Rapid Molecular Assay Research Articles

Abstract 2098: Molecular based blood screening assay for metastatic adenoid cystic carcinoma

Abstract Adenoid cystic carcinoma (ACC) is a rare, yet highly lethal, tumor that originates in the excretory glands of the head and neck. It is a locally aggressive disease, with more than half of patients presenting with perineural invasion and/or bone erosion. The current standard of care consists of surgical excision followed by radiation. Many healthy patients are left with positive margins after surgery and receive systemic therapy in addition to adjuvant radiation without evidence showing added benefit for that approach. Despite extensive surgery, long-term survival rates remain low due to a high incidence of locoregional disease progression and metastatic disease to the lung, liver, brain, or bones, sometimes several years after definitive treatment. Extensive genome analysis of ACC has uncovered a high mutation rate of the proto-oncogene MYB, typically as a genomic translocation resulting in an MYB fusion event with one of the following gene loci: nuclear factor I/B (NFIB), transforming growth factor beta 3 (TGFBR3) or RAD51B. The translocation approximates an enhancer to the MYB regulatory element which can be transactivated by the MYB fusion product, thus leading to the overexpression of MYB via a positive feedback mechanism. Currently, detection of systemic disease relies on diagnostic imaging, which can be imprecise, costly and time-consuming. Years of routine imaging is very impractical, attrition rates are high, and patients often present years later with symptomatic metastatic disease. A rapid and sensitive molecular-based blood screening tool would allow for continuous monitoring and earlier detection of metastatic ACC, ideally improving survival outcomes for these patients. In this study, we hypothesized that the dysregulation in MYB, and the reliance of ACC cells on MYB for survival and progression could be harnessed to develop a molecular screening tool to monitor for metastatic disease of ACC. We developed a blood-based PCR screening probes for metastatic disease in patients with a history of ACC. By analyzing blood samples from four cohorts: healthy individuals (no history of cancer) (n=6), patients with a history of ACC but with no evidence of disease for longer than 3yrs (NED) (n=16), patients with newly diagnosed local disease (n=8), and patients with confirmed metastatic disease (n=7), we demonstrate that our assay was able to detect significantly elevated levels of aberrant MYB expression in all blood specimens from our metastatic disease cohort (p < 0.01). Additionally, our probes were able to detect aberrant levels of MYB in the blood of a patient 11 months before imaging techniques identified metastatic lung lesions. These findings indicate that we can harness a specific ACC genetic trait to develop a sensitive, rapid, and inexpensive molecular screening assay from blood to detect metastatic ACC. We continue to expand and validate these findings by screening existing and new ACC patients every 6 months. Citation Format: Acadia H. Moeyersoms, Ryan A. Gallo, Zoukaa B. Sargi, Jason M. Leibowitz, Andrew J. Rong, David T. Tse, Daniel Pelaez. Molecular based blood screening assay for metastatic adenoid cystic carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2098.

Drug-Resistant Tuberculosis on the Balkan Peninsula: Determination of Drug Resistance Mechanisms with Xpert MTB/XDR and Whole-Genome Sequencing Analysis.

The new molecular assay Xpert MTB/XDR (Cepheid, Sunnyvale, CA, USA) was launched in 2021 to detect Mycobacterium tuberculosis (MT) complex with mutations conferring resistance to isoniazid (INH), ethionamide (ETH), fluoroquinolone (FQ), and second-line injectable drugs (SLIDs). The aim of our study was to evaluate the performance of the Xpert MTB/XDR rapid molecular assay on rifampicin-resistant, multidrug-resistant, and pre-extensively resistant tuberculosis (TB) isolates in a clinical laboratory in the Balkan Peninsula compared to a phenotypic drug susceptibility test (pDST). Xpert MTB/XDR was used to test positive Bactec MGIT 960 (Becton, Dickinson and Co., Franklin Lakes, NJ, USA) cultures or DNA isolates. In the case of discrepant results between Xpert MTB/XDR and pDST, the usefulness of whole-genome sequencing (WGS) was emphasized. In our study, 80 MT isolates from different Balkan countries were selectively chosen from the National Mycobacterial Strain Collection in Golnik, Slovenia. Isolates were tested with the Xpert MTB/XDR assay, conventional pDST, and WGS. Xpert MTB/XDR showed high sensitivities of 91.9%, 100%, and 100% for detecting INH, FQ, and SLID resistance, respectively, compared to pDST. In contrast, low sensitivity (51.9%) for ETH resistance was achieved because isolates harbored widespread mutations across the ethA gene. The specificity of Xpert MTB/XDR was 100% for all drugs except for INH (66.7%). Further investigation with WGS revealed -57c→t mutations in the oxyR-ahpC region marked with uncertain significance, which caused the low specificity for detecting INH resistance with the new assay. Xpert MTB/XDR can be used in clinical laboratories for the rapid detection of INH, FQ, and SLID resistance. Moreover, it can be used to rule in resistance to ETH. Additional use of WGS is recommended in cases of discrepant results between pDST and Xpert MTB/XDR. Future improvements of Xpert MTB/XDR with the inclusion of additional genes may increase the usefulness of the assay. IMPORTANCE The Xpert MTB/XDR was tested on drug-resistant Mycobacterium tuberculosis complex isolates from the Balkan Peninsula. Positive Bactec MGIT 960 cultures or DNA isolates were tested as starting material. According to the results of our study with Xpert MTB/XDR, sensitivities for the detection of SLID, FQ, and INH resistance were sufficient (>90%) for the assay to be implemented into diagnostic algorithms. In our study, WGS revealed lesser-known mutations in genes conferring INH and ETH resistance, and their impact on resistance is still unknown. Mutations in the ethA gene causing resistance to ETH were scattered along structural gene without high-confidence markers for resistance. Therefore, resistance to ETH should be reported based on a combination of methods. Because the Xpert MTB/XDR assay was found to have good performance, we propose that it should be the method of choice for confirming resistance to INH, FQ, and SLID and conditionally for resistance to ETH.

A Colorimetric LAMP Detection of Xylella fastidiosa in Crude Alkaline Sap of Olive Trees in Apulia as a Field-Based Tool for Disease Containment

Xylella fastidiosa subsp. pauca (Xfp) infects olive trees and other hosts in Southern Apulia (Italy), devastating agriculture and landscape. A containment strategy of the disease requires quick and sensitive detection tools. Therefore, a colorimetric LAMP protocol was developed using as a template a crude alkaline sap obtained from incubation of 50–60 mg of thin slices of olive twigs in a NaOH-containing buffer. This rapid molecular assay can be performed directly in the field, as it needs only a portable isothermal block. Tissues of the same olive trees analysed by this technique were also compared to qPCR (using purified total plant DNA as template) as well as digital droplet PCR (on the same crude alkaline extracts used in cLAMP). A titration of the cLAMP reaction with healthy olive sap, spiked with dilutions of in vitro cultivated Xfp cells and plasmid DNA containing the target sequence, gave positive detection results as low as 102 CFU/mL and up to 169.2 target copies/µL, equivalent to about 0.9 pg of the genomic DNA. A portable, sensitive and target-specific Xfp field test was developed, which has a 40 min sample-to-answer time and does not require any DNA isolation procedure or laboratory equipment. The application of this detection assay could help the monitoring and containment of the disease spread.

Development of a simple, rapid, and sensitive molecular diagnostic assay for cholera

Cholera continues to inflict high rates of morbidity and mortality. Prompt identification of cholera cases facilitates rapid outbreak responses in the short term while providing reliable surveillance data to guide long-term policies and interventions. Microbiological stool culture, the current recognized gold standard for diagnosing cholera, has significant limitations. Rapid diagnostic tests (RDTs) represent promising alternatives for diagnosing cholera in areas with limited laboratory infrastructure. However, studies conducted with the current cholera RDTs demonstrated wide variations in sensitivity and specificity. To address this gap in the diagnosis of cholera, we developed a simple, rapid, and sensitive diagnostic assay, "Rapid LAMP based Diagnostic Test (RLDT)." With a novel, simple sample preparation method directly from the fecal samples along with lyophilized reaction strips and using established Loop-mediated Isothermal Amplification (LAMP) platform, cholera toxin gene (ctxA) and O1 (O1rfb) gene could be detected in less than an hour. Cholera RLDT assay is cold chain and electricity-free. To avoid any end-user bias, a battery-operated, handheld reader was used to read the RLDT results. The performance specifications of the cholera RLDT assay, including analytical sensitivity and specificity, were evaluated using direct fecal samples, dried fecal samples on filter paper, and environmental water samples spiked with cholera strain. The limit of detection (LOD) was ~104 CFU/gm of stool for both ctxA and O1 genes, corresponding to about 1 CFU of Vibrio cholerae per reaction within 40 minutes. The LOD was 10 bacteria per ml of environmental water when tested with RLDT directly, without enrichment. Being simple, RLDT has the potential to be applied in resource-poor endemic settings for rapid, sensitive, and reliable diagnosis of cholera.

Consequences of rpoB mutations missed by the GenoType MTBDRplus assay in a programmatic setting in South Africa.

Rifampicin resistance missed by commercial rapid molecular assays but detected by phenotypic assays may lead to discordant susceptibility results and affect patient management. This study was conducted to evaluate the causes of rifampicin resistance missed by the GenoType MTBDRplus and its impact on the programmatic management of tuberculosis in KwaZulu-Natal, South Africa. We analysed routine tuberculosis programme data from January 2014 to December 2014 on isolates showing rifampicin susceptibility on the GenoType MTBDRplus assay but resistance on the phenotypic agar proportion method. Whole-genome sequencing was performed on a subset of these isolates. Out of 505 patients with isoniazid mono-resistant tuberculosis on the MTBDRplus, 145 (28.7%) isolates showed both isoniazid and rifampicin resistance on the phenotypic assay. The mean time from MTBDRplus results to initiation of drug-resistant tuberculosis therapy was 93.7 days. 65.7% of the patients had received previous tuberculosis treatment. The most common mutations detected in the 36 sequenced isolates were I491F (16; 44.4%) and L452P (12; 33.3%). Among the 36 isolates, resistance to other anti-tuberculosis drugs was 69.4% for pyrazinamide, 83.3% for ethambutol, 69.4% for streptomycin, and 50% for ethionamide. Missed rifampicin resistance was mostly due to the I491F mutation located outside the MTBDRplus detection area and the L452P mutation, which was not included in the initial version 2 of the MTBDRplus. This led to substantial delays in the initiation of appropriate therapy. The previous tuberculosis treatment history and the high level of resistance to other anti-tuberculosis drugs suggest an accumulation of resistance.

Validation of the efficacy of air purifiers using molecular techniques.

The importance of air purifiers has increased in recent years, especially with the "coronavirus disease 2019" pandemic. The efficacy of air purifiers is usually determined under laboratory conditions before widespread application. The standard procedure for testing depends on virus cultivation and titration on cell culture. This, however, requires several days to deliver results. The aim of this study was to establish a rapid molecular assay which can differentiate between intact infectious and distorted non-infectious virus particles. Feline Coronavirus was selected as model for screening. First the samples were pretreated with enzymes (universal nuclease and RNase cocktail enzyme mixture) or viability dye (propidium monoazide) to eliminate any free nucleic acids. The ribonucleic acid (RNA) from intact virus was released via magnetic beads-based extraction, then the amount of the RNA was determined using real-time reverse transcription polymerase chain reaction (RT-PCR) or reverse transcription recombinase-aided amplification (RT-RAA). All results were compared to the infectivity assay based on the calculation of the 50% tissue culture infectious dose (TCID50). The nuclease has eliminated 100% of the free Feline Coronavirus RNA, while propidium monoazide underperformed (2.3-fold decrease in free RNA). Both RT-RAA and real-time RT-PCR produced similar results to the infectivity assay on cell culture with limit of detection of 102 TCID50/mL. Two UV-C air purifiers with prosperities of 100% inactivation of the viruses were used to validate the established procedure. Both real-time RT-PCR and RT-RAA were able to differentiate between intact virus particles and free RNA. To conclude, this study revealed a promising rapid method to validate the efficacy of air purifiers by combining enzymatic pretreatment and molecular assays.

Utility of the BioFire® FilmArray® Pneumonia Panel plus assay for syndromic testing of lower respiratory tract infections in a low/middle-income setting.

Determining lower respiratory tract infection (LRTI) aetiology is complex. Culture-based methods are laborious with poor sensitivity. Molecular assays improve detection of potential pathogens, but incorrect interpretation of results may lead to inappropriate antimicrobial therapy. The utility of the BioFire® FilmArray® Pneumonia Panel plus (FA-PP) to detect LRTI pathogens, and the potential impact on antimicrobial stewardship in a low-resource setting, were assessed. Routine LRT samples were included from adult patients with clinically suspected LRTI or with a concomitant blood culture at Groote Schuur Hospital and referring facilities. Culture and FA-PP results were compared, and pharmacy data analysed to determine appropriateness of antibiotic therapy. There was an 80% correlation between cultured LRTI pathogens and the FA-PP bin ≥107 results. Compared with culture, the FA-PP detected substantially more pathogens (86.6% versus 17.9%) and produced a combined 100% positive percent agreement, and 88% negative percent agreement. The FA-PP detected bacterial/viral coinfections in 27% of samples. Correlation of FA-PP results with pharmacy data (n = 69) indicated a potential antibiotic change in 75% of cases, but this is difficult to accurately characterize without a 'gold standard' for treatment or complete clinical data. The FA-PP increased the number of positive samples with typical bacteria, but the semi-quantitative reporting algorithm does not describe the correlation between the different bin values and colonization versus infection. This complicates result interpretation and may lead to inappropriate antimicrobial treatment. This study highlights the potential positive impact of rapid molecular assays for routine care in lower-income settings, but also underscores the interpretive challenges associated with these tests.

Rapid molecular assays for the detection of the four dengue viruses in infected mosquitoes.

The pantropic emergence of severe dengue disease can partly be attributed to the co-circulation of different dengue viruses (DENVs) in the same geographical location. Effective monitoring for circulation of each of the four DENVs is critical to inform disease mitigation strategies. In low resource settings, this can be effectively achieved by utilizing inexpensive, rapid, sensitive and specific assays to detect viruses in mosquito populations. In this study, we developed four rapid DENV tests with direct applicability for low-resource virus surveillance in mosquitoes. The test protocols utilize a novel sample preparation step, a single-temperature isothermal amplification, and a simple lateral flow detection. Analytical sensitivity testing demonstrated tests could detect down to 1,000 copies/µL of virus-specific DENV RNA, and analytical specificity testing indicated tests were highly specific for their respective virus, and did not detect closely related flaviviruses. All four DENV tests showed excellent diagnostic specificity and sensitivity when used for detection of both individually infected mosquitoes and infected mosquitoes in pools of uninfected mosquitoes. With individually infected mosquitoes, the rapid DENV-1, -2 and -3 tests showed 100% diagnostic sensitivity (95% CI = 69% to 100%, n=8 for DENV-1; n=10 for DENV 2,3) and the DENV-4 test showed 92% diagnostic sensitivity (CI: 62% to 100%, n=12) along with 100% diagnostic specificity (CI: 48-100%) for all four tests. Testing infected mosquito pools, the rapid DENV-2, -3 and -4 tests showed 100% diagnostic sensitivity (95% CI = 69% to 100%, n=10) and the DENV-1 test showed 90% diagnostic sensitivity (55.50% to 99.75%, n=10) together with 100% diagnostic specificity (CI: 48-100%). Our tests reduce the operational time required to perform mosquito infection status surveillance testing from > two hours to only 35 minutes, and have potential to improve accessibility of mosquito screening, improving monitoring and control strategies in low-income countries most affected by dengue outbreaks.

172. Does Passive Reporting of Streptococcus spp Identified by Rapid Molecular Assay Change Antibacterial Prescribing Compared to Gram Stain (GS) Alone?

Abstract Background At University of Virginia Health, the detection of Streptococcus spp via rapid molecular assay from blood cultures (BCx) was not historically reported in the EMR until the isolate was further identified. However this was reconsidered following anecdotal observation of inappropriate vancomycin (VAN) prescribing with GS alone. The purpose of this study was to investigate the impact of implementing passive reporting of Streptococcus spp on antibacterial prescribing patterns with a focus on VAN. Methods This was a single-center, pre-post quasi-experimental study of adult inpatients with non-duplicate Streptococcus spp identified via rapid molecular assay (ePlex®; GenMark Diagnostics) from BCx before (pre-intervention, Jul 2020 – Sep 2021) and after (post-intervention, Oct 2021 - Apr 2022) implementing passive reporting of Streptococcus spp. The primary outcome was any antibacterial switch within 24 hours of GS. VAN days of therapy (DOT) within 7 days and rates of Streptococcus spp deemed contamination by the primary team were also assessed. Results A total of 83 patients were included (58 pre- and 25 post-intervention). Baseline characteristics were similar including immunocompromised status (45% vs 52%), febrile neutropenia (28% vs 28%), and admission to a medical service (90% vs 96%). An antibacterial switch occurred within 24 hours of GS in 59% and 64% of the pre- and post-intervention groups, respectively, and was most commonly the addition of VAN (66% [25/38] vs 40% [8/20]; p=0.09). Fewer post-intervention patients continued VAN beyond 24 hours (66% vs 48%; p=0.15), but this was not statistically significant. The median VAN DOT remained unchanged at 2 days. A non-significant decrease in Streptococcus spp deemed contaminants was noted in the post-intervention arm (28% vs 16%; p=0.16). Finally, a non-streptococcal organism was isolated from concomitant BCx in approximately one-third of patients in each arm. Conclusion Passively reporting Streptococcus spp identified by rapid molecular assay without paired stewardship intervention did not impact antibacterial prescribing. The identification of Streptococcus spp may be characterized by heterogeneous circumstances requiring nuanced real-time stewardship intervention. Disclosures All Authors: No reported disclosures.

2188. A Quality Improvement Initiative for Influenza Diagnosis and Treatment within Health Systems Serving American Indian/Alaskan Native Patients

Abstract Background This project aimed to identify practices, knowledge and confidence in diagnosing and treating influenza in health systems serving American Indian/Alaskan Native communities and to provide education to support alignment with IDSA guidelines. Methods Anonymous baseline surveys were completed electronically by healthcare professionals (HCPs) from Cherokee Nation Health Services (CNHS) and the Northwest Portland Area Indian Health Board’s teleECHO program. Responses informed a 2-part series of live virtual educational trainings featuring pre- and post-program surveys and team-based action planning. Results Baseline surveys were completed by 247 HCPs. 149 attended the 1st meeting, and 90 attended the 2nd. For outpatient influenza diagnosis, 72% of baseline survey respondents reported primarily using rapid antigen testing, and 11% reported using molecular testing. Similarly, 15% of learners in the live meetings correctly identified rapid molecular assay as the guideline-recommended test, which improved to 72% afterwards. All 9 CNHS outpatient health centers switched from rapid antigen to molecular testing for influenza diagnosis after the training. Influenza treatment decision-making was assessed using a case vignette. For a patient at high risk for complications with flu-like symptoms but stable for outpatient care, 23% of learners in the live meetings correctly chose to start antiviral treatment immediately, which improved to 54% after the program, and more learners felt confident applying diagnostic test results to treatment, from 31% before to 68% after the training. Learner confidence also improved from 19% feeling confident in selecting antiviral treatment and 33% feeling confident in identifying high risk patients before the program to 60% and 70% afterwards, respectively. Action plans prioritized by the HCPs included: patient engagement (61%), reviewing guidelines and data (57%), improving patient adherence to treatment (50%), and developing criteria for immediate antiviral treatment (50%). Conclusion Knowledge and confidence in influenza management improved after the training, as did system-wide alignment with IDSA-recommended diagnostic testing. Studies evaluating the clinical impact of this activity is warranted. Disclosures Jorge Mera, MD, FACP, Abbvie: Grant/Research Support|Gilead Sciences: Grant/Research Support|Gilead Sciences: Received one time honorarium for participating as a panelist in a COVID-19 Webinar|Merck: Grant/Research Support.

Pediatric Tuberculosis Diagnostics: Present and Future.

The current diagnostic abilities for the detection of pediatric tuberculosis are suboptimal. Multiple factors contribute to the under-diagnosis of intrathoracic tuberculosis in children, namely the absence of pathognomonic features of the disease, low bacillary loads in respiratory specimens, challenges in sample collection, and inadequate access to diagnostic tools in high-burden settings. Nonetheless, the 2020s have witnessed encouraging progress in the area of novel diagnostics. Recent WHO-endorsed rapid molecular assays hold promise for use in service decentralization strategies, and new policy recommendations include stools as an alternative, child-friendly specimen for testing with the GeneXpert assay. The pipeline of promising assays in mid/late-stage development is expanding, and novel pediatric candidate biomarkers based on the host immune response are being identified for use in diagnostic and triage tests. For a new test to meet the pediatric target product profiles prioritized by the WHO, it is key that the peculiarities and needs of the hard-to-reach pediatric population are considered in the early planning phases of discovery, validation, and implementation studies.

Comparative Performance of Line Probe Assay and GeneXpert in the Detection of Rifampicin Monoresistance in a TB-Endemic African Country.

Rapid, accurate and reliable assays are required for timely detection of drug-resistant tuberculosis and early initiation of second-line TB treatment as well as to minimize transmission of resistant strains. This study assessed diagnostic performance characteristics of two rapid molecular assays, line probe assay (LPA) and GeneXpert (MTB/RIF), in the detection rifampicin monoresistance using the phenotypic proportion method on Lowenstein-Jensen media as the gold standard. This study involved a total of 357 isolates, 74 rifampicin-resistant and 283 rifampicin-susceptible, collected at the Central Tuberculosis Reference Laboratory (CTRL) in Dar es Salaam, Tanzania, between 2016 and 2019. Sensitivity, specificity and positive and negative predictive values were used to assess the performance characteristics of the two assays while kappa coefficient was used to determine agreement of test results. The receiver operating curve (ROC) was used to determine the discriminatory ability of the test in distinguishing resistant and susceptible TB isolates. Our results showed that GeneXpert had sensitivity, specificity and positive and negative predictive values of 93.2, 82.7, 58.5 and 97.9%, respectively; the corresponding performance for LPA was 86.5, 97.5, 90.1 and 96.5%, respectively. Compared with conventional phenotypic DST results, GeneXpert had a moderate agreement (kappa 0.621, p < 0.001), while LPA had high agreement (0.853, p < 0.001). LPA showed an accuracy of 95.2% compared to GeneXpert's 84.9%. ROC curve depicted the ability of the tests to distinguish rifampicin-sensitive and rifampicin-resistant strains to be 87.9% for GeneXpert and 92.0% for LPA. Our results indicate the superiority of LPA over GeneXpert regarding detection of rifampicin monoresistance. However, logistic challenges such as longer turnaround time and need for skilled laboratory personnel may limit its use.

Accessible and Adaptable Multiplexed Real-Time PCR Approaches to Identify SARS-CoV-2 Variants of Concern.

ABSTRACTRapid identification and continuous surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants are critical for guiding the response to the COVID-19 pandemic. Whole-genome sequencing (WGS) is a preferred tool for this aim, but many laboratories suffer from a lack of resources to support population-level sequencing. Here, we describe two PCR strategies targeting spike protein mutations to identify the Alpha, Delta, and Omicron variants. Signature mutations were selected using a dedicated bioinformatic program. The selected mutations in Alpha and Delta variants were detected using multicolor melting curve analysis (MMCA). Thirty-two mutations of the Omicron variant were targeted using the MeltArray approach in one reaction, which was able to detect the Omicron subvariants BA.1, BA.2, BA.3, and BA.4/5. The limits of detection varied from five to 50 copies of RNA templates/reactions. No cross-reactivity was observed with nine other respiratory viruses, including other coronaviruses. We validated the MMCA and MeltArray assays using 309 SARS-CoV-2 positive samples collected at different time points. These assays exhibited 98.3% to 100% sensitivity and 100% specificity compared with WGS. Multiplexed real-time PCR strategies represent an alternative tool capable of identifying current SARS-CoV-2 VOCs, adaptable for emerging variants and accessible for laboratories using existing equipment and personnel.IMPORTANCE Rapid detection and mutation surveillance of SARS-CoV-2 VOCs is crucial for COVID-19 control, management, and prevention. We developed two rapid molecular assays based on the real-time PCR platform to identify important variants of concern, including the Omicron variant with a large number of mutations. Signature mutations were selected by an R program. Then, MMCA assays were established for Alpha and Delta variants, and a MeltArray assay targeting 32 mutations was developed for Omicron variant. These multiplexed PCR assays could be performed in a 96-well real-time PCR instrument within 2.5 h, offering a high-throughput choice for dynamic monitoring of SARS-CoV-2 VOCs in a standard microbiology laboratory.

Passengers screening for SARS-CoV-2 infection at Rome Fiumicino Airport: a strategy aimed at limiting the spreading of the infection through COVID-free air flights revealed the airport role as a formidable sentinel center for monitoring pandemic trends and viral variants circulation

Background and aims: At the Fiumicino Rome Airport, passengers can undergo the antigen or molecular test for SARS-CoV-2 at the COVID-19 Test Area (CTA), in compliance with the boarding regulations on air flights and those for entering the destination countries. The aim of this study was to describe the adaptability and usefulness of using rapid molecular tests designed for point of care (POC) in a context such as the CTA of Rome Fiumicino airport, by describing the volume and activities performed in the last period of the state of emergency in Italy, July 10, 2021- March 31, 2022.
 
 Materials and methods: Rapid molecular tests for SARS-CoV-2 were carried out with ID NOWTM COVID-19 assay on the ID NOWTM platform using dry swabs collected from passengers (or airport staff) at the CTA. Swabs were stored at room temperature and analyzed within 1-2 hours of collection. Sequencing for SARS-CoV-2 variants of concern (VOC) identification was carried out on swabs dipped in universal transport medium (UTM), after new swab gathering from consenting passengers resulted positive with the rapid molecular assay. A proficiency panel prepared with the viral isolate 2019-nCoV/Italy INMI1 was used to evaluate analytical sensitivity; clinical sensitivity was tested by performing real-time polymerase chain reaction (RT-PCR) tests on 50 swabs obtained from passengers who previously resulted positive to ID NOWTM COVID-19 assay.
 
 Results: In total, 14632 rapid molecular tests for SARS-CoV-2 were performed at CTA and 5.6% resulted positive. Sequence analysis of samples with Ct value <25 (61,2 %) with real-time RT-PCR test allowed detection of all the main VOCs: B.1.617.2 (Delta) in 18% of samples, BA.1 (Omicron-1) in 34% of positive swabs and BA.2 (Omicron-2) lineage in 9% of samples. The BA.2 variant was detected in the Lazio region for the first time at CTA, in one sample obtained from an Italian passenger coming from Doha, Qatar. A proficiency panel for SARS-CoV-2 detection was used to establish the limit of detection of ID NOWTM COVID-19 assay. For clinical sensitivity evaluation, 50 subjects with positive results by ID NOWTM COVID-19 assay were tested with routine real-time RT-PCR method in the laboratory and confirmed for SARS-CoV-2 detection.
 
 Conclusions: The great success among passengers, demonstrated by the large number of rapid molecular tests performed, confirmed the appreciation of this service by the users. Our experience at CTA showed that airports, railways and maritime stations should represent strategic surveillance centers for monitoring of circulation of respiratory pathogens, and may have a direct impact on the implementation of prevention and control strategies for public health against the spreading of infectious diseases. Indeed, SARS-CoV-2 data obtained at the CTA faithfully reflected the Italian epidemic curve trend of SARS-CoV-2 and its variants. The rapid molecular ID NOWTM COVID-19 assay used at CTA has proven to be suitable for use in a special context, such as the Rome Fiumicino Airport, though it was designed for use in POC. Due to the simplicity of use, robustness of method and technology that allows the return of results in a few minutes, this system revealed itself to be an optimal tool at the CTA, ensuring passengers boarding on COVID-free flights in a short time and security.

Roadmap to incorporating group A Streptococcus molecular point-of-care testing for remote Australia: a key activity to eliminate rheumatic heart disease.

Roadmap to incorporating group A Streptococcus molecular point-of-care testing for remote Australia: a key activity to eliminate rheumatic heart disease.

Tuberculosis in pregnancy.

Due to COVID-19 pandemic, the latest progress of the End Tuberculosis (TB) Strategy was far from optimal and services for TB needs to be quickly restored. Pregnancy is a unique opportunity to screen and manage TB, and it is an essential step in TB eradication. Early diagnosis and treatment for active disease can reduce maternal and neonatal morbidities and mortality. The more widespread utilization of newer rapid molecular assays with drug-susceptibility testing has significantly shortened the diagnostic process for active TB disease. First-line anti-TB drugs are proven to be safe in pregnancy. Management of latent TB infection (LTBI) during pregnancy is controversial, but puerperium is a period of increased susceptibility to progress to active disease. Extrapulmonary TB (EPTB), multidrug-resistant TB (MDR-TB) and HIV co-infection remain significant issues surrounding TB management during pregnancy and often require input from a multidisciplinary team including TB experts.

Rapid molecular assays for the detection of the four dengue viruses in infected mosquitoes.

The pantropic emergence of severe dengue disease can partly be attributed to the co-circulation of different dengue viruses (DENVs) in the same geographical location. Effective monitoring for circulation of each of the four DENVs is critical to inform disease mitigation strategies. In low resource settings, this can be effectively achieved by utilizing inexpensive, rapid, sensitive and specific assays to detect viruses in mosquito populations. In this study, we developed four rapid DENV tests with direct applicability for low-resource virus surveillance in mosquitoes. The test protocols utilize a novel sample preparation step, a single-temperature isothermal amplification, and a simple lateral flow detection. Analytical sensitivity testing demonstrated tests could detect down to 1,000 copies/µL of virus-specific DENV RNA, and analytical specificity testing indicated tests were highly specific for their respective virus, and did not detect closely related flaviviruses. All four DENV tests showed excellent diagnostic specificity and sensitivity when used for detection of both individually infected mosquitoes and infected mosquitoes in pools of uninfected mosquitoes. With individually infected mosquitoes, the rapid DENV-1, -2 and -3 tests showed 100% diagnostic sensitivity (95% CI = 69% to 100%, n=8 for DENV-1; n=10 for DENV 2,3) and the DENV-4 test showed 92% diagnostic sensitivity (CI: 62% to 100%, n=12) along with 100% diagnostic specificity (CI: 48-100%) for all four tests. Testing infected mosquito pools, the rapid DENV-2, -3 and -4 tests showed 100% diagnostic sensitivity (95% CI = 69% to 100%, n=10) and the DENV-1 test showed 90% diagnostic sensitivity (55.50% to 99.75%, n=10) together with 100% diagnostic specificity (CI: 48-100%). Our tests reduce the operational time required to perform mosquito infection status surveillance testing from > two hours to only 35 minutes, and have potential to improve accessibility of mosquito screening, improving monitoring and control strategies in low-income countries most affected by dengue outbreaks.

Rapid syndromic testing for respiratory viral infections in children attending the emergency department during COVID-19 pandemic in Lille, France, 2021-2022.

Objectives: Viral respiratory infections are common in children, and usually associated with non-specific symptoms. Respiratory panel-based testing was implemented during the COVID-19 pandemic, for the rapid differentiation between SARS-CoV-2 and other viral infections, in children attending the emergency department (ED) of the teaching hospital of Lille, northern France, between February 2021 and January 2022.Methods: Samples were collected using nasopharyngeal swabs. Syndromic respiratory testing was performed with two rapid multiplex molecular assays: the BioFire® Respiratory Panel 2.1 - plus (RP2.1 plus) or the QIAstat-Dx Respiratory SARS-CoV-2 Panel. SARS-CoV-2 variant was screened using mutation-specific PCR-based assays and genome sequencing.Results: A total of 3517 children were included in the study. SARS-CoV-2 was detected in samples from 265 children (7.5%). SARS-CoV-2 infected patients were younger than those without SARS-CoV-2 infection (median age: 6 versus 12 months, p < 0.0001). The majority of infections (61.5%) were associated with the Omicron variant. The median weekly SARS-CoV-2 positivity rate ranged from 1.76% during the Alpha variant wave to 24.5% with the emergence of the Omicron variant. Most children (70.2%) were treated as outpatients, and seventeen patients were admitted to the intensive care unit. Other respiratory viruses were more frequently detected in SARS-CoV-2 negative children than in positive ones (82.1% versus 37.4%, p < 0.0001). Human rhinovirus/enterovirus and respiratory syncytial virus were the most prevalent in both groups.Conclusions: We observed a low prevalence of SARS-CoV-2 infection in children attending pediatric ED, despite the significant increase due to Delta and Omicron variants, and an important circulation of other respiratory viruses. Severe disease was overall rare in children.

A Rapid Molecular Assay for Detecting the Mediterranean Fanworm Sabella spallanzanii Trialed by Non-Scientist Users

The invasive Mediterranean fanworm Sabella spallanzanii (Gmelin, 1791) is a notifiable organism under New Zealand’s Biosecurity Act and is recognized as a marine pest of particular concern, that must be reported to the Ministry for Primary Industries (MPI), New Zealand. Since its first detection in 2008, great effort and financial resources are put into surveillance and removal of individuals to contain population growth and spread. Sensitive molecular detection techniques gain great interest and are being increasingly tested for the fanworm detection in marine high-risk sites (i.e., ports and marinas) around New Zealand. However, conventional molecular detection via PCR assays from environmental DNA (eDNA) samples requires specific laboratory resources and technical expertise. This restricts the wider applicability of this approach by biosecurity practitioners or communities willing to be engaged in biosecurity surveillance. To provide end-users with a fast, easy and highly specific way to detect S. spallanzanii directly at the site of interest, a species-specific recombinase polymerase amplification (RPA) assay was designed to be read-out with lateral flow strips (RPA-LF). The RPA generates amplification within 20 minutes at 37-39°C, with a detection limit of 10 pg of the target DNA and was matching the detection limit of digital droplet PCR (ddPCR) when performed on eDNA samples. A simplified visual protocol for non-scientist users of the assay was developed and improved through independent trials with different end-user groups. The assay applicability was verified in a final validation trial with participants without scientific background resulting in 50 percent of the participants successfully detecting S. spallanzanii. Participants rated the ease of use and performance and read-out mostly as easy-to-very easy with overall positive written feedback on its usability for citizen science applications.

Abbott® ID NOW™ COVID-19 rapid molecular assay versus Hologic® Panther Aptima™ SARS-CoV-2 assay in nasopharyngeal specimens: results from 1-year retrospective study in an emergency department

We compared ID Now™ and Hologic® Panther Aptima™ for the detection of SARS-COV-2. ID Now™ showed a positive and negative percent agreement of 86.9% and 99.7% respectively. This facilitates faster clinical decision-making, along with the rapid implementation of infection control measures, and improvement of patient flow in the emergency department toward inpatient wards.