Impact of the Reverse 2-Step Algorithm for Clostridioides difficile Testing in the Microbiology Laboratory on Hospitalized Patients.

Background: Multistep laboratory testing is recommended for the diagnosis of Clostridioides difficile infection (CDI). The aim of this study was to present the impact of multistep CDI diagnostic testing in an academic hospital system and evaluate the toxin B gene polymerase chain reaction (PCR) cycle threshold (Ct) values of PCR-positive tests. Methods: In October 2022, our system began reflex testing all PCR-positive stool samples with the C. DIFF QUIK CHEK COMPLETE (Techlab), an enzyme immunoassay-based test with results for the glutamate dehydrogenase antigen (GDH) and C difficile toxin A/B. Hospital-onset (HO) CDI and CDI antibiotic use before and after testing were tracked. Ct values were obtained from the Infectious Diseases Diagnostic Laboratory. Receiver operating curve analysis was used to examine the sensitivity and specificity for identifying GDH+/toxin+ and GDH-/toxin- at various Ct thresholds. Results: The HO-CDI rate decreased from 0.352 cases per 1000 patient-days to 0.115 cases per 1000 patient-days post-reflex testing (P < .005). Anti-CDI antibiotics use decreased, but the decrease was not commensurate with CDI rates following reflex testing. PCR+/GDH+/toxin+ samples had a lower mean Ct value than PCR+/GDH-/toxin- samples (23.3 vs 33.5, P < .0001). A Ct value of 28.65 could distinguish between those 2 groups. Fifty-four percent of PCR+/GDH+/toxin- samples had a Ct value below that cut-off, suggesting the possibility of CDI with a negative toxin test. Conclusions: Reflex testing for a laboratory diagnosis of CDI results in rapid, systemwide decreases in the rate of HO-CDI. Additional research is needed to distinguish CDI from C difficile colonization in patients with discordant testing.

Paper-based loop-mediated isothermal amplification and CRISPR integrated platform for on-site nucleic acid testing of pathogens.

We report the development and initial validation of a paper-based nucleic acid testing platform that integrates Loop-mediated isothermal amplification (LAMP) with clustered regularly interspaced short palindromic repeats (CRISPR) technology, referred to as PLACID (Paper-based LAMP-CRISPR Integrated Diagnostics). LAMP eliminates the need for thermal cycling, resulting in simplified instrumentation, and the CRISPR-associated protein (Cas 12a) system eliminates false positive signals from LAMP products, resulting in highly selective and sensitive assays. We optimized the assay to perform both amplification and detection entirely on paper, eliminating the need for complex fluid handling steps and lateral flow assay transfers. Additionally, we engineered a smartphone-operated system that includes a low-powered, non-contact IR heating chamber to actuate paper-based LAMP and CRISPR reactions and enable the detection of fluorescent signals from the paper. The platform demonstrates high specificity and sensitivity in detecting nucleic acid targets with a limit of detection of 50 copies/µL. We integrate an equipment-free sample preparation separation technology designed to streamline the preparation of crude samples prior to nucleic acid testing. The practical utility of our platform is demonstrated by the successful detection of spiked SARS-CoV-2 RNA fragments in saliva, E. Coli in soil, and pathogenic E. Coli in clinically fecal samples of infected patients. Furthermore, we demonstrate that the paper-based LAMP CRISPR chips employed in our assays possess a shelf life of several weeks, establishing them as viable candidates for on-site diagnostics.

Group A Streptococcus pharyngitis in Children: New Perspectives on Rapid Diagnostic Testing and Antimicrobial Stewardship.

The most common cause of bacterial pharyngitis is Group A Streptococcus (GAS). Accurate diagnosis of GAS pharyngitis is crucial to identify children who would benefit from antibiotic treatment. Rapid diagnosis has the potential to reduce antibiotic overuse. Current national guidelines differ in their recommendations for GAS testing. While rapid antigen detection tests (RADTs) are widely used, their sensitivity is considered too low for stand-alone testing by several expert bodies. Newer molecular tests using nucleic acid amplification show higher accuracy and fast results, but their cost, complexity, and very high sensitivity may limit widespread adoption. This review provides up-to-date evidence regarding rapid diagnostic testing and antimicrobial stewardship in children with sore throat. We discuss discrepancies across GAS testing guidelines at the international level, patient selection for testing for GAS, rapid test accuracy, and the potential role of rapid GAS tests to promote antibiotic stewardship, with emphasis on emerging rapid molecular tests.

Competency of malaria laboratory diagnosis at national and provincial levels at the beginning of malaria post-elimination phase, China.

BACKGROUND: Qualified malaria diagnosis competency has contributed to the great achievement of malaria elimination in China. After eliminating malaria, it is still critical to the prevention of re-establishment of malaria transmission in China. This study was aimed to assess the malaria detection competency at national and provincial levels in China at the beginning of malaria post-elimination phase. METHODS: In the present study, different competency assessment activities on the laboratory malaria diagnosis were carried out for national and provincial malaria diagnostic laboratories based on the WHO scoring schedules, including malaria microscopy or nucleic acid amplification tests (NAAT), at the beginning of malaria post-elimination phase (2021-2022) in China. RESULTS: A total of 60 slides for malaria microscopy and 10 specimen for NAAT were included into the WHO External Quality Assessments of malaria parasite qualitative detection and species identification, and the scoring rate was 96.6% (microscopy: 171/177) and 85.0% (NAAT: 17/20), respectively. Moreover, 124 samples were included into the national NAAT quality assessment, and an accuracy of 87.9% (109/124) was found without significance among reference laboratories and non-reference laboratories. CONCLUSIONS: The findings suggest that there is still a need for sustained strengthening of malaria detection competency, particularly in the areas of parasite counting and detection of low-density parasitemia, to ensure prompt detection of the sources of infection and accurate identification of Plasmodium species, and contribute to case management and focus disposal, thereby effectively preventing the malaria re-establishment.

Detection of Plasmodium spp. in asymptomatic blood donors by the new Brazilian NAT PLUS HIV/HBV/HCV/Malaria Bio-Manguinhos kit.

BACKGROUND: Transfusion-transmitted malaria (TTM) is a public health problem in endemic and nonendemic areas. The Brazilian Ministry of Health (MH) requested the development of a nucleic acid amplification test (NAT) for the detection of Plasmodium spp. in public blood centers to increase blood safety. STUDY DESIGN AND METHODS: The new Brazilian NAT kit named NAT PLUS HIV/HBV/HCV/Malaria Bio-Manguinhos was first implemented in HEMORIO, a public blood center in the city of Rio de Janeiro. Since October 1, 2022, this blood center has been testing all its blood donations for malaria in a pool of six plasma samples to detect Plasmodium spp. by real-time polymerase chain reaction (PCR). RESULTS: Since the implementation of the NAT PLUS platform until February 2023, HEMORIO has successfully received and tested 200,277 donations. The platform detected two asymptomatic donors in the city of Rio de Janeiro, which is a nonendemic region for malaria. Our analyses suggested a malaria from the Amazon region caused by Plasmodium vivax, in the first case, while an autochthonous transmission case by Plasmodium malariae was identified in the rural area of Rio de Janeiro state. DISCUSSION: The NAT PLUS platform detects Plasmodium spp. in plasma samples with sensitivity capable of detecting subpatent infections. This is the first time worldwide that a group developed and implemented molecular diagnosis for Plasmodium spp. to be used by public blood centers to avoid TTM.

The Infectious Diseases Society of America Guidelines on the Diagnosis of COVID-19: Molecular Diagnostic Testing (December 2023).

Accurate molecular diagnostic tests are necessary for confirming a diagnosis of coronavirus disease 2019 (COVID-19) and for identifying asymptomatic carriage of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The number of available SARS-CoV-2 nucleic acid detection tests continues to increase as does the COVID-19 diagnostic literature. Thus, the Infectious Diseases Society of America (IDSA) developed an evidence-based diagnostic guideline to assist clinicians, clinical laboratorians, patients, and policymakers in decisions related to the optimal use of SARS-CoV-2 nucleic acid amplification tests. In addition, we provide a conceptual framework for understanding molecular diagnostic test performance, discuss nuances of test result interpretation in a variety of practice settings, and highlight important unmet research needs related to COVID-19 diagnostic testing. IDSA convened a multidisciplinary panel of infectious diseases clinicians, clinical microbiologists, and experts in systematic literature review to identify and prioritize clinical questions and outcomes related to the use of SARS-CoV-2 molecular diagnostics. Grading of Recommendations Assessment, Development and Evaluation (GRADE) methodology was used to assess the certainty of evidence and make testing recommendations. The panel agreed on 12 diagnostic recommendations. Access to accurate SARS-CoV-2 nucleic acid testing is critical for patient care, hospital infection prevention, and the public health response to COVID-19 infection. Information on the clinical performance of available tests continues to grow, but the quality of evidence of the current literature to support this updated molecular diagnostic guideline remains moderate to very low. Recognizing these limitations, the IDSA panel weighed available diagnostic evidence and recommends nucleic acid testing for all symptomatic individuals suspected of having COVID-19. In addition, testing is suggested for asymptomatic individuals with known or suspected contact with a COVID-19 case when the results will impact isolation/quarantine/personal protective equipment (PPE) usage decisions. Evidence in support of rapid testing and testing of upper respiratory specimens other than nasopharyngeal swabs, which offer logistical advantages, is sufficient to warrant conditional recommendations in favor of these approaches.

Testing for COVID-19: a 2023 update.

Nucleic acid amplification tests (NAATs), including polymerase chain reaction (PCR) assays, are more sensitive for the detection of SARS-CoV-2 than rapid antigen tests (RATS), and are the gold standard for diagnosis of acute COVID-19. However NAATs can remain positive for weeks following infection due to low-level shedding of non-viable viral fragments. RATs (in particular self-testing) are the mainstay of COVID-19 diagnosis due to their convenience, speed and high specificity. The sensitivity of RATs is highest within seven days of symptom onset. A negative RAT result may warrant a NAAT or repeat RAT for confirmation. The presence of spike antibodies is consistent with either vaccination or infection. Nucleocapsid antibodies suggest a previous infection. Serological tests measuring neutralising antibodies that infer immunity are not readily available.

Treponema pallidum PCR screening at mucosal sites of asymptomatic men who have sex with men taking HIV pre-exposure prophylaxis.

Early detection and treatment of syphilis will reduce the infectious period and transmission. We aimed to determine whether screening men who have sex with men (MSM) taking HIV pre-exposure prophylaxis (PrEP) for syphilis using Treponema pallidum polymerase chain reaction (PCR) could detect syphilis before the appearance of syphilis antibodies in serology. MSM attending 3-monthly PrEP clinic visits in Melbourne, Australia, were screened with a PCR assay targeting the polA gene of T. pallidum from an anal swab and an oral rinse between November 2019 and March 2020. Participants were serologically screened for syphilis using chemiluminescence immunoassay. A total of 309 asymptomatic participants provided an anal swab and oral rinse sample for T. pallidum PCR screening. Two syphilis cases (0.6%) were detected: one man had a positive serology only; another man had T. pallidum detected by PCR from an anal swab and a positive serology. PCR positivity was 0.3% (n = 1) for anal swabs and 0% (n = 0) for oral rinse. In this study, T. pallidum PCR screening at routine PrEP clinic visits did not identify additional cases of early syphilis over serological screening performed at these visits. IMPORTANCE With the ongoing syphilis epidemic in men who have sex with men (MSM), we investigated the role of using Treponema pallidum polymerase chain reaction (PCR) testing at the oral cavity and anus in MSM taking pre-exposure prophylaxis for the early detection of syphilis. We evaluated whether the PCR tests from these mucosal sites can detect syphilis infection early, before the development of syphilis antibodies in serology. Our study found two syphilis cases among 309 MSM, and only one syphilis case had a positive anal PCR swab, although serology was positive. We conclude that additional PCR testing is likely to be expensive and would not be cost effective for individuals who regularly screen for syphilis. However, future studies with a larger sample size are required.

Treponema pallidum Detection at Asymptomatic Oral, Anal, and Vaginal Sites in Adults Reporting Sexual Contact with Persons with Syphilis.

We investigated Treponema pallidum PCR positivity at mucosal sites (oral, anal, and vaginal sites) among adults who had sexual contact with a person with syphilis (syphilis contacts). All syphilis contacts had oral rinse and swab samples collected for testing. Men who have sex with men had anal swab and women had vaginal swab samples collected for testing, regardless of the presence of lesions. Of 407 persons tested, 42 (10%) had early syphilis diagnosed; of those, 19 (45%) tested positive by PCR from any anatomic site and had a positive serologic test. T. pallidum was positive from vaginal samples in 3 women, anal samples in 3 men, and oral cavity samples in 2 women and 3 men, without symptoms at those sites. Three women had no prior syphilis serologic test. T. pallidum detection at asymptomatic mucosal sites suggests early syphilis infections, particularly in cases that would conventionally be staged as latent syphilis of unknown duration.

Diagnostic Yield of Nucleic Acid Amplification Tests in Oral Samples for Pulmonary Tuberculosis: A Systematic Review and Meta-analysis.

The diagnostic accuracy of oral specimen nucleic acid amplification tests (NAATs) for pulmonary tuberculosis (PTB) remains controversial. We performed a systematic review according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines, including studies that reported the diagnostic yield of NAATs in oral samples for PTB diagnosis. The pooled estimates, including those of sensitivity and specificity, were calculated, and a meta-regression was performed to investigate heterogeneity, which was determined using χ2 and I² tests. A total of 23 articles were included, and the pooled sensitivity, specificity, and area under the curve of NAATs in oral samples for PTB diagnosis were 50% (95% CI, 37%-63%), 97% (95% CI, 93%-99%), and 0.89 (95% CI, 86%-92%; I 2 = 99%; chi-square, 169.61; P < .001), respectively. Our data demonstrated that NAATs using oral samples have a less satisfactory sensitivity and high specificity for PTB diagnosis. However, due to significant heterogeneity, such as heterogeneity in age, the results should be interpreted with caution.

Development of a validated molecular analytical method to determine the viral safety of F(AB´)2 products: A novel application for a well-known technique.

The immunotherapy agents derived from horses are biological products that allow the neutralization of clinically relevant immunogens, such as the SARS-CoV-2 virus that causes COVID-19, or the neutralization of toxins present in the venoms of snakes, spiders, and other poisonous animals. Due to their importance, detecting adventitious viruses in equine hyperimmune serum (raw material in industrial processes) is a critical step to support the safety of products for human use, and, in consequence, it is a requirement for commercialization and distribution. The safety of the finished product is based on three complementary approaches: (i) testing of the source material (horse serum) donations, (ii) release of the starting material (i.e., pool of horse serum) based on non-reactivity for a range of human infectious or pathogenic viruses, and (iii) validate (selected) steps of the manufacturing process for their capacity to inactivate and/or remove a wide range of viruses potentially present in the starting material. Orthogonal approaches to reduce viral contamination risk include implementing a reliable and validated system for detecting adventitious viruses. Thus, it is necessary to establish trustworthy and sufficiently sensitive analytical methods to evidence the lack of viruses to assure the safety of the therapeutic product. Therefore, in this research, an analytical method based on end-point Reverse Transcription Polymerase Chain Reaction (RT-PCR) was developed, implemented, and validated in hyperimmune equine serum samples to detect Venezuelan equine encephalitis virus, West Nile virus, and Rabies virus.

Authorized SARS-CoV-2 molecular methods show wide variability in the limit of detection.

On February 29th, 2020, the U.S. Food and Drug Administration issued the first Emergency Use Authorization (EUA) for a SARS-CoV-2 assay outside of the U.S. Centers for Disease Control and Prevention. As of May 3rd, 2021, 289 total EUAs have been granted. Like influenza, there is no standard for defining limit of detection (LoD), but rather guidance that analytical sensitivity/LoD be established as the level that gives a 95% detection rate in at least 20 replicates. Here we compare the performance characteristics of SARS-CoV-2 tests receiving EUA by standardizing sensitivity to a common unit of measure and assess the variability in LoD between tests. Additionally, we looked at factors that may impact sensitivities due to lack of standardization of the test development process and compare results for a standardized reference panel for comparative analysis within a subset of EUA tests offered by the U.S. Food and Drug Administration.

Direct capture and amplification of nucleic acids using a universal, elution-free magnetic bead-based method for rapid pathogen detection in multiple types of biological samples.

Nucleic acid amplification tests (NAATs) have become an attractive approach for pathogen detection, and obtaining high-quality nucleic acid extracts from biological samples plays a critical role in ensuring accurate NAATs. In this work, we established an elution-free magnetic bead (MB)-based method by introducing polyethylene-polypropylene glycol (PEPPG) F68 in lysis buffer and using NaOH solution instead of alcohols as the washing buffer for rapid nucleic acid extraction from multiple types of biological samples, including nasopharyngeal swabs, serum, milk, and pork, which bypassed the nucleic acid elution step and allowed the nucleic acid/MB composite to be directly used as the template for amplification reactions. The entire extraction process was able to be completed in approximately 7 min. Even though the nucleic acid/MB composite could not be used for quantitative real-time PCR (qPCR) assays, this elution-free MB-based method significantly improved the sensitivity of the loop-mediated isothermal amplification (LAMP) assay. The sensitivity of the quantitative real-time LAMP (qLAMP) assays combined with this elution-free MB-based method showed an improvement of one to three orders of magnitude compared with qLAMP or qPCR assays combined with the traditional MB-based method. In addition to manual operation, like the traditional MB-based method, this universal, rapid, and facile nucleic acid extraction method also has potential for integration into automated robotic processing, making it particularly suitable for the establishment of an analysis platform for ultrafast and sensitive pathogen detection in various biological samples both in centralized laboratories and at remote sites.

Impact of molecular diagnostic tests on diagnostic and treatment delays in tuberculosis: a systematic review and meta-analysis.

BACKGROUND: Countries with high TB burden have expanded access to molecular diagnostic tests. However, their impact on reducing delays in TB diagnosis and treatment has not been assessed. Our primary aim was to summarize the quantitative evidence on the impact of nucleic acid amplification tests (NAAT) on diagnostic and treatment delays compared to that of the standard of care for drug-sensitive and drug-resistant tuberculosis (DS-TB and DR-TB). METHODS: We searched MEDLINE, EMBASE, Web of Science, and the Global Health databases (from their inception to October 12, 2020) and extracted time delay data for each test. We then analysed the diagnostic and treatment initiation delay separately for DS-TB and DR-TB by comparing smear vs Xpert for DS-TB and culture drug sensitivity testing (DST) vs line probe assay (LPA) for DR-TB. We conducted random effects meta-analyses of differences of the medians to quantify the difference in diagnostic and treatment initiation delay, and we investigated heterogeneity in effect estimates based on the period the test was used in, empiric treatment rate, HIV prevalence, healthcare level, and study design. We also evaluated methodological differences in assessing time delays. RESULTS: A total of 45 studies were included in this review (DS = 26; DR = 20). We found considerable heterogeneity in the definition and reporting of time delays across the studies. For DS-TB, the use of Xpert reduced diagnostic delay by 1.79 days (95% CI - 0.27 to 3.85) and treatment initiation delay by 2.55 days (95% CI 0.54-4.56) in comparison to sputum microscopy. For DR-TB, use of LPAs reduced diagnostic delay by 40.09 days (95% CI 26.82-53.37) and treatment initiation delay by 45.32 days (95% CI 30.27-60.37) in comparison to any culture DST methods. CONCLUSIONS: Our findings indicate that the use of World Health Organization recommended diagnostics for TB reduced delays in diagnosing and initiating TB treatment. Future studies evaluating performance and impact of diagnostics should consider reporting time delay estimates based on the standardized reporting framework.

The Past, Present, and Future of Kingella kingae Detection in Pediatric Osteoarthritis.

As a result of the increasing use of improved detection methods, Kingella kingae, a Gram-negative component of the pediatric oropharyngeal microbiota, is increasingly appreciated as the prime etiology of septic arthritis, osteomyelitis, and spondylodiscitis in children aged 6 to 48 months. The medical literature was reviewed to summarize the laboratory methods required for detecting the organism. Kingella kingae is notoriously fastidious, and seeding skeletal system samples onto solid culture media usually fails to isolate it. Inoculation of synovial fluid aspirates and bone exudates into blood culture vials enhances Kingella kingae recovery by diluting detrimental factors in the specimen. The detection of the species has been further improved by nucleic acid amplification tests, especially by using species-specific primers targeting Kingella kingae's rtxA, groEL, and mdh genes in a real-time PCR platform. Although novel metagenomic next-generation technology performed in the patient's plasma sample (liquid biopsy) has not yet reached its full potential, improvements in the sensitivity and specificity of the method will probably make this approach the primary means of diagnosing Kingella kingae infections in the future.

A novel method for inward fluid displacement in centrifugal microdevices for highly integrated nucleic acid processing with long-term reagent storage.

Rotationally-driven lab-on-a-disc (LoaD) microfluidic systems are among the most promising methods for realizing complex nucleic acid (NA) testing at the point-of-need (PoN). However, despite significant advancements in NA amplification methods, very few sample-to-answer centrifugal microfluidic platforms have been realized due, in part, to a lack of on-disc sample preparation. In many instances, NA extraction (NAE) and/or lysis must be performed off-disc using conventional laboratory equipment and methods, thus tethering the assay to centralized facilities. Omission of in-line cellular lysis and NAE can be partially attributed to the nature of centrifugally-driven fluidics. Since flow is directed radially outward relative to the center of rotation (CoR), the number of possible sequential unit operations is limited by the disc radius. To address this, we report a simple, practical, automatable, and easy-to-implement method for inward fluid displacement (IFD) compatible with downstream nucleic acid amplification tests (NAATs). This approach leverages carbon dioxide (CO2) gas generated from on-board acid-base neutralization to drive liquid from the disc periphery towards the CoR. Large architectural features or highly corrosive chemicals required in other approaches were replaced with safe-to-handle IFD reagents that maintained their reactivity for at least six months of storage on-disc. Further, spatiotemporal control over neutralization initiation and containment of the resultant pneumatic pressure head was reliably achieved using a single diode for both laser-actuated valve opening and channel sealing, which eliminated the need for manual intervention (e.g., taping over vents) required in other IFD methods. Following initial characterization via dye recovery studies, we demonstrated for the first time that CO2-driven displacement does not inhibit downstream NAATs; NAs isolated direct-from-swab on disc were compatible with both 'gold standard' polymerase chain reaction (PCR) techniques and loop-mediated isothermal amplification (LAMP). The IFD approach described here stands to significantly ease integration of an increased number of sequential on-board processes, including cellular lysis, nucleic acid extraction, amplification, and detection, to greatly lower barriers towards automatable sample-to-answer LoaDs amenable for use on-site operation by non-technical personnel.

Antimicrobial Stewardship Strategies Including Point-of-Care Testing (POCT) for Pediatric Patients with Upper-Respiratory-Tract Infections in Primary Care: A Systematic Review of Economic Evaluations.

Upper-respiratory-tract infections (URTIs) are among the main causes of antibiotic prescriptions in pediatric patients. Over one-third of all antibiotic prescriptions for URTIs in children are estimated to be inappropriate, as the majority of URTIs are caused by viral agents. Several strategies, including clinical scoring algorithms and different point-of-care tests (POCTs) have been developed to help discriminate bacterial from viral URTIs in the outpatient clinical setting. A systematic review of the literature was conducted following PRISMA guidelines with the objective of summarizing evidence from health-economic evaluations on the use of POCT for URTIs in pediatric outpatients. A total of 3375 records identified from four databases and other sources were screened, of which 8 met the inclusion criteria. Four studies were classified as being of high reporting quality, and three were of medium quality. Five out of eight studies concluded in favor of strategies that included POCTs, with an additional study finding several POCTs to be cost-effective compared to usual care but over an acceptable WTP threshold. This review found POCT could be a valuable tool for antimicrobial stewardship strategies targeted towards childhood URTIs in primary care.

Accuracy of Xpert MTB/RIF Ultra for the diagnosis of tuberculosis in adult patients: a retrospective cohort study.

OBJECTIVES: The value of the "trace" result in Xpert Ultra for diagnosing active tuberculosis (TB) remains unclear. Our study evaluated the diagnostic accuracy of Xpert MTB/RIF Ultra (Xpert Ultra) (Cepheid, Sunnyvale, USA) over Xpert MTB/RIF (Xpert) (Cepheid, Sunnyvale, USA) and mycobacterial culture when compared with a composite reference standard (CRS). METHODS: A retrospective single-center observational study was conducted in a tertiary care hospital in South India. Over three months, patients (aged ≥15 years) data on Xpert Ultra tests and mycobacterial culture of pulmonary and extrapulmonary samples were extracted from their electronic medical records. Patients were defined as TB cases based on the CRS criteria. Sensitivity, specificity, positive and negative predictive values of diagnostic tests were calculated by comparing them to the CRS. RESULTS: Xpert Ultra was more sensitive (87.8%) than Xpert (72.1%) and culture (44.1%). The specificity of Xpert Ultra was lower (98.1%) than those of Xpert (100%) and culture (100%). The sensitivity (92%) and specificity (100%) of Xpert Ultra were highest when performed on pus samples. CONCLUSIONS: Xpert Ultra with the trace category is superior to the conventional Xpert, and mycobacterial culture in identifying TB.

Clinical Diagnostic Point-of-Care Molecular Assays for SARS-CoV-2.

Laboratories faced many challenges throughout the COVID-19 pandemic. Point-of-care (POC) SARS-CoV-2 nucleic acid amplification tests (NAATs) provided a key solution to the need for rapid turnaround time in select patient populations and were implemented at the POC but also within laboratories to supplement traditional molecular assays. Clinical Laboratory Improvement Amendments-waived rapid POC SARS-CoV-2 NAATs offer the benefit of reduced educational requirements for operators and can be performed by non-laboratory-trained individuals. However, these methods must be validated to ensure the manufacturer's performance specifications are met and they are found to be fit-for-purpose in the clinical workflows they are implemented.

Exploring beyond the limit: How comparative stochastic performance affects retesting outcomes in six commercial SARS CoV-2 nucleic acid amplification tests.

Objectives: To examine the comparative stochasticity profile of six commercial SARS-CoV-2 nucleic acid amplification tests (NAATs) and how this may affect retesting paradigms. Methods: Commercial quality control (QC) material was serially diluted in viral transport media to create a panel covering 10-10,000 copies/ml. The panel was tested across six commercial NAATs. A subset of high cycle threshold results was retested on a rapid PCR assay to simulate retesting protocols commonly used to discriminate false positives. Results: Performance beyond the LOD differed among assays, with three types of stochasticity profiles observed. The ability of the rapid PCR assay to reproduce a true weak positive specimen was restricted to its own stochastic performance at the corresponding viral concentration. Conclusion: Stochastic performance of various NAATs overlap across low viral concentrations and affect retesting outcomes. Relying on retesting alone to discriminate false positives risk missing true positives even when a more sensitive assay is deployed for confirmatory testing.