diseases to control in the blueberry cropping system. These latent pathogens can cause significant losses both in the field, and especially during transport and marketplace storage. Although both diseases result in rotted fruit, the biology and infection strategies of the causal pathogens are very different, and the management strategies differ. Innovations for management, such as improved molecular detection assays for fungicide resistance, postharvest imaging, breeding resistant cultivars, and biopesticides have been developed for improved fruit quality. Development and integration of new strategies is critical for the long-term success of the blueberry industry.

Combined Use of Phenotypic Screening and of a Novel Commercial Assay (REALQUALITY Carba-Screen) for the Rapid Molecular Detection of Carbapenemases: A Single-Center Experience.

Carbapenem resistance is a serious public health threat, causing numerous deaths annually primarily due to healthcare-associated infections. To face this menace, surveillance programs in high-risk patients are becoming a widespread practice. Here we report the performance of the combined use of a recently approved commercial multiplex real-time PCR assay (REALQUALITY Carba-Screen kit) with conventional phenotypic screening. In this three-month study, 479 rectal swabs from 309 patients across high-risk units were evaluated by combining the two approaches. Although the molecular assay showed a higher positivity rate than phenotypic screening (7.1% vs. 5%), it should be noted that the molecular method alone would have missed eight carbapenem-resistant isolates, while using only phenotypic screening would not have detected sixteen isolates. This demonstrates the complementary strengths of each method. Our study confirms the need for a combined approach to maximize the possible clinical impact of this kind of screening, ensuring a more comprehensive detection of resistant strains.

Real-world diagnostic potential of bacterial biomarkers of canine periodontitis.

The objective of this study was to investigate the diagnostic potential of bacterial biomarkers by comparing the performance of molecular detection assays with clinical assessments of dog's oral health performed by veterinarians. Supragingival and subgingival plaque samples were collected from 127 client-owned dogs, pre-booked for procedures under general anesthesia, visiting veterinary practices in the United States. DNA was extracted and bacterial biomarkers quantified using quantitative polymerase chain reaction. Gingivitis and periodontitis were recorded by a trained clinician using the Weighted Gingivitis Periodontitis Score which involved assessing the buccal surfaces of 18 teeth while under general anesthesia. Intraoral dental radiographs of the left and right mandibular first molar teeth were also obtained. These data were then used to establish the diagnostic performance of the molecular assay to detect periodontitis. An initial conscious, visual oral examination performed by the veterinarian identified 67.7% of dogs as having periodontitis, but examination under general anesthesia indicated a higher proportion (86.6%). Analysis of supragingival plaque samples collected by veterinarians from conscious and unconscious dogs demonstrated the assay had an accuracy of 77.7 to 80.9%, a sensitivity of 77.6 to 81.0%, and a specificity of 80.0%. Use of this molecular screening tool in conscious dogs has the potential to improve early periodontal disease detection and support veterinary decision making, ultimately improving the oral health of dogs and consequently their quality of life.

Challenging the gold standard: the limitations of molecular assays for detection of Mycobacterium tuberculosis heteroresistance

ObjectivesHeteroresistant infections are defined as infections in which a mixture of drug-resistant and drug-susceptible populations are present. In Mycobacterium tuberculosis (M. tb), heteroresistance poses a challenge in diagnosis and has...

Influenza A, Influenza B, human respiratory syncytial virus and SARSCoV-2 molecular diagnostics and epidemiology in the post COVID-19 era

BackgroundThe concurrent circulation of SARS-CoV-2 with other respiratory viruses is unstoppable and represents a new diagnostic reality for clinicians and clinical microbiology laboratories. Multiplexed molecular testing on automated platforms that focus on the simultaneous detection of multiple respiratory viruses in a single tube is a useful approach for current and future diagnosis of respiratory infections in the clinical setting.MethodsTwo time periods were included in the study: from February to April 2022, an early 2022 period, during the gradual lifting of COVID-19 prevention measures in the country, and from October 2022 to April 2023, the 2022/23 respiratory infections season. We analysed a total of 1,918 samples in the first period and 18,131 respiratory samples in the second period using a multiplex molecular assay for the simultaneous detection of Influenza A (Flu-A), Influenza B (Flu-B), Human Respiratory Syncytial Virus (HRSV) and SARS-CoV-2.ResultsThe results from early 2022 showed a strong dominance of SARS-CoV-2 infections with 1,267/1,918 (66.1%) cases. Flu-A was detected in 30/1,918 (1.6%) samples, HRSV in 14/1,918 (0.7%) samples, and Flu-B in 2/1,918 (0.1%) samples. Flu-A/SARS-CoV-2 co-detections were observed in 11/1,267 (0.9%) samples, and HRSV/SARS-CoV-2 co-detection in 5/1,267 (0.4%) samples. During the 2022/23 winter respiratory season, SARS-CoV-2 was detected in 1,738/18,131 (9.6%), Flu-A in 628/18,131 (3.5%), Flu-B in 106/18,131 (0.6%), and HRSV in 505/18,131 (2.8%) samples. Interestingly, co-detections were present to a similar extent as in early 2022.ConclusionThe results show that the multiplex molecular approach is a valuable tool for the simultaneous laboratory diagnosis of SARS-CoV-2, Flu-A/B, and HRSV in hospitalized and outpatients. Infections with Flu-A/B, and HRSV occurred shortly after the COVID-19 control measures were lifted, so a strong reoccurrence of various respiratory infections and co-detections in the post COVID-19 period was to be expected.