Multiplex PCR Assay Research Articles

Molecular uncovering of important helminth species in wild ruminants in the Czech Republic

Monitoring gastrointestinal helminth infections in wild ruminants poses significant challenges for managing wildlife health, particularly regarding invasive species. Traditional coprological methods are often limited by their labor-intensive nature and potential for erroneous identification due to morphological similarities among parasite species. This study employed advanced molecular techniques to assess the prevalence and distribution of several helminth taxa, including the invasive nematode Ashworthius sidemi and the trematode Fascioloides magna, in wild ruminant populations in the Czech Republic (CR). A comprehensive and extensive survey on parasite occurrence, unique in its nationwide scope, was conducted on 983 fecal samples collected from red deer (Cervus elaphus), roe deer (Capreolus capreolus), fallow deer (Dama dama), and mouflon (Ovis musimon) across various regions of the CR. The samples were analyzed using multiplex real-time PCR assays specifically designed to detect the DNA of six helminth representatives: the nematodes A. sidemi and Haemonchus spp., as well as the trematodes F. magna, Dicrocoelium dendriticum, Fasciola hepatica, and Calicophoron daubneyi (and representatives of the family Paramphistomidae, respectively). These assays targeted regions of ribosomal DNA (rDNA) and were designed to exhibit high sensitivity and specificity, enabling accurate detection of helminth parasites directly in fecal samples. The molecular assays revealed that invasive nematode A. sidemi was the most prevalent helminth species, detected in 15.8% of all samples (155/983), with the highest infection rate observed in red deer at 30.7% (124/404). Haemonchus spp. were also frequently detected, identified in 14.9% of samples (146/983), particularly in roe deer, with a prevalence of 23.2% (86/371). Spatial analysis of these nematodes across various regions of the CR revealed the extensive distribution of both A. sidemi and Haemonchus spp. in nearly all regions. In contrast, trematode infections were less common, with F. magna and D. dendriticum each found in only 1.5% of samples (15/983). Members of the family Paramphistomidae were detected in 0.2% of the samples (2/983) and were confirmed through sequencing as C. daubneyi. The geographical distribution patterns identified in this study indicate potential hotspots for specific helminth species. These findings are critical for planning health management and conservation strategies to mitigate the impacts of helminth infections, especially in areas affected by invasive species.

Harnessing Non-standard Nucleic Acids for Highly Sensitive Icosaplex (20-Plex) Detection of Microbial Threats for Environmental Surveillance.

Environmental surveillance and clinical diagnostics heavily rely on the polymerase chain reaction (PCR) for target detection. A growing list of microbial threats warrants new PCR-based detection methods that are highly sensitive, specific, and multiplexable. Here, we introduce a PCR-based icosaplex (20-plex) assay for detecting 18 enteropathogen and two antimicrobial resistance genes. This multiplexed PCR assay leverages the self-avoiding molecular recognition system (SAMRS) to avoid primer dimer formation, the artificially expanded genetic information system (AEGIS) for amplification specificity, and next-generation sequencing for amplicon identification. Using parallelized multitarget TaqMan Array Cards (TAC) to benchmark performance of the 20-plex assay on wastewater, soil, and human stool samples, we found 90% agreement on positive calls and 89% agreement on negative calls. Additionally, we show how long-read and short-read sequencing information from the 20-plex can be used to further classify allelic variants of genes and distinguish subspecies. The strategy presented offers sensitive, affordable, and robust multiplex detection that can be used to support efforts in wastewater-based epidemiology, environmental monitoring, and human/animal diagnostics.

Clostridioides difficile hypervirulent strain ST1 isolated from clinical stool specimens obtained from three Provinces in South Africa

Clostridioides difficile infection is a serious healthcare-associated infection linked to antimicrobial use. The severity of the disease can be associated with hypervirulent ribotypes such as RT027. The study aimed to investigate the molecular epidemiology and genomic characteristics of C. difficile isolates from private and public healthcare settings in South Africa. One hundred clinical stool specimens were cultured on cycloserine-cefoxitin-fructose agar. Conventional multiplex polymerase chain reaction (M-PCR) assays were conducted for isolate identification and detection of toxin genes. Genomic characteristics of the isolates were determined using whole genome sequencing (WGS) and data was analysed using pubMLST, EnteroBase, Pathogenwatch and CARD. One hundred clinically presumptive C. difficile positive stool specimens were collected, of which 62% (62/100) were confirmed as C. difficile by M-PCR assay. Among the 62 identified C. difficile isolates, 97% (60/62) were toxigenic, with the most dominant toxin profile being A+B+CDT+according to the M-PCR assay. The results showed that 93% (40/43) of the WGS analysed C. difficile strains clustered into clades 1 to 5. These 40 strains were categorized into 16 sequence types (STs), with ST1 (clade 2) being the most prevalent, representing 45% (18/40), this strain is an RT027-associated strain previously epidemic hypervirulent strain. One major cluster (n=18) comprising ST1 strains was identified in Gauteng Province and all the isolates associated with this cluster showed the same resistome (antimicrobial resistance genes and mutations: CDD-1, aac (6')-Ie-aph (2″)-Ia, PnimBG and Thr82Ile). The study also identified one strain as ST11, this strain is well known for its zoonotic potential, and two strains were identified as ST37 known as an epidemic strain. Strains from public healthcare settings exhibited genetic similarity, while those from private settings showed greater genetic diversity. The study reported, for the first time, hypervirulent strains ST1 in Africa and ST11 in South Africa, with a minimum spanning tree indicating an ongoing ST1 outbreak.

Developing and validating a multiplex hydrolysis probe-based quantitative PCR assay for the detection of four pathogens in chelonians.

Many wildlife conservation efforts focus on the effects of one pathogen, but for many conservation efforts to be successful, researchers require an understanding of ecological processes that may include multiple co-occurring pathogens. We developed a multiplex quantitative PCR (qPCR) assay to detect four pathogens in eastern box turtles (Terrapene carolina carolina), including frog virus 3 (FV3), Terrapene herpesvirus 1 (TerHV1), box turtle Mycoplasma sp. (BTMyco), and Terrapene adenovirus (TerAdv). TaqMan™ primer probes were designed using previously published assays with four different fluorophores. Multiplex Cq values plotted against singleplex Cq values demonstrated slopes of 0.967, 1.00, 0.980, and 0.973 for TerHV1, TerAdv, FV3, and BTMyco, respectively, and R2 values of 0.999 for all four pathogens. The assay was highly consistent with the intra-assay variation of all four pathogen targets, ranging from 0.05-1.826 % across all concentrations, while inter-assay variation ranged from 0.031-4.569 % among all four targets at all concentrations. Clinical samples were tested using previously collected samples from eastern box turtles and red-eared sliders (Trachemys scripta elegans) and performed similarly to singleplex assays. This multiplex assay is an effective, time-efficient diagnostic tool to quickly monitor chelonian pathogens by detecting FV3, TerHV1, BTMyco, and TerAdv within a single reaction. A validated and clinically utilized multiplex assay will be beneficial to characterizing a more complex pathogen profile for future chelonian epidemiological studies to better describe pathogen dynamics and their impacts on individual and population health.

Development and evaluation of pan-mucorales and multiplex PCR assays

Development and evaluation of pan-mucorales and multiplex PCR assays

P-1629. Reduction in Clostridioides difficile Over-diagnosis Following Suppression of C. difficile Target from Multiplex Molecular Stool Assay in a Pediatric Healthcare System

Abstract Background Clostridioides difficile testing by polymerase chain reaction (PCR) does not differentiate asymptomatic colonization from infection, and children are frequently colonized with toxigenic strains of C. difficile. Over-diagnosis of C. difficile infections can lead to adverse patient outcomes and poorer quality metrics for hospitals.Figure 1:C. difficile PCR orders following change in GI PCR Panel, Interrupted Time Series Methods In January 2023 our pediatric health system enacted multimodal changes to the electronic ordering of stool tests for C. difficile and other gastrointestinal pathogens. A multiplex PCR test for stool was updated to suppress testing for toxigenic C. difficile. The only remaining method to test for C. difficile by PCR in house was a C. difficile toxin gene specific PCR. Additionally, appropriate clinical criteria with a hard stop were added to the C. difficile PCR order (all of: no laxatives in the past 24 hours, at least 3 liquid stools in the past 24 hours, at least one risk factor for C. difficile infection, and patient at least one year old). If providers still sought C. difficile testing without meeting these criteria, infectious disease specialist approval was required. We analyzed the effect of this intervention using interrupted time series analysis and pre-post aggregate rates from January 2019 through February 2024.Figure 2:C. difficile positive rate following change in GI PCR Panel, Interrupted Time Series Results There were 4426 stool PCR tests performed for C. difficile in the pre-intervention period and 622 in the post-intervention period. The mean (± sd) age of those tested was 7.8 years (± 6.1 years). After the intervention, there was an immediate 61% decrease in C. difficile testing incidence (95% CI [53% - 67%], Figure 1) and 51% decrease in C. difficile diagnosis incidence (95% CI [26% - 67%], Figure 2). There was no significant immediate change or change in slope in the C. difficile antibiotic treatment incidence or HFO C. difficile incidence, but there was a trend towards lower rates of healthcare facility onset C. difficile after the change (aggregate rate 4.13 per 10,000 non-NICU patient-days prior to intervention vs. 3.39 after, P = .28). Conclusion Removing C. difficile from multiplex PCR assays and adding required clinical criteria to the electronic order reduces the number of C. difficile tests and diagnoses in a pediatric health system. Disclosures Laura Filkins, PhD, Avsana Labs: Board Member|Avsana Labs: Stocks/Bonds (Private Company)|Biofire Diagnostics: Grant/Research Support

P-1190. Viral Codetection in Infants with Respiratory Syncytial Virus (RSV) Infection Is Associated with Decreased Viral Loads, Reduced Mucosal Cytokine Concentrations and Longer Hospitalization

Abstract Background Studies have analyzed the contribution of host and viral factors to RSV disease pathogenesis and severity in children, but the role of RSV/viral co-detection is poorly understood. We investigated the relationship between viral co-detection, RSV viral loads, mucosal cytokine concentrations and disease severity in young children with RSV acute respiratory infection (ARI). Methods We enrolled a convenience sample of previously healthy children < 2 years of age with RSV ARI that were either hospitalized or managed as outpatients at a large pediatric hospital. Nasopharyngeal samples were obtained to assess RSV viral loads by rt-PCR, viral codetection using a multiplex PCR assay, and mucosal cytokine concentrations. We assessed the impact of viral co-detection on RSV viral loads, mucosal cytokine concentrations, and clinical outcomes stratified by age (0-6 and >6-24 months).Figure 1.Mucosal cytokine concentrations according to single RSV detection versus RSV/viral co-detection . The Y axis depicts individual concentrations of mucosal antiviral cytokines (IFN-β and CXCL-10) and of IL-6 expressed as log10 pg/ml, and the x axis the two study groups: single RSV infections and RSV/viral co-detections. Comparisons between groups were made with Mann-Whitney test and statistical significance was set at p< 0.05. Results From February 2013 to March 2020, 483 children with RSV ARI were enrolled (inpatients, n=383; outpatients, n=95). Of those, 108 (22.4%) had viral co-detection. Rhinovirus/Enterovirus (RV/EV) followed by endemic coronaviruses (CoV) were the most common co-detected viruses. Median age was 3.9 months for infants with RSV/viral co-detection vs 2.8 months for with single RSV ARI (p=0.02); with no significant differences in other demographic characteristics (Table 1). RSV loads were higher in single RSV vs RSV/viral co-detection (7.55 vs 7.16 log10 copies/ml, p=0.0009), particularly in infants 0-6 months. Mucosal concentrations of IL-6, CXCL-10, and IFN-β were significantly higher in children with single RSV vs RSV/viral co-detection (Figure 1). Analysis by age showed significant differences of IL-6 and CXCL-10 concentrations in infants 0-6 months, but not in older children. There were no significant differences with respect to need for hospitalization, clinical disease severity or PICU care in infants with single RSV vs RSV/viral codetections, but RSV/viral co-detection was associated with longer hospitalizations (3 vs 2.3 days respectively, p=0.022). Conclusion Concomitant detection of other respiratory viruses in children with RSV ARI was common and associated with reduced RSV viral loads, decreased mucosal proinflammatory and antiviral cytokines, and longer length of stay. Disclosures Diego R. Hijano, MD, MSc, FDA: Grant/Research Support|Merck: Grant/Research Support|National Institute of Health: Grant/Research Support Octavio Ramilo, MD, AstraZeneca: Honoraria|Bill & Melinda Gates Foundation: Grant/Research Support|Merck: Advisor/Consultant|Merck: Grant/Research Support|Merck: Honoraria|NIH/NIAID: Board Member|NIH/NIAID: Grant/Research Support|NIH/NICHD: Grant/Research Support|Pfizer: Advisor/Consultant|Pfizer: Honoraria|Sanofi: Advisor/Consultant|Sanofi: Honoraria Asuncion Mejias, MD, PhD, MsCS, Astra-Zeneca: Advisor/Consultant|Astra-Zeneca: Honoraria|Enanta: Advisor/Consultant|Janssen: Advisor/Consultant|Janssen: Grant/Research Support|Merck: Advisor/Consultant|Merck: Grant/Research Support|Moderna: Advisor/Consultant|Pfizer: Advisor/Consultant|Pfizer: Honoraria|Sanofi-Pasteur: Advisor/Consultant|Sanofi-Pasteur: Honoraria

P-40. Impact of case and control selection on influenza vaccine effectiveness (VE) among adults aged 40 years and older hospitalized with acute respiratory illness (ARI) during 2022-2023 using a test negative design (TND): secondary analysis of the North America multi-specimen study

Abstract Background Case and control definitions may impact VE estimates in observational studies. Using a TND design, we evaluated how influenza vaccine effectiveness against ARI hospitalizations changed when 1) influenza cases were detected through nasopharyngeal swabs (NPS) only versus multiple specimens and 2) controls included or excluded other vaccine preventable diseases (VPD). Methods Adults aged ≥40 years hospitalized for ARI had NPS and saliva or sputum tested using Luminex RSV/influenza multiplex PCR assay. Influenza was defined as either positive by NPS alone or positive by saliva, sputum, or NPS. Controls were defined as either all persons who were influenza negative or those negative for influenza and positive for respiratory syncytial virus (RSV). Exposure was self-reported seasonal influenza vaccination, defined as receipt of vaccine after 1 July 2022 and prior to 14 days of ARI illness onset. VE was calculated as 1 - ratio of odds of influenza vaccination among cases over controls. Results Among 2,999 patients with NPS and ≥1 other specimen, influenza detection increased by 40%, 74% and 91% when sputum, saliva, or both were added to NPS results, respectively (Table 1). Among 2,954 (98.5%) patients with influenza vaccine data, and when controls were defined as any influenza-negative ARI hospitalization, VEs were -3.6% (95%CI -49.5–28.3) and -0.9% (95% CI -32.5–23.2), using cases identified by NPS versus any specimen type, respectively (Table 2). When controls were persons who were RSV positive and influenza negative, VEs were 38.5% (95% CI -3.5–63.4) and 43.2% (95% CI 14.9–62.1) using cases identified by NPS versus any specimen type, respectively. Conclusion Adding saliva and sputum nearly doubles influenza detection compared to NPS alone but does not substantially alter VE estimates. By contrast, VE was substantially increased when defining controls as those who were RSV+, yielding a result closer to the US Centers for Disease Control and Prevention values of 39-43% for the same season. The latter increase in VE may have occurred because RSV positivity reduces confounding caused by inclusion of other vaccine preventable infections (ie, SARS-CoV-2) in the control group due to correlation between vaccination against influenza and SARS-CoV-2. Disclosures Negar Aliabadi, MD, MS, Pfizer Inc: employment|Pfizer Inc: Stocks/Bonds (Public Company) Qing Liu, M.S., Pfizer Inc.: Stocks/Bonds (Public Company) Julio A. Ramirez, MD, FACP, Pfizer: Julio Ramirez is an employee of Norton Healthcare (Louisville, KY), which received fees from Pfizer in relation to this study. Allison McGeer, MD, AstraZeneca: Honoraria|GSK: Honoraria|Merck: Honoraria|Moderna: Honoraria|Novavax: Honoraria|Pfizer: Grant/Research Support|Pfizer: Honoraria|Roche: Honoraria|Seqirus: Grant/Research Support|Seqirus: Honoraria Ruth Carrico, PhD, DNP, APRN, Pfizer: Advisor/Consultant|Pfizer: Grant/Research Support|Pfizer: Honoraria|Sanofi: Advisor/Consultant|Seqirus: Advisor/Consultant Samira Mubareka, MD, Pfizer: Grant/Research Support Verna Welch, PhD, MPH, Pfizer Inc.: Stocks/Bonds (Public Company) Malak Elsobky, MD, Pfizer: Stocks/Bonds (Public Company) Bradford D. Gessner, M.D., M.P.H., Pfizer: Employee|Pfizer: Stocks/Bonds (Public Company) Elizabeth Begier, MD, M.P.H., Pfizer Vaccines: Employee|Pfizer Vaccines: Stocks/Bonds (Private Company)

P-2139. Evaluation of a Multiplex Real-time PCR for the Detection and Differentiation of Sporothrix schenckii and Sporothrix brasiliensis

Abstract Background Sporothrix spp is a thermally dimorphic fungi known to cause subacute or chronic subcutaneous lesions in humans and animals and is the cause of increasing public health concern due to spread of feline-associated cases.Figure 1.Limit of detection and genomic DNA standard curve of S. schenckii and S. brasiliensis Methods A recently described Real-time PCR assay targeting the β-tubulin gene to detect and differentiate two related species, S. schenckii and S. brasiliensis, was evaluated and adapted by adding lambda primers and probe for internal control amplification. Isolates, fresh tissues and suspected sporotrichosis Formalin-Fixed, Paraffin Embedded (FFPE) tissues from humans and animals were received in the Mycotic Diseases Branch laboratory at the U.S. Centers for Disease Control and Prevention (CDC) for the routine fungal identification by conventional PCR and sequencing, using the primers D1/D2, ITS5/ITS4 and ITS3/ITS4. Ten-fold serial dilutions from 1 ng/μl to 1 fg/μl of DNA were tested with 20 replicates to establish limit of detection (LOD), and DNA from four fresh tissues extracted on three different days was used to assess reproducibility.Table 1.Ct values and coefficient of variation (CV) of the reproducibility test using fresh tissues on three different days Results The assay was tested with 55 S. brasiliensis, 19 S. schenckii, and 85 isolates from other clinically relevant fungi, showing 100% concordance with sequencing methods. The test was also able to amplify S. schenckii DNA from 10 fresh tissues, which were previously confirmed by culture and sequencing as S. schenckii (100% sensitivity), with Ct values ranging from 19 to 38. Among 9 FFPE samples, S. schenckii was amplified in 6 samples (67% sensitivity) with Ct values ranging from 29 to 38. LOD was 1pg/μl of DNA. Results of the 20 replicates showed that at this concentration all the DNAs had a positive signal for both targets (fig 1). For reproducibility, the results showed consistent Ct values over the 3 days of the test with the four fresh tissue samples with a coefficient of variation (CV) of 4% (table 1). Conclusion This multiplex Real-time PCR assay successfully detected DNA from the S. brasiliensis and S. schenckii isolates as well as S. schenckii from fresh and FFPE tissues. Our results demonstrate this molecular assay performs well and could be a helpful molecular tool to support rapid identification and differentiation at species level from cultures and primary human and animal specimens. Disclosures All Authors: No reported disclosures

P-604. Preliminary real-world Abrysvo vaccine effectiveness (VE) against Respiratory Syncytial Virus (RSV)-related lower respiratory tract disease (LRTD) hospitalizations and emergency department (ED) visits—Kaiser Permanente of Southern California (KPSC), November 2023-April 2024

Abstract Background While the pivotal randomized clinical trial documented the efficacy of the RSVpreF vaccine (Abrysvo) against RSV-related LRTD, the limited number of hospitalizations and ED visits hampered VE assessment for these clinical outcomes. We evaluated Abrysvo’s effectiveness against 1st occurrence of RSV-related inpatient or ED LRTD in KPSC, a large US healthcare system.Table 1.Characteristics of study population, KPSC Methods In this test-negative case control study, we assessed VE among adults ≥60 years of age by analyzing KPSC members’ electronic health records and enhancing specimen testing from 11/24/2023-4/9/2024. Events from inpatient or ED settings meeting an ICD-10 code-based LRTD case definition with a respiratory specimen tested on GenMark RP2 expanded multiplex PCR assay were included. Cases were defined as RSV+ episodes. Analyses were conducted using two sets of pre-specified controls: 1) ‘Strict’: RSV- and positive for a non-vaccine preventable disease (VPD) etiology, and 2) ‘Broad’: all RSV-. The strict approach accounted for lower sensitivity of RSV testing among adults and potential bias associated with VPD controls. Exposure was defined as Abrysvo receipt ≥21 days before encounter. Adjusted odds ratios (OR) and 95% confidence intervals (CI) were estimated from a multivariable logistic regression model with adjustment for patient demographic and clinical characteristics. VE was calculated as 1−OR multiplied by 100%.Table 1.Characteristics of study population, KPSC, cont. Results The overall study population included 4,469 KPSC members: >50% were ≥75 years, 93% had ≥1 comorbidities, and 15% immunocompromised. In the strict analysis, there were 425 cases and 522 controls who received Abrysvo a median of 59 days before illness onset; VE compared to no vaccine receipt was 84% (95% CI: 29–97). In the broad analysis, there were 425 cases and 4,044 controls who received Abrysvo a median of 53 days before illness onset; adjusted VE was 82% (95% CI: 28–96).Table 2.Abrysvo Vaccine Effectiveness against RSV LRTD hospitalizations and ED visits, KPSC Conclusion This study demonstrated real world VE of Abrysvo during its first 5 months of use against severe LRTD in the hospital and ED settings among US adults ≥60 years of age. These results expand on efficacy results from clinical trial populations by including a substantial number of persons age 75+ years and with comorbidities and immunocompromising conditions. Disclosures Sara Y. Tartof, PhD MPH, GSK: Grant/Research Support|Pfizer Inc: Grant/Research Support Negar Aliabadi, MD, MS, Pfizer Inc: employment|Pfizer Inc: Stocks/Bonds (Public Company) Jeff Slezak, MS, Dynavax Technologies: Grant/Research Support|Pfizer: Grant/Research Support Vennis Hong, MPH, Pfizer: Grant/Research Support Bradley Ackerson, MD, Dynavax: Grant/Research Support|GlaxoSmithKline: Grant/Research Support|Moderna: Grant/Research Support|Pfizer: Grant/Research Support Qing Liu, M.S., Pfizer Inc.: Stocks/Bonds (Public Company) Sally Shaw, DrPH, MPH, Pfizer: Grant/Research Support Sabrina Welsh, MPH, Pfizer Inc: Stocks/Bonds (Public Company) Julie Stern, MPH, GlaxoSmithKline: Grant/Research Support|Pfizer: Grant/Research Support|Sanofi: Grant/Research Support Michael Dutro, PharmD, Pfizer: Stocks/Bonds (Public Company) Erica Chilson, PharmD, Pfizer Inc: Employee|Pfizer Inc: Stocks/Bonds (Public Company) Elisa Gonzalez, MS, Pfizer: Stocks/Bonds (Private Company) Robin Hubler, MS, Pfizer, Inc.: Employee of Pfizer Inc.|Pfizer, Inc.: Stocks/Bonds (Private Company) Luis Jodar, PhD, Pfizer Inc: Employment|Pfizer Inc: Stocks/Bonds (Public Company) Bradford D. Gessner, M.D., M.P.H., Pfizer: Employee|Pfizer: Stocks/Bonds (Public Company) Elizabeth Begier, MD, M.P.H., Pfizer Vaccines: Employee|Pfizer Vaccines: Stocks/Bonds (Private Company)

165. Real-world Abrysvo Vaccine Effectiveness (VE) against Respiratory Syncytial Virus (RSV)-related Severe Acute Respiratory Infection (ARI) Hospitalizations and Emergency Department (ED) Visits—Kaiser Permanente of Southern California (KPSC), November 2023–April 2024

Abstract Background Abrysvo’s pivotal trial analysis of efficacy against severe RSV-related respiratory disease was hampered by accrual of few hospitalizations/ED events. In this post-licensure study, we evaluated Abrysvo VE against 1st RSV-related severe ARI in KPSC, a large healthcare system. Methods We assessed Abrysvo VE among adults aged ≥ 60 years through KPSC members’ electronic health records plus enhanced specimen testing using a test-negative case-control study (study period: 11/24/2023–4/9/2024). ARI was defined as ED/hospitalizations with an ARI ICD-10 code, with respiratory specimens tested on GenMark RP2 expanded multiplex PCR assay; severe ARI required supplemental oxygen use. Cases were defined as RSV+ ARIs. Two sets of pre-specified controls were used: ‘Strict’: RSV- and positive for a non-vaccine preventable disease (VPD), and ‘Broad’: all RSV-. The strict group accounted for lower sensitivity of RSV testing in adults and potential bias with VPD controls. Exposure was Abrysvo receipt ≥ 21 days before encounter. Odds ratios (OR) and 95% confidence intervals (CI) were estimated from multivariable logistic regression adjusted for demographic and clinical characteristics. VE (1−OR X 100%) was estimated for severe ARI, ARI hospitalization/ED visit (separately and combined), and ARI among those with high-risk conditions. Results Among 5,120 KPSC members in the study population, > 50% were ≥ 75 years, 95% had ≥ 1 underlying comorbidities, 15% were immunocompromised. For RSV-related severe ARI (Table 1), the strict analysis included 372 cases and 496 controls; the vaccinated received Abrysvo a median of 69 days before the ARI. Adjusted VE was 91% (95% CI: 27–99). The broad analysis included 372 cases and 4,748 controls; the vaccinated received Abrysvo a median of 62.5 days before the ARI. Adjusted VE was 90% (95% CI: 26–99). Additional VE estimates ranged from 88–91% among different subsets of the ARI hospitalization/ED visit population (Table 2). Conclusion This study demonstrated real-world Abrysvo VE during its first 5 months of use against severe ARI in hospital/ED settings among US adults ≥ 60 years of age. These results expand on clinical trial results by including severe disease endpoints and a substantial proportion of high-risk persons. Disclosures Sara Y. Tartof, PhD MPH, GSK: Funding paid directly to institution; vaccines provided directly to institution|Pfizer Inc.: Funding paid directly to institution; vaccines provided directly to institution Negar Aliabadi, MD, MS, Pfizer: I am an employee of Pfizer and own stocks in the company Jeff Slezak, MS, Dynavax Technologies: Grant/Research Support|Pfizer, Inc.: Grant/Research Support Qing Liu, M.S., Pfizer Inc: Stocks/Bonds (Public Company) Michael Dutro, PharmD, Pfizer: Employee|Pfizer: Stocks/Bonds (Public Company) Erica Chilson, PharmD, Pfizer, Inc: Employee|Pfizer, Inc: Stocks/Bonds (Public Company) Elisa Gonzalez, MS, Pfizer: Stocks/Bonds (Private Company) Ashley Miller, MA, CCRP, Pfizer: Stocks/Bonds (Public Company) Bradford D. Gessner, M.D., M.P.H., Pfizer: Employee|Pfizer: Stocks/Bonds (Public Company) Elizabeth Begier, MD, M.P.H., Pfizer: Employee|Pfizer: Stocks/Bonds (Public Company)

Development and utilization of a multiplex PCR assay for detecting three feline enteroviruses.

Feline diarrhea is a common digestive tract disease in clinical practice, with watery feces as the main clinical manifestation. There are numerous pathogenic factors causing feline diarrhea, among which viral infections are prevalent, and feline panleukopenia virus (FPV) is the most common pathogen. In recent years, a variety of novel viruses have been detected in the intestines of cats with diarrhea. For example, feline kobuvirus (FKoV) and feline norovirus (FNoV) have been identified. These viruses may have a direct relationship with feline diarrhea or the connection has yet to be discovered. However, with the continuous emergence of these novel viruses and the frequent contact between pet cats and humans, it is prone to large-scale epidemics and outbreaks of viruses. Therefore, developing an accurate, rapid, and simple method to detect novel enteric viruses is of great significance for the early warning of emerging feline enteric viral infectious diseases. A detailed comparison of the genome sequences of the three aforementioned feline enteroviruses was conducted. Subsequently, three pairs of specific primers were designed by selecting the conserved gene regions, and the single and multiplex PCR amplification reaction systems as well as reaction conditions were repeatedly optimized. The target fragment sizes detected by the multiplex PCR method were 650bp for FPV, 500bp for FKoV, and 340bp for FNoV. Sensitivity tests demonstrated that the lower detection limit was one-tenth of that of single PCR. Meanwhile, the detection results for feline calicivirus (FCV), feline herpesvirus (FHV), and feline coronavirus (FCoV) were all negative. Testing of a total of 209 clinical samples from various regions in Shandong Province revealed that the detection rates of the three viruses were 13.4%, 4.8%, and 3.8%, respectively, and mixed infections were present. In this study, an epidemiological investigation of the three feline enteroviruses was performed, and a sensitive, specific, and reproducible multiplex PCR assay was developed, which can be utilized for the detection of clinical samples.

Detection of Francisellaceae and the differentiation of main European F. tularensis ssp. holarctica strains (Clades) by new designed qPCR assays

BackgroundThe zoonotic and highly infectious pathogen Francisella tularensis is the etiological agent of tularemia. Tularemia in humans is mainly caused by F. tularensis subspecies tularensis and holarctica, but Francisella species like F. novicida, F. philomiragia, F. hispaniensis and others are known to cause tularemia-like infections in immunocompromised humans. In addition to these Francisella species, further genera of the family Francisellaceae have been described, such as Allofrancisella, Parafrancisella and Pseudofrancisella, but less is known about the distribution and putative virulence of these genera. The methods currently available were not made for a fast and easy detection of all these strains and genera of Francisellaceae.ResultsWe developed a multiplex quantitative real-time PCR assay that can accurately detect all genera of Francisellaceae, including Francisella, Francisella-like endosymbionts, Allofrancisella, Parafrancisella and Pseudofrancisella. In addition, we developed a qPCR assay to differentiate the major clades (B.4, B.6 and B.12 [B.71 and B.72]) of F. tularensis ssp. holarctica strains. Both primer sets were shown to work on isolated DNA out of human and tick samples.ConclusionSince the developed qPCRs are able to detect all genera of Francisellaceae tested, an easy and fast identification of opportunistic Francisella strains causing tularemia-like symptoms in humans or animals is possible now. The application of these qPCR assays will thus improve the capability for clinical diagnostics and molecular typing during epidemiological investigations.

Targeted sputum sequencing for rapid and broad drug resistance of Mycobacterium tuberculosis.

Rapid detection of drug resistance in Mycobacterium tuberculosis (Mtb) from clinical samples facilitates the timely provision of optimal treatment regimens for tuberculosis (TB) patients. In November, 2023, the WHO released its second catalogue of resistance-conferring mutations in Mtb. Utilizing this information, we developed a single 17-plex PCR assay covering 16 key resistance genes and modified thermo-protection buffer to amplify 30 kbp DNA directly from sputum samples for nanopore sequencing. We implemented our protocol using rapid barcoding for sequencing with both a Flongle and a MinION flow cell. The single multiplex PCR assay was successfully validated on clinical sputum samples using the thermo-protection buffer. The protocol was applied to both Flongle and MinION flow cells, analyzing 12 and 40 samples, respectively. Data analysis suggested that optimal performance could be achieved by processing 6 and 12 samples with similar microscope staining scores on these two platforms. This approach facilitated rapid antimicrobial resistance (AMR) predictions directly from sputum on the day of collection or the following day, with a cost of less than $35 per sample. Compared to AMR predictions based on whole-genome sequencing (WGS) using Mykrobe and TBProfiler, our amplicon-based analysis tool, ARapidTb, demonstrated superior resistance detection capabilities. When analyzing publicly available nanopore WGS datasets for 442 isolates, ARapidTb achieved agreement rates of 95.8% and 98.0%, outperforming Mykrobe (89.4% and 98.3%) and TBProfiler (75.6% and 89.8%). Our study significantly reduces the time required for drug resistance detection, enabling quicker initiation of appropriate treatments and potentially improving patient outcomes and TB management.

Multiplex one-step RT‒qPCR assays for simultaneous detection of AMDV, MEV and CDV

BackgroundAleutian mink disease, mink viral enteritis and canine distemper are known as the three most serious diseases that cause great economic loss in the mink industry. In clinical practice, aleutian mink disease virus (AMDV), mink enteritis virus (MEV) and canine distemper virus (CDV) are common mixed infections, and they have similar clinical clinical signs, such as diarrhoea. Therefore, a rapid and accurate differential diagnosis method for use on mink ranches is essential for the control of these three pathogens. Here, we developed multiplex one-step real-time quantitative PCR (RT‒qPCR) assays for the simultaneous detection and quantification of AMDV, MEV and CDV by using three primers and probes based on the conserved NS1, VP2 and N genes, respectively.ResultsThe results showed that the established method can not cross-react with other mink pathogens, with a detection sensitivity of 25 copies/µL and a coefficient of variation less than 3.51%. Moreover, the interference experiment showed that the presence of AMDV, MEV and CDV templates at different concentrations would not interfere with the detection results. Furthermore, two hundred clinical samples of mink with diarrhoea were simultaneously analysed using multiplex RT‒qPCR and single RT‒qPCR, the Kappa values were all greater than 0.921, indicating that there was a high degree of coincidence between the two detection methods.ConclusionsIn conclusion, multiplex RT‒qPCR exhibited high specificity, sensitivity, and reproducibility, indicating that this method can be used as a reliable and specific tool for the differential detection and quantification of AMDV, MEV and CDV.

Status of IKZF1 Deletions in Diagnose and Relapsed Pediatric B-ALL Patients.

IKZF1 deletions (ΔIKZF1) are common in precursor B-cell acute lymphoblastic leukemia (B-ALL) and are assumed to have a prognostic impact. We aimed to determine the prognostic implications ofΔIKZF1 and CRLF2 overexpression in pediatric B-ALL. Furthermore, we sought to compare the multiplex polymerase chain reaction (PCR) assay with standard multiplex ligand-dependent probe amplification (MLPA) methods to ascertain IKZF1 status in a clinical context. Seventy-nine diagnoses and 43 relapse B-ALL samples were evaluated for deletions of IKZF1 Δ2-7, Δ4-7, and Δ4-8 by conventional PCR and then sequenced by targeted sequencing. Subsequently, MLPA analysis was performed for ΔIKZF1 detection, and CRLF2 expression was evaluated in 42 diagnose time B-ALL patients by QRT-PCR. ΔIKZF1 was detected in 10 out of 79 diagnose samples (12.66%) and eight of the 43 first relapsed materials (18.60%). Our results revealed no association between survival outcomes with ΔIKZF1 or CRLF2 overexpression status in pediatric B-ALL patients. However, we found ΔIKZF1was more frequent among relapsed samples, and the deletions showed consistency between diagnose-first/second relapse pairs of samples. These results suggest that ΔIKZF1 may contribute to the development of treatment failure in B-ALL. Furthermore, we demonstrated methodological adjustments in conventional PCR and MLPA for selected alterations in ΔIKZF1.

A Multiplex Polymerase Chain Reaction Assay for the Detection of Herpes Simplex Virus, Cytomegalovirus, and Varicella-Zoster Virus in Cerebrospinal Fluid.

Viral meningitis poses a significant clinical challenge due to its rapid onset and potential progression to life-threatening encephalitis. Early detection of treatable viral pathogens such as Herpes simplex virus (HSV), Cytomegalovirus (CMV), and Varicella-zoster virus (VZV) is essential for initiating appropriate therapies. However, multiplex PCRs for the rapid and simultaneous detection of these pathogens are scarce due to the complex PCR design and the elaborate validation process using cerebrospinal fluid samples. In this study, we established and validated a novel multiplex PCR assay for detecting HSV, CMV, and VZV in cerebrospinal fluid samples and implemented the assay on a fully automated platform.

A novel approach for detecting Salmonella enterica strains frequently attributed to human illness-development and validation of the highly pathogenic Salmonella (HPS) multiplex PCR assay.

Non-typhoidal Salmonella enterica (NTS) are leading bacterial agents of foodborne illnesses and a global concern for human health. While there are over 2,600 different serovars of NTS, epidemiological data suggests that certain serovars are better at causing disease than others, resulting in the majority of reported human illnesses in the United States. To improve food safety, there is a need to rapidly detect these more pathogenic serovars to facilitate their removal from the food supply. Addressing this need, we conducted a comparative analysis of 23 closed Salmonella genomic sequences of five serotypes. The analysis pinpointed eight genes (sseK2, sseK3, gtgA/gogA, avrA, lpfB, SspH2, spvD, and invA) that in combination, identify 7 of the 10 leading Salmonella serovars attributed to human illnesses in the US each year (i.e., Serovars of Concern or SoC). A multiplex PCR assay was developed to detect the presence of these genes, with strains amplifying five or more targets designated Highly Pathogenic Salmonella, or HPS. The utility of the resulting HPS assay for identifying SoC was examined in silico, using BLAST to determine the distribution of gene targets among closed Salmonella genome sequences in GenBank (n = 2,192 representing 148 serotypes) and by assaying 1,303 Salmonella (69 serotypes), isolated from FSIS regulatory samples. Comparison of serotypes identified by the assay as HPS, with those identified as SoC, produced an Area Under the Curve (AUC) of 92.2% with a specificity of 96% and a positive predictive value of 97.4%, indicating the HPS assay has strong ability to identify SoC. The data presented lay the groundwork for development of rapid commercial assays for the detection of SoC.

Development and Validation of Multiplex-PCR Assay for β-Carotene hydroxylase and γ-Tocopherol methyl transferase Genes Governing Enhanced Multivitamins in Maize for Its Application in Genomics-Assisted Breeding.

Traditional maize possesses low concentrations of provitamin-A and vitamin-E, leading to various health concerns. Mutant alleles of crtRB1 and vte4 that enhance β-carotene (provitamin-A) and α-tocopherol (vitamin-E), respectively, in maize kernels have been explored in several biofortification programs. For genetic improvement of these target nutrients, uniplex-PCR assays are routinely used in marker-assisted selection. However, due to back-to-back breeding seasons, the time required for genotyping individually for each target gene in large backcross populations becomes a constraint for advancing the generations. Additionally, multiple PCR assays for various genes increase the required costs and resources. Here, we aimed to develop a multiplex-PCR assay to simultaneously identify different allelic forms of crtRB1 and vte4 genes and validate them in a backcross-based segregating population. The PCR assay was carried out using newly developed primers for crtRB1 and a gene-specific primer for vte4. The uniplex-PCR assay was standardized for selected primer pairs in the BC1F1 population segregating for crtRB1 and vte4 genes. Subsequently, a multiplex-PCR assay for crtRB1 and vte4 genes was developed and employed for genotyping in the BC1F1 population. The assay differentiated among four possible genotypic classes, namely crtRB1+crtRB1/vte4+vte4, crtRB1crtRB1/vte4+vte4, crtRB1+crtRB1/vte4+vte4+, and crtRB1crtRB1/vte4+vte4+. This newly developed multiplex-PCR assay saved 41.7% of the cost and 35.6% of the time compared to two individual uniplex-PCR assays. The developed assay could accelerate maize nutritional quality breeding programs through rapid and cost-effective genotyping for the target genes. This is the first report of a multiplex-PCR assay specific to crtRB1 and vte4 genes for its use in genomics-assisted breeding in maize.

Evaluation of the efficacy of herbal (neem) and chemical (chlorhexidine) chips as local drug delivery agents in the treatment of chronic periodontitis: A clinical and microbiological study

Context: Herbal remedies can be effective in controlling periodontal disease. Neem is gaining attention as a reliable alternative to pharmaceuticals. Aims: The purpose of this study was to compare the efficacy of chlorhexidine (CHX) and neem chips (NCs) when used with scaling and root planing (SRP). Settings and Design: The study design involves interventional prospective study. Methods: A total of 45 periodontal pockets were divided into three groups of 15 sites each. Included both males and females of age group 35–55 years. Group I–SRP alone, Group II–SRP and CHXC, and Group III–SRP and NC. Parameters evaluated were plaque index (PI), gingival index (GI), probing pocket depth (PPD), and clinical attachment level (CAL). A qualitative multiplex polymerase chain reaction assay aided in detecting red complex organisms. Statistical Analysis Used: Evaluated at baseline, 10th day, and 1 month. Kruskal–Wallis test and Mann–Whitney U-test were used for the analysis. Results: There was a noticeable variation in the PPD at the start between the SRP, NC, and CHX groups. However, no significant differences were observed in other aspects such as the PI, GI, CAL, and the presence of Porphyromonas, Treponema, and Tannerella bacteria at the baseline, 10th day, and 30th day. Conclusions: All groups improved clinically, but no statistical difference was found in all clinical and microbiological parameters from baseline to day 30. Trial registration: https://ctri.nic.in/Clinicaltrials/login.php.Date registered 21/10/2020.reg no.CTRI/2020/10/028532.