RT-PCR negative COVID-19.

BACKGROUND: COVID-19 is a multi-system infection with emerging evidence-based antiviral and anti-inflammatory therapies to improve disease prognosis. However, a subset of patients with COVID-19 signs and symptoms have repeatedly negative RT-PCR tests, leading to treatment hesitancy. We used comparative serology early in the COVID-19 pandemic when background seroprevalence was low to estimate the likelihood of COVID-19 infection among RT-PCR negative patients with clinical signs and/or symptoms compatible with COVID-19. METHODS: Between April and October 2020, we conducted serologic testing of patients with (i) signs and symptoms of COVID-19 who were repeatedly negative by RT-PCR ('Probables'; N = 20), (ii) signs and symptoms of COVID-19 but with a potential alternative diagnosis ('Suspects'; N = 15), (iii) no signs and symptoms of COVID-19 ('Non-suspects'; N = 43), (iv) RT-PCR confirmed COVID-19 patients (N = 40), and (v) pre-pandemic samples (N = 55). RESULTS: Probables had similar seropositivity and levels of IgG and IgM antibodies as propensity-score matched RT-PCR confirmed COVID-19 patients (60.0% vs 80.0% for IgG, p-value = 0.13; 50.0% vs 72.5% for IgM, p-value = 0.10), but multi-fold higher seropositivity rates than Suspects and matched Non-suspects (60.0% vs 13.3% and 11.6% for IgG; 50.0% vs 0% and 4.7% for IgM respectively; p-values < 0.01). However, Probables were half as likely to receive COVID-19 treatment than the RT-PCR confirmed COVID-19 patients with similar disease severity. CONCLUSIONS: Findings from this study indicate a high likelihood of acute COVID-19 among RT-PCR negative with typical signs/symptoms, but a common omission of COVID-19 therapies among these patients. Clinically diagnosed COVID-19, independent of RT-PCR positivity, thus has a potential vital role in guiding treatment decisions.

Highly versatile antibody binding assay for the detection of SARS-CoV-2 infection and vaccination.

Monitoring the burden and spread of infection with the new coronavirus SARS-CoV-2, whether within small communities or in large geographical settings, is of paramount importance for public health purposes. Serology, which detects the host antibody response to the infection, is the most appropriate tool for this task, since virus-derived markers are most reliably detected during the acute phase of infection. Here we show that our ELISA protocol, which is based on antibody binding to the Receptor Binding Domain (RBD) of the S1 subunit of the viral Spike protein expressed as a novel fusion protein, detects antibody responses to SARS-CoV-2 infection and vaccination. We also show that our ELISA is accurate and versatile. It compares favorably with commercial assays widely used in clinical practice to determine exposure to SARS-CoV-2. Moreover, our protocol accommodates use of various blood- and non-blood-derived biospecimens, such as breast milk, as well as dried blood obtained with microsampling cartridges that are appropriate for remote collection. As a result, our RBD-based ELISA protocols are well suited for seroepidemiology and other large-scale studies requiring parsimonious sample collection outside of healthcare settings.

Highly versatile antibody binding assay for the detection of SARS-CoV-2 infection.

Monitoring the burden and spread of infection with the new coronavirus SARS-CoV-2, whether within small communities or in large geographical settings, is of paramount importance for public health purposes. Serology, which detects the host antibody response to the infection, is the most appropriate tool for this task, since virus-derived markers are most reliably detected during the acute phase of infection. Here we show that our ELISA protocol, which is based on antibody binding to the Receptor Binding Domain (RBD) of the S1 subunit of the viral Spike protein expressed as a novel fusion protein, detects antibody responses to SARS-CoV-2 infection and COVID-19 vaccination. We also show that our ELISA is accurate and versatile. It compares favorably with commercial assays widely used in clinical practice to determine exposure to SARS-CoV-2. Moreover, our protocol accommodates use of various blood- and non-blood-derived biospecimens, such as breast milk, as well as dried blood obtained with microsampling cartridges that are appropriate for remote collection. As a result, our RBD-based ELISA protocols are well suited for seroepidemiology and other large-scale studies requiring parsimonious sample collection outside of healthcare settings.

Analytical performance of the Xpert MTB/XDR® assay for tuberculosis and expanded resistance detection.

In a manufacturer-independent laboratory validation study, the Xpert MTB/XDR® assay demonstrated equivalent limit of detection to Xpert MTB/RIF®, detected 100% of tested resistance mutations and showed some utility for resistance detection in strain mixtures. The Xpert MTB/XDR assay is a reliable, sensitive assay for tuberculosis and expanded resistance detection.

A Pilot Study of Human Papillomavirus Detection in Urine Using a Novel Nucleic Acid Amplification Test.

INTRODUCTION: Human papillomavirus (HPV) testing is the standard-of-care for cervical cancer screening globally. Urine is a promising alternative to collecting a cervical specimen during a pelvic exam for HPV testing. There are no studies to date of HPV testing of urine using the Xpert HPV test. METHODS: We conducted a pilot study of 40 women; 30 women undergoing colposcopy because of a previous abnormality and 10 undergoing routine screening, to evaluate HPV detection in urine by the Xpert HPV test on the GeneXpert platform. Xpert HPV testing of urine was done according to the manufacturer's instructions for testing cervical specimens. These results were compared to a reference of combined results of 2 research HPV genotyping tests conducted on cervical specimens and to repeat Xpert HPV testing of urine. RESULTS: Analytic sensitivity and specificity of Xpert testing of urine for any high-risk HPV versus the cervical sample, categorized as HPV positive if at least 1 test was positive, were 64.3% (95% confidence interval [95%CI] = 42.1-76.1%) and 100% (97.5%CI = 71.5-100%), respectively. Analytic sensitivity and specificity of Xpert testing of urine for any high-risk HPV versus the cervical sample, categorized as positive if both tests were positive, were 66.7% (95%CI = 44.7-84.4%) and 86.7% (95%CI = 59.5-98.3%), respectively. Kappa values for first vs. second and first vs. third testing of urine by Xpert were 0.89 (95%CI = 0.79-1.00) and 0.90 (95%CI = 0.81-1.00), respectively. DISCUSSION: Given the call for global elimination of cervical cancer and widespread availability of GeneXpert, optimizing Xpert HPV testing of urine may be warranted.

Xpert MTB/XDR: a 10-Color Reflex Assay Suitable for Point-of-Care Settings To Detect Isoniazid, Fluoroquinolone, and Second-Line-Injectable-Drug Resistance Directly from Mycobacterium tuberculosis-Positive Sputum.

We describe the design, development, analytical performance, and a limited clinical evaluation of the 10-color Xpert MTB/XDR assay (CE-IVD only, not for sale in the United States). This assay is intended as a reflex test to detect resistance to isoniazid (INH), fluoroquinolones (FLQ), ethionamide (ETH), and second-line injectable drugs (SLIDs) in unprocessed sputum samples and concentrated sputum sediments which are positive for Mycobacterium tuberculosis The Xpert MTB/XDR assay simultaneously amplifies eight genes and promoter regions in M. tuberculosis and analyzes melting temperatures (Tm s) using sloppy molecular beacon (SMB) probes to identify mutations associated with INH, FLQ, ETH, and SLID resistance. Results can be obtained in under 90 min using 10-color GeneXpert modules. The assay can differentiate low- versus high-level resistance to INH and FLQ as well as cross-resistance versus individual resistance to SLIDs by identifying mutation-specific Tm s or Tm patterns generated by the SMB probes. The assay has a limit of detection comparable to that of the Xpert MTB/RIF assay and successfully detected 16 clinically significant mutations in a challenge set of clinical isolate DNA. In a clinical study performed at two sites with 100 sputum and 214 clinical isolates, the assay showed a sensitivity of 94% to 100% and a specificity of 100% for all drugs except for ETH compared to that of sequencing. The sensitivity and specificity were in the same ranges as those of phenotypic drug-susceptibility testing. Used in combination with a primary tuberculosis diagnostic test, this assay should expand the capacity for detection of drug-resistant tuberculosis near the point of care.

Automatic Identification of Individual rpoB Gene Mutations Responsible for Rifampin Resistance in Mycobacterium tuberculosis by Use of Melting Temperature Signatures Generated by the Xpert MTB/RIF Ultra Assay.

Molecular surveillance of rifampin-resistant Mycobacterium tuberculosis can help to monitor the transmission of the disease. The Xpert MTB/RIF Ultra assay detects mutations in the rifampin resistance-determining region (RRDR) of the rpoB gene by the use of melting temperature (Tm ) information from 4 rpoB probes which can fall in one of the 9 different assay-specified Tm windows. The large amount of Tm data generated by the assay offers the possibility of an RRDR genotyping approach more accessible than whole-genome sequencing. In this study, we developed an automated algorithm to specifically identify a wide range of mutations in the rpoB RRDR by utilizing the pattern of the Tm of the 4 probes within the 9 windows generated by the Ultra assay. The algorithm builds a RRDR mutation-specific "Tm signature" reference library from a set of known mutations and then identifies the RRDR genotype of an unknown sample by measuring the Tm distances between the test sample and the reference Tm values. Validated using a set of clinical isolates, the algorithm correctly identified RRDR genotypes of 93% samples with a wide range of rpoB single and double mutations. Our analytical approach showed a great potential for fast RRDR mutation identification and may also be used as a stand-alone method for ruling out relapse or transmission between patients. The algorithm can be further modified and optimized for higher accuracy as more Ultra data become available.

The New Xpert MTB/RIF Ultra: Improving Detection of Mycobacterium tuberculosis and Resistance to Rifampin in an Assay Suitable for Point-of-Care Testing.

The Xpert MTB/RIF assay (Xpert) is a rapid test for tuberculosis (TB) and rifampin resistance (RIF-R) suitable for point-of-care testing. However, it has decreased sensitivity in smear-negative sputum, and false identification of RIF-R occasionally occurs. We developed the Xpert MTB/RIF Ultra assay (Ultra) to improve performance. Ultra and Xpert limits of detection (LOD), dynamic ranges, and RIF-R rpoB mutation detection were tested on Mycobacterium tuberculosis DNA or sputum samples spiked with known numbers of M. tuberculosis H37Rv or Mycobacterium bovis BCG CFU. Frozen and prospectively collected clinical samples from patients suspected of having TB, with and without culture-confirmed TB, were also tested. For M. tuberculosis H37Rv, the LOD was 15.6 CFU/ml of sputum for Ultra versus 112.6 CFU/ml of sputum for Xpert, and for M. bovis BCG, it was 143.4 CFU/ml of sputum for Ultra versus 344 CFU/ml of sputum for Xpert. Ultra resulted in no false-positive RIF-R specimens, while Xpert resulted in two false-positive RIF-R specimens. All RIF-R-associated M. tuberculosis rpoB mutations tested were identified by Ultra. Testing on clinical sputum samples, Ultra versus Xpert, resulted in an overall sensitivity of 87.5% (95% confidence interval [CI], 82.1, 91.7) versus 81.0% (95% CI, 74.9, 86.2) and a sensitivity on sputum smear-negative samples of 78.9% (95% CI, 70.0, 86.1) versus 66.1% (95% CI, 56.4, 74.9). Both tests had a specificity of 98.7% (95% CI, 93.0, 100), and both had comparable accuracies for detection of RIF-R in these samples. Ultra should significantly improve TB detection, especially in patients with paucibacillary disease, and may provide more-reliable RIF-R detection.IMPORTANCE The Xpert MTB/RIF assay (Xpert), the first point-of-care assay for tuberculosis (TB), was endorsed by the World Health Organization in December 2010. Since then, 23 million Xpert tests have been procured in 130 countries. Although Xpert showed high overall sensitivity and specificity with pulmonary samples, its sensitivity has been lower with smear-negative pulmonary samples and extrapulmonary samples. In addition, the prediction of rifampin resistance (RIF-R) in paucibacillary samples and for a few rpoB mutations has resulted in both false-positive and false-negative results. The present study is the first demonstration of the design features and operational characteristics of an improved Xpert Ultra assay. This study also shows that the Ultra format overcomes many of the known shortcomings of Xpert. The new assay should significantly improve TB detection, especially in patients with paucibacillary disease, and provide more-reliable detection of RIF-R.