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.

Cepheid Xpert Xpress Flu/RSV evaluation performed by minimally trained non-laboratory operators in a CLIA-waived environment.

The COVID-19 pandemic highlighted the significance of readily available and easily performed viral testing for surveillance during future infectious pandemics. The objectives of this study were: to assess the performance of the Xpert Xpress Flu and/or RSV test, a multiplex PCR assay for detecting influenza A and B virus and respiratory syncytial virus nucleic acids in respiratory tract specimens, relative to the Quidel Lyra Influenza A+B assay and the Prodesse ProFlu+ assay, and the system's ease of use by minimally trained operators. Overall, the Xpert Xpress Flu/RSV test demonstrated a high positive and negative percent agreement with the comparator assays, and was easy to use and interpret results, based on the operators' feedback. We concluded that the Xpert Xpress Flu/RSV test is sensitive, specific, and easy to use for the diagnosis of influenza and RSV by minimally trained operators and can be a valuable tool in future infectious clusters or pandemics.

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.

Pathologic Inflammation in Malnutrition Is Driven by Proinflammatory Intestinal Microbiota, Large Intestine Barrier Dysfunction, and Translocation of Bacterial Lipopolysaccharide.

Acute malnutrition, or wasting, is implicated in over half of all deaths in children under five and increases risk of infectious disease. Studies in humans and preclinical models have demonstrated that malnutrition is linked to an immature intestinal microbiota characterized by increased prevalence of Enterobacteriaceae. Observational studies in children with moderate acute malnutrition (MAM) have also observed heightened systemic inflammation and increased circulating bacterial lipopolysaccharides (LPS; endotoxin). However, the mechanisms that underpin the systemic inflammatory state and endotoxemia, and their pathophysiological consequences, remain uncertain. Understanding these pathophysiological mechanisms is necessary to design targeted treatments that will improve the unacceptable rate of failure or relapse that plague current approaches. Here we use a mouse model of MAM to investigate the mechanisms that promote inflammation in the malnourished host. We found that mice with MAM exhibited increased systemic inflammation at baseline, increased translocation of bacteria and bacterial LPS, and an exaggerated response to inflammatory stimuli. An exaggerated response to bacterial LPS was associated with increased acute weight loss. Remarkably, intestinal inflammation and barrier dysfunction was found in the cecum and colon. The cecum showed a dysbiotic microbiota with expansion of Gammaproteobacteria and some Firmicutes, and contraction of Bacteroidetes. These changes were paralleled by an increase in fecal LPS bioactivity. The inflammatory phenotype and weight loss was modulated by oral administration of non-absorbable antibiotics that altered the proportion of cecal Gammaproteobacteria. We propose that the heightened inflammation of acute malnutrition is the result of changes in the intestinal microbiota, intestinal barrier dysfunction in the cecum and colon, and increased systemic exposure to LPS.

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.

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.

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.

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.

Cutaneous nontuberculous mycobacteria infections: A retrospective case series of 78 patients from the Texas Gulf Coast region.

BACKGROUND: The incidence of cutaneous nontuberculous mycobacteria (NTM) infections is increasing. These infections are a diagnostic and therapeutic challenge. OBJECTIVE: We investigated the clinical features, diagnosis, and management of cutaneous NTM infections. METHODS: A retrospective case series studied 78 patients from a Gulf Coast tertiary referral center diagnosed with cutaneous NTM infection by culture or stain of a skin biopsy specimen. RESULTS: A history of trauma, procedure, or environmental exposure was common. The mean time between the initial evaluation and diagnosis was 12 weeks. Only 15% of acid-fast bacillus-positive cultures had a positive acid-fast bacillus smear, and only 43% of those accompanied by skin biopsy specimen had a positive Fite stain. Immunosuppressed patients were more likely to have a positive Fite stain. Treatment included surgery and multiple antibiotics. Immunosuppressed patients and Mycobacterium abscessus group infections were more likely to have persistent disease. LIMITATIONS: M chelonae and M abscessus isolates were indistinguishable and therefore were reported together. Five cases were not confirmed by culture. CONCLUSIONS: Even with clinical suspicion, the diagnosis of NTM infection can be difficult. Results of acid-fast bacillus smears and special stains are frequently negative. Antibiotic resistance is common. Multidrug treatment is often required, and surgical therapy may be needed.

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.