A pilot study for the evaluation of an interferon-gamma release assay (IGRA) to measure T-cell immune responses after SARS-CoV-2 infection or vaccination in a unique cloistered cohort

Background: Assessment of T-cell responses to SARS-CoV-2 antigens may be of value to determine long-lasting protection to breakthrough infections or reinfections. Interferon-gamma release assay is a validated method to test cellular immunity in mycobacterial infections and has been proposed for patients with SARS-CoV-2 infection or vaccination. Methods: Quantitative IgG to spike and qualitative IgG to nucleocapsid antigens were determined by chemiluminescence microparticle immunoassay using the Architect® platform (Abbott®), and interferon-gamma release assay against two Qiagen® proprietary mixes of SARS-CoV-2 spike protein (antigen-1 and antigen-2) were performed for a selected group of subjects. Results: A total of 121 subjects in a cloistered institution after a COVID-19 outbreak were studied. IgG-spike levels and interferon-gamma concentration were highest among subjects after two doses of vaccine, followed by patients with a longer history of past COVID-19 and no vaccination. Best cut-off for interferon-gamma assay was 25 IU/μL for all subgroups of individuals and the two sets of SARS-CoV-2 antigens studied. Conclusions: Testing T-cell response may be of clinical utility to determine immunity after exposure to SARS-CoV-2 antigens, with the interferon-gamma concentration of 25 IU/μL as the best cut-off either after infection or vaccination.

Tools for detecting a “superbug”: updates on Candida auris testing

Candida auris is an emerging yeast species that has the unique characteristics of patient skin colonization and rapid transmission within healthcare facilities and the ability to rapidly develop antifungal resistance. When C. auris first started appearing in clinical microbiology laboratories, it could only be identified using DNA sequencing. In the decade since its first identification outside of Japan there have been many improvements in the detection of C. auris . These include the expansion of MALDI-TOF databases to include C. auris , the development of both laboratory-developed tests and commercially available kits for its detection, and special CHROMagar for identification from laboratory specimens. Here we discuss the current tools and resources that are available for C. auris identification and detection.

Biographical Feature: Susan E. Sharp, Ph.D., D(ABMM), F(AAM)

“…You have brains in your head. You have feet in your shoes.… You can steer yourself any direction you choose. You're on your own. And you know what you know. And YOU are the [one] who'll decide where to go…

Combination of mycological criteria: a better surrogate to identify COVID-19 associated pulmonary aspergillosis patients and evaluate prognosis?

Introduction: Diagnosis of COVID-19 associated pulmonary aspergillosis remains unclear especially in non-immunocompromised patients. The aim of this study was to evaluate seven mycological criteria and their combination in a large homogenous cohort of patients. Methods: All successive patients (n=176) hospitalized for COVID-19 requiring mechanical ventilation and who clinically worsened despite appropriate standard of care were included over a one-year period. Direct examination, culture, Aspergillus qPCR ( Af -qPCR) and galactomannan was performed on all respiratory samples (n=350). Serum galactomannan, ß-D-glucan and plasma Af -qPCR were also assessed. Criteria were analyzed alone or in combination in relation to mortality rate. Results: Mortality was significantly different in patients with 0, ≤2 and ≥3 positive criteria (logrank test, p=0.04) with death rate of 43.1, 58.1 and 76.4% respectively. Direct examination, plasma qPCR and serum galactomannan were associated with a 100% mortality rate. Bronchoalveolar lavage (BAL) galactomannan and positive respiratory sample culture were often found as isolated markers (28.1 and 34.1%) and poorly repeatable when a second sample was obtained. Aspergillus DNA was detected in 13.1% of samples (46/350) with significantly lower Cq when associated with at least one other criteria (30.2 vs 35.8) (p<0.001). Conclusion: Combination of markers and/or blood biomarkers and/or direct respiratory sample examination seems more likely to identify patients with CAPA. Af -qPCR may help identifying false positive results of BAL galactomannan testing and culture on respiratory samples while quantifying fungal burden accurately.

Clinical Validation of the Xpert MTB/RIF test for Identification of the MTB Complex in AFB Smear-Positive MGIT broth cultures

The identification of the M. tuberculosis complex (MTBC) from smear positive broth cultures can be achieved using several methods including both lab-developed and commercially available molecular assays. In the United States, a commercially available probe-based assay has been used for over a decade by many laboratories for identification of MTBC directly from AFB smear positive broth cultures, including those recovered from the MGIT 960 system. However, recent difficulties in obtaining probe kits for identification resulted in mycobacteriology laboratories looking for alternative platforms to provide for rapid identification of MTBC and detection of rifampicin resistance. The Xpert® MTB/RIF test (Cepheid, Sunnyvale, Ca) has shown high sensitivity for the diagnosis of MTBC from pulmonary specimens but is not often used for identification directly from smear positive, MGIT 960 broth cultures (Becton Dickinson, Sparks, Md). We sought to validate the Xpert MTB/RIF test for use with AFB smear positive MGIT 960 cultures in a clinical hospital setting. Overall, the assay showed a categorical agreement of 100% for identification of MTBC and detection of rifampin resistance. No false positive results or cross-reactivity were noted. Findings indicate that the Xpert MTB/RIF test may be suitable as a rapid replacement for identification of MTBC and detection of rifampicin resistance from AFB smear positive MGIT 960 broth cultures.

A survey of current activities and technologies used to detect carbapenem resistance in bacteria isolated from companion animals at veterinary diagnostic laboratories—United States, 2020

Carbapenems are antimicrobial drugs reserved for the treatment of severe multidrug-resistant Gram-negative bacterial infections. Carbapenem-resistant organisms (CROs) are an urgent public health threat and have been made reportable to public health authorities in many jurisdictions. Recent reports of CROs in companion animals and veterinary settings suggest that CROs are a One Health problem. However, standard practices of U.S. veterinary diagnostic laboratories (VDLs) to detect CROs are unknown. We assessed the capacity of VDLs to characterize carbapenem resistance in isolates from companion animals. Among 74 VDLs surveyed in 42 states, 23 laboratories (31%) from 22 states responded. Most (22/23, 96%) include ≥1 carbapenem on their primary antimicrobial susceptibility testing panel; approximately one-third (9/23, 39%) perform phenotypic carbapenemase production testing or molecular identification of carbapenemase genes. Overall, 35% (8/23) of VDLs across eight states reported they would notify public health if a CRO was detected. Most (17/21, 81%) VDLs were not aware of CRO reporting mandates; some expressed uncertainty about whether the scope of known mandates included CROs from veterinary sources. Although nearly all surveyed VDLs tested for carbapenem resistance, fewer had capacity for mechanism testing or awareness of public health reporting requirements. Addressing these gaps is critical to monitoring CRO incidence and trends in veterinary medicine, preventing spread in veterinary settings, and mounting an effective One Health response. Improved collaboration and communication between public health and veterinary medicine is critical to inform infection control practices in veterinary settings and conduct public health response when resistant isolates are detected.

Collaboration between clinical and academic laboratories for sequencing SARS-CoV-2 genomes

Genomic sequencing of SARS-CoV-2 continues to provide valuable insight into the ever-changing variant makeup of the COVID-19 pandemic. More than three million SARS-COV-2 genomes have been deposited in GISAID, but contributions from the United States, particularly through 2020, lagged behind the global effort. The primary goal of clinical microbiology laboratories is seldom rooted in epidemiologic or public health testing and many labs do not contain in-house sequencing technology. However, we recognized the need for clinical microbiologists to lend expertise, share specimen resources, and partner with academic laboratories and sequencing cores to assist in SARS-COV-2 epidemiologic sequencing efforts. Here we describe two clinical and academic laboratory collaborations for SARS-COV-2 genomic sequencing. We highlight roles of the clinical microbiologists and the academic labs, outline best practices, describe two divergent strategies in accomplishing a similar goal, and discuss the challenges with implementing and maintaining such programs.

Detection of the hepatitis B surface antigen in patients with occult hepatitis B using an assay with enhanced sensitivity

Patients with occult hepatitis B infection (OBI) have undetectable hepatitis B surface antigen (HBsAg) by conventional assays but detectable hepatitis B virus (HBV) DNA in blood/liver. We evaluated the key performance characteristics of a sensitive HBsAg assay (ARCHITECT HBsAg Next Qualitative Assay, referred as NEXT) with respect to HBsAg detection. Assay precision, sample carryover and seroconversion sensitivity of NEXT were evaluated. HBsAg was measured by NEXT in 1,138 individuals, including 1,038 patients who attended liver clinics in a tertiary hospital (200 HBV DNA-positive blood donors whose HBsAg was undetectable by conventional assays, and 38 patients receiving immunosuppressive therapy, 800 chronic hepatitis B patients with HBsAg seroclearance) and 100 HBsAg-negative subjects recruited from a community project. The within-run and within-laboratory coefficients of variation were <6% for the positive sample pools. In 9 seroconversion panels tested, NEXT allowed an earlier HBsAg detection than conventional assays. NEXT detected HBsAg in 10/200 (5%) HBsAg-negative blood donors, 1/20 (5%) and 0/18 HBsAg-negative patients with and without HBV reactivation respectively, and 59/800 (7.3%) patients with HBsAg seroclearance. HBsAg was detectable by NEXT in 27.8%, 8.2%, 6.9%, 3.8% and 1.9% samples at <3, 3–5, >5–8, >8–11, and >11 years after HBsAg seroclearance, respectively. Seven out of 100 HBsAg-negative community identified subjects was tested positive by NEXT. Comparing with conventional HBsAg assays, NEXT demonstrated a higher sensitivity and conferred an increment of 5–7% detection rate in patients with OBI, thereby helping in identifying HBV carriers and prevention of OBI-associated HBV transmission and reactivation.

Assessment of the analytical sensitivity of ten lateral flow devices against the SARS-CoV-2 omicron variant

Timely and accurate diagnostic testing is a critical component of the public health response to COVID-19. Antigen tests are used widely in many countries to provide rapid, economical and accessible point-of-care testing (1). The vast majority of antigen tests detect nucleocapsid (N) protein, a structural protein that displays less variation than the spike (S) protein across different SARS-CoV-2 lineages. Although antigen tests are less sensitive than RT-PCR tests, their ability to quickly detect individuals with high viral loads provides clinical and public health utility in many countries, including Australia, where antigen tests have recently been approved for self-testing (2). As new variants arise, including the recent emergence of the SARS-CoV-2 omicron variant, it is essential to rapidly assess the performance of diagnostic assays. Here, in order to assess and compare the ability of antigen tests to detect delta and omicron variants, we performed a rapid assessment of ten commercially available antigen tests.

Quantitative SARS-CoV-2 viral-load curves in paired saliva and nasal swabs inform appropriate respiratory sampling site and analytical test sensitivity required for earliest viral detection

Early detection of SARS-CoV-2 infection is critical to reduce asymptomatic and pre-symptomatic transmission, curb the spread of variants, and maximize treatment efficacy. Low-analytical-sensitivity nasal-swab testing is commonly used for surveillance and symptomatic testing, but the ability of these tests to detect the earliest stages of infection has not been established. In this study, conducted between September 2020 and June 2021 in the greater Los Angeles County, California area, initially-SARS-CoV-2-negative household contacts of individuals diagnosed with COVID-19 prospectively self-collected paired anterior-nares nasal-swab and saliva samples twice daily for viral-load quantification by high-sensitivity RT-qPCR and digital-RT-PCR assays. We captured viral-load profiles from the incidence of infection for seven individuals and compared diagnostic sensitivities between respiratory sites. Among unvaccinated persons, testing saliva with a high-analytical-sensitivity assay detected infection up to 4.5 days before viral loads in nasal swabs reached concentrations detectable by low-analytical-sensitivity nasal-swab tests. For most participants, nasal swabs reached higher peak viral loads than saliva, but were undetectable or at lower loads during the first few days of infection. High-analytical-sensitivity saliva testing was most reliable for earliest detection. Our study illustrates the value of acquiring early (within hours after a negative high-sensitivity test) viral-load profiles to guide the appropriate analytical sensitivity and respiratory site for detecting earliest infections. Such data are challenging to acquire but critical to design optimal testing strategies with emerging variants in the current pandemic and to respond to future viral pandemics.