Determination of the cycle threshold value of the Xpert Xpress SARS-CoV-2/Flu/RSV test that corresponds to the presence of infectious SARS-CoV-2 in anterior nasal swabs.

Despite having high analytical sensitivities and specificities, qualitative SARS-CoV-2 nucleic acid amplification tests (NAATs) cannot distinguish infectious from non-infectious virus in clinical samples. In this study, we determined the highest cycle threshold (Ct) value of the SARS-CoV-2 targets in the Xpert Xpress SARS-CoV-2/Flu/RSV (Xpert 4plex) test that corresponded to the presence of detectable infectious SARS-CoV-2 in anterior nasal swab samples. A total of 111 individuals with nasopharyngeal swab specimens that were initially tested by the Xpert Xpress SARS-CoV-2 test were enrolled. A healthcare worker subsequently collected anterior nasal swabs from all SARS-CoV-2-positive individuals, and those specimens were tested by the Xpert 4plex test, viral culture, and laboratory-developed assays for SARS-CoV-2 replication intermediates. SARS-CoV-2 Ct values from the Xpert 4plex test were correlated with data from culture and replication intermediate testing to determine the Xpert 4plex assay Ct value that corresponded to the presence of infectious virus. Ninety-eight of the 111 (88.3%) individuals initially tested positive by the Xpert Xpress SARS-CoV-2 test. An anterior nasal swab specimen collected from positive individuals a median of 2 days later (range, 0-9 days) tested positive for SARS-CoV-2 by the Xpert 4plex test in 39.8% (39/98) of cases. Of these samples, 13 (33.3%) were considered to contain infectious virus based on the presence of cultivable virus and replication intermediates, and the highest Ct value observed for the Xpert 4plex test in these instances was 26.3. Specimens that yielded Ct values of ≤26.3 when tested by the Xpert 4plex test had a likelihood of containing infectious SARS-CoV-2; however, no infectious virus was detected in specimens with higher Ct values.IMPORTANCEUnderstanding the correlation between real-time PCR test results and the presence of infectious SARS-CoV-2 may be useful for informing patient management and workforce return-to-work or -duty. Further studies in different patient populations are needed to correlate Ct values or other biomarkers of viral replication along with the presence of infectious virus in clinical samples.

Maintaining point of care testing capacity and pandemic preparedness in the post-COVID-19 era.

INTRODUCTION: Testing at the point of care (we also refer to the 'point of need'), with rapid, actionable results reported to the patient and provider within hours can impact the individual as well as public health. Faster testing is good for patients and public health outcomes during 'peace time' (outside of the pandemic setting). AREAS COVERED: Testing at the point of need was important during the COVID-19 pandemic to meet testing capacity demands, providing actionable results, and for providing testing within communities to increase access for all populations. Resources were acquired and built up dramatically during the pandemic as part of the response. With the end of the COVID-19 public health emergency and transition back to 'peace time' some testing sites have successfully shifted to using this capacity for testing for other critical needs, like sexually transmitted infection (STI) testing, and response to other seasonal diseases and for outbreak response. EXPERT OPINION: The increased testing capacity added to handle unprecedented testing volume during the COVID-19 pandemic can be repurposed for other critical infectious diseases during 'peace time' (post-COVID-19 pandemic). This maintains testing capacity for the next pandemic.

Practical Challenges of Point-of-Care Testing.

The practical challenges of point-of-care testing (POCT) include analytical performance and quality compared with testing performed in a central laboratory and higher cost per test compared with laboratory-based tests. These challenges can be addressed with new test technology, consensus, and practice guidelines for the use of POCT, instituting a quality management system and data connectivity in the POCT setting, and studies that demonstrate evidence of clinical and economic value of POCT.

Taxonomic Changes for Human Viruses, 2020 to 2022.

The classification of viruses remains relevant to several disciplines, including clinical virology. Since the original publication of this review in 2019, many known viruses have undergone taxonomic revisions, and several novel human and animal viruses have been described. Here, we provide an update to our previous reviews of taxonomic changes for disease-causing viruses of humans, covering changes that occurred between 2020 and 2022. As with previous editions, this update was informed by recent advances in virus taxonomy made by the International Committee on Taxonomy of Viruses; the changes and additions noted herein are not all-inclusive.

Design and Implementation of Improved SARS-CoV-2 Diagnostic Assays To Mitigate the Impact of Genomic Mutations on Target Failure: the Xpert Xpress SARS-CoV-2 Experience.

In 2020, the U.S. Food and Drug Administration (FDA) enabled manufacturers to request emergency use authorization (EUA) to facilitate the rapid authorization of in vitro diagnostic (IVD) platforms for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Uncommon SARS-CoV-2 point mutations could cause nucleocapsid (N) gene target failure (NGTF) when using first-generation Xpert Xpress assays, so improvements were designed and implemented. In response to NGTF reports and with consideration of viral genomic information in public databases, the Xpress assays were redesigned to mitigate the impact of SARS-CoV-2 mutations on qualitative assay performance. The second-generation assays include a third gene target (RNA-dependent RNA polymerase [RdRp]) and redundant oligonucleotide probes for the N2 target. First- and second-generation assay performances were evaluated using a challenge set of samples. A second-generation assay with updated oligonucleotide chemistry received FDA EUA in September 2021. A prototype assay with oligonucleotide chemistry similar to that of the second-generation assay with FDA EUA successfully detected all three gene targets (N2, envelope [E], and RdRp) in all challenge samples (100%; 50/50), including variants with N gene mutations (g.29197C>T or g.29200C>T), which caused NGTF in the first-generation assays. Investigation and reporting of IVD target failures, public sharing of viral genomic sequence data, and the FDA EUA pathway were essential components in facilitating a short cycle time from the identification of mutations that impact the performance of an IVD assay to the design and implementation of an improved IVD assay. IMPORTANCE The SARS-CoV-2 genome has mutated during the coronavirus disease 2019 (COVID-19) pandemic. Some of these mutations have impacted the performance of nucleic acid amplification tests like PCR, which are commonly used as diagnostic tools to detect an infection. The U.S. Food and Drug Administration (FDA) emergency use authorization (EUA) process enables the rapid reformulation and regulatory authorization of improved PCRs. In our experience, the identification of SARS-CoV-2 mutations that impact PCR performance, the subsequent development of improved PCR chemistry, and the use of the FDA EUA regulatory pathway led to improved diagnostic performance during the SARS-CoV-2 pandemic that is able to keep pace with the rapidly evolving genome of SARS-CoV-2.

Considerations regarding Interpretation of Positive SARS-CoV-2 Molecular Results with Late Cycle Threshold Values.

COVID-19 disease lies on a spectrum, ranging from completely asymptomatic to mild disease to severe and critical disease. Studies have shown that prolonged shedding or sporadic detection of SARS-CoV-2 RNA can occur long after symptom resolution. Adding to these clinical complexities is the demand for testing for SARS-CoV-2 at all stages of diseases, frequently driven by screening of asymptomatic persons, something that traditionally has not been performed for other viral respiratory diseases. This can lead to positive results from nucleic acid amplification tests (NAATs), such as RT-PCR, with late cycle threshold (CT) values near the test's limit of detection. In this commentary, we review unique attributes of COVID-19 and causes of NAAT late CT values. We provide interpretation considerations as well as strategies to aid in test interpretation.

Detection of SARS-CoV-2 at the point of care.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiologic agent of COVID-19. Testing for SARS-CoV-2 infection is a critical element of the public health response to COVID-19. Point-of-care (POC) tests can drive patient management decisions for infectious diseases, including COVID-19. POC tests are available for the diagnosis of SARS-CoV-2 infections and include those that detect SARS-CoV-2 antigens as well as amplified RNA sequences. We provide a review of SARS-CoV-2 POC tests including their performance, settings for which they might be used, their impact and future directions. Further optimization and validation, new technologies as well as studies to determine clinical and epidemiological impact of SARS-CoV-2 POC tests are needed.

Taxonomic Changes for Human and Animal Viruses, 2018 to 2020.

The classification of viruses is relevant to a number of scientific and clinical disciplines, including the practice of diagnostic virology. Here, we provide an update to our previous review of taxonomic changes for disease-causing viruses in humans and vertebrate animals, covering changes between 2018 and 2020. Recent advances in virus taxonomy structure by the International Committee on Taxonomy of Viruses inform this update.

Multicenter Evaluation of the Cepheid Xpert Xpress SARS-CoV-2/Flu/RSV Test.

With the approach of respiratory virus season in the Northern Hemisphere, clinical microbiology and public health laboratories will need rapid diagnostic assays to distinguish severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from influenza virus and respiratory syncytial virus (RSV) infections for diagnosis and surveillance. In this study, the clinical performance of the Xpert Xpress SARS-CoV-2/Flu/RSV test (Cepheid, Sunnyvale, CA, USA) for nasopharyngeal swab specimens was evaluated in four centers: Johns Hopkins Medical Microbiology Laboratory, Northwell Health Laboratories, NYC Public Health Laboratory, and Los Angeles County/University of Southern California (LAC+USC) Medical Center. A total of 319 nasopharyngeal swab specimens, positive for SARS-CoV-2 (n = 75), influenza A virus (n = 65), influenza B virus (n = 50), or RSV (n = 38) or negative (n = 91) by the standard-of-care nucleic acid amplification tests at each site, were tested using the Cepheid panel test. The overall positive percent agreement for the SARS-CoV-2 target was 98.7% (n = 74/75), and the negative agreement was 100% (n = 91), with all other analytes showing 100% total agreement (n = 153). Standard-of-care tests to which the Cepheid panel was compared included the Cepheid Xpert Xpress SARS-CoV-2, Cepheid Xpert Xpress Flu/RSV, GenMark ePlex respiratory panel, BioFire respiratory panel 2.1 and v1.7, DiaSorin Simplexa COVID-19 Direct, and Hologic Panther Fusion SARS-CoV-2 assays. The Xpert Xpress SARS-CoV-2/Flu/RSV test showed high sensitivity and accuracy for all analytes included in the test. This test will provide a valuable clinical diagnostic and public health solution for detecting and differentiating SARS-CoV-2, influenza A and B virus, and RSV infections during the current respiratory virus season.

Meta-analysis of diagnostic performance of serology tests for COVID-19: impact of assay design and post-symptom-onset intervals.

Serology detection is recognized for its sensitivity in convalescent patients with COVID-19, in comparison with nucleic acid amplification tests (NAATs). This article aimed to evaluate the diagnostic accuracy of serologic methods for COVID-19 based on assay design and post-symptom-onset intervals. Two authors independently searched PubMed, Cochrane library, Ovid, EBSCO for case-control, longitudinal and cohort studies that determined the diagnostic accuracy of serology tests in comparison with NAATs in COVID-19 cases and used QUADAS-2 for quality assessment. Pooled accuracy was analysed using INLA method. A total of 27 studies were included in this meta-analysis, with 4 cohort, 16 case-control and 7 longitudinal studies and 4565 participants. Serology tests had the lowest sensitivity at 0-7 days after symptom onset and the highest at >14 days. TAB had a better sensitivity than IgG or IgM only. Using combined nucleocapsid (N) and spike(S) protein had a better sensitivity compared to N or S protein only. Lateral flow immunoassay (LFIA) had a lower sensitivity than enzyme-linked immunoassay (ELISA) and chemiluminescent immunoassay (CLIA). Serology tests will play an important role in the clinical diagnosis for later stage COVID-19 patients. ELISA tests, detecting TAB or targeting combined N and S proteins had a higher diagnostic sensitivity compared to other methods.

Evaluation of High-Throughput Serological Tests for SARS-CoV-2.

Serologic methods are an important part of a clinical laboratory's portfolio of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) tests and are essential to the broader response to coronavirus infectious disease 2019 (COVID-19), including epidemiological studies and vaccine development. There are currently a number of commercial SARS-CoV-2 antibody tests with emergency use authorization (EUA) from the U.S. Food and Drug Administration. In this issue of the Journal of Clinical Microbiology, H. E. Prince, T. S. Givens, M. Lapé-Nixon, N. J. Clarke, et al. (J Clin Microbiol 58:e01742-20, https://doi.org/10.1128/JCM.01742-20, 2020) report the results of their evaluation of the agreement of 4 high-throughput EUA tests for SARS-CoV-2 IgG: Abbott Architect, DiaSorin Liaison, Euroimmun, and Ortho Vitros. They showed excellent agreement between the tests and rare false-positive reactivity for all tests.

Point-of-Care Testing for Sexually Transmitted Infections: A Review of Recent Developments.

CONTEXT.­: Sexually transmitted infections (STIs) are among the most common communicable diseases globally and are associated with significant morbidity and mortality worldwide. Point-of-care tests have the potential to revolutionize the prevention and control of STIs by enabling rapid diagnosis and early treatment of infections, thus interrupting transmission and preventing the sequelae of untreated infections. Currently, there are several point-of-care (POC) tests available for the diagnosis of Treponema pallidum, Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis infections, although these tests differ with regard to their performance, turnaround time, and cost. OBJECTIVE.­: To provide an updated review of the POC tests available and under development for the diagnosis of T pallidum, C trachomatis, N gonorrhoeae, and T vaginalis infections, to discuss the context for which these tests might be used, and to highlight future directions for test development. DATA SOURCES.­: We reviewed the literature pertaining to the recent development and performance evaluations of POC tests for the diagnosis of syphilis, chlamydia, gonorrhea, and trichomonas. CONCLUSIONS.­: Recently, there has been rapid development of new POC tests for STIs. Although there are inexpensive, rapid, and accurate POC tests available for syphilis, there are few such tests available for the diagnosis of chlamydia, gonorrhea, or trichomonas, and currently none with the ability to detect antimicrobial resistance in N gonorrhoeae. Research evaluating implementation strategies for the currently available tests and the development of additional POC tests that are rapid, accurate, and affordable are urgently needed to address the rising number of STIs worldwide.

The Traditional or Reverse Algorithm for Diagnosis of Syphilis: Pros and Cons.

We reviewed relevant syphilis diagnostic literature to address the question "What diagnostic considerations should be taken into account when screening for syphilis using the traditional or reverse algorithm?" Improved laboratory diagnosis of syphilis is an important element of the effort to reduce syphilis rates. Screening for syphilis is performed using either a nontreponemal or treponemal test (part of the traditional or reverse algorithm, respectively). Both syphilis algorithms are used by laboratories. However, there are limited data on the performance and cost-effectiveness of the algorithms. An expert panel generated "key questions" in the laboratory diagnosis of syphilis. This paper pertains to the key factors that should be considered when deciding whether to screen for syphilis using either the traditional or the reverse algorithm. A systematic literature review was performed, and tables of evidence were created to address this question.

Multicenter Evaluation of the Cepheid Xpert Xpress SARS-CoV-2 Test.

Nucleic acid amplification tests (NAATs) are the primary means of identifying acute infections caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Accurate and fast test results may permit more efficient use of protective and isolation resources and allow rapid therapeutic interventions. We evaluated the analytical and clinical performance characteristics of the Xpert Xpress SARS-CoV-2 (Xpert) test, a rapid, automated molecular test for SARS-CoV-2. Analytical sensitivity and specificity/interference were assessed with infectious SARS-CoV-2; other infectious coronavirus species, including SARS-CoV; and 85 nasopharyngeal swab specimens positive for other respiratory viruses, including endemic human coronaviruses (hCoVs). Clinical performance was assessed using 483 remnant upper- and lower-respiratory-tract specimens previously analyzed by standard-of-care (SOC) NAATs. The limit of detection of the Xpert test was 0.01 PFU/ml. Other hCoVs, including Middle East respiratory syndrome coronavirus, were not detected by the Xpert test. SARS-CoV, a closely related species in the subgenus Sarbecovirus, was detected by a broad-range target (E) but was distinguished from SARS-CoV-2 (SARS-CoV-2-specific N2 target). Compared to SOC NAATs, the positive agreement of the Xpert test was 219/220 (99.5%), and the negative agreement was 250/261 (95.8%). A third tie-breaker NAAT resolved all but three of the discordant results in favor the Xpert test. The Xpert test provided sensitive and accurate detection of SARS-CoV-2 in a variety of upper- and lower-respiratory-tract specimens. The high sensitivity and short time to results of approximately 45 min may impact patient management.

Laboratory diagnosis of emerging human coronavirus infections - the state of the art.

The three unprecedented outbreaks of emerging human coronavirus (HCoV) infections at the beginning of the twenty-first century have highlighted the necessity for readily available, accurate and fast diagnostic testing methods. The laboratory diagnostic methods for human coronavirus infections have evolved substantially, with the development of novel assays as well as the availability of updated tests for emerging ones. Newer laboratory methods are fast, highly sensitive and specific, and are gradually replacing the conventional gold standards. This presentation reviews the current laboratory methods available for testing coronaviruses by focusing on the coronavirus disease 2019 (COVID-19) outbreak going on in Wuhan. Viral pneumonias typically do not result in the production of purulent sputum. Thus, a nasopharyngeal swab is usually the collection method used to obtain a specimen for testing. Nasopharyngeal specimens may miss some infections; a deeper specimen may need to be obtained by bronchoscopy. Alternatively, repeated testing can be used because over time, the likelihood of the SARS-CoV-2 being present in the nasopharynx increases. Several integrated, random-access, point-of-care molecular devices are currently under development for fast and accurate diagnosis of SARS-CoV-2 infections. These assays are simple, fast and safe and can be used in the local hospitals and clinics bearing the burden of identifying and treating patients.

Streptococcus agalactiae Strains with Chromosomal Deletions Evade Detection with Molecular Methods.

Surveillance of circulating microbial populations is critical for monitoring the performance of a molecular diagnostic test. In this study, we characterized 31 isolates of Streptococcus agalactiae (group B Streptococcus [GBS]) from several geographic locations in the United States and Ireland that contain deletions in or adjacent to the region of the chromosome that encodes the hemolysin gene cfb, the region targeted by the Xpert GBS and GBS LB assays. PCR-negative, culture-positive isolates were recognized during verification studies of the Xpert GBS assay in 12 laboratories between 2012 and 2018. Whole-genome sequencing of 15 GBS isolates from 11 laboratories revealed four unique deletions of chromosomal DNA ranging from 181 bp to 49 kb. Prospective surveillance studies demonstrated that the prevalence of GBS isolates containing deletions in the convenience sample was <1% in three geographic locations but 7% in a fourth location. Among the 15 isolates with chromosomal deletions, multiple pulsed-field gel electrophoresis types were identified, one of which appears to be broadly dispersed across the United States.

Taxonomic Changes for Human and Animal Viruses, 2016 to 2018.

The classification of viruses provides the structure necessary to appreciate their biological diversity. Herein, we provide an update to our previous review of changes in viral taxonomy, covering changes between 2016 and 2018.

Comparison of the Alere i Strep A Test and the BD Veritor System in the Detection of Group A Streptococcus and the Hypothetical Impact of Results on Antibiotic Utilization.

Rapid detection of group A Streptococcus (GAS) is an integral component of treatment decisions in the clinic, especially in the pediatric population. We prospectively collected 216 specimens from symptomatic, predominantly pediatric patients and evaluated the performance of the Alere i Strep A test (Alere i; Alere Inc., Scarborough, ME) and the BD Veritor system (BD Veritor; Becton, Dickinson and Company, Sparks, MD), with culture results being used as a comparator. Real-time PCR (RT-PCR) was performed as an arbiter in discordant cases. Comprehensive chart review was also done to determine the hypothetical impact of the results on antibiotic use. Alere i had a sensitivity and a specificity of 100% and 91.3%, respectively, and BD Veritor had a sensitivity and a specificity of 76.2% and 93.6%, respectively, when the results were compared to those of GAS culture. Further analysis of discordant results using RT-PCR revealed that while BD Veritor missed 13 confirmed positive cases, Alere i detected 100% (n = 13) of the same cases. Analysis of assay agreement showed that Alere i and BD Veritor had only moderate agreement (agreement = 0.888 [95% confidence interval {CI}, 0.838 to 0.927]; kappa index = 0.689 [95% CI, 0.91 to 0.974]). We also found both the underutilization and the overutilization of antibiotics based on the results of molecular testing. Overall, Alere i showed superior performance over BD Veritor in the detection of GAS pharyngitis and could potentially assist in better antibiotic utilization.

Evaluation of the Lumipulse G TP-N Chemiluminescent Immunoassay as a Syphilis Screening Test.

A syphilis diagnosis is often aided by the detection of treponemal and nontreponemal antibodies. Automated treponemal antibody detection systems enable high-volume clinical laboratories to perform syphilis screening at a faster pace with lower labor costs. The Lumipulse G TP-N chemiluminescent immunoassay is an automated system that qualitatively detects IgG and IgM antibodies against Treponema pallidum antigens in human serum and plasma. To assess performance characteristics and workflow efficiency, the Lumipulse G TP-N assay was compared to the Bioplex 2200 Syphilis IgG multiplex flow immunoassay. Among the 4,134 routine and HIV samples tested by the two automated assays, the percentage of agreement was excellent at 99.0% (95% confidence interval [CI], 98.6% to 99.2%; κ, 0.89), with the Lumipulse G TP-N having a shorter time to first and subsequent results. All specimens with reactive syphilis screening results were further tested by rapid plasma reagin (RPR) and Treponema pallidum particle agglutination (TP·PA) testing (n = 231). The results from the RPR-reactive samples (n = 82) showed complete concordance with the two automated assays, while the TP·PA assay displayed some discrepancies. The positive percent agreement (PPA) and negative percent agreement (NPA) between the TP·PA test and the Lumipulse G TP-N test were 98.9% and 77.3%, respectively. The Bioplex 2200 Syphilis IgG immunoassay displayed a similar PPA (100%) but a substantially lower NPA (15.9%). Patient chart reviews of discrepant results suggested that the Lumipulse G TP-N assay produced 27 fewer falsely reactive results and can reduce the amount of additional confirmatory RPR and TP·PA testing needed. The analogous performance characteristics of the two automated systems indicate that the Lumipulse G TP-N assay is suitable for high-throughput syphilis screening.