The foundation for the microbiology laboratory's essential role in diagnostic stewardship: an ASM Laboratory Practices Subcommittee report.

Diagnostic stewardship (DxS) has gained traction in recent years as a cross-disciplinary method to improve the quality of patient care while appropriately managing resources within the healthcare system. Clinical microbiology laboratorians have been highly engaged in DxS efforts to guide best practices with conventional microbiology tests and more recently with molecular infectious disease diagnostics. Laboratories can experience resistance to their role in DxS, especially when the clinical benefits, motivations for interventions, and underlying regulatory requirements are not clearly conveyed to stakeholders. Clinical laboratories must not only ensure ethical practices but also meet obligatory requirements to steward tests responsibly. In this review, we aim to support clinical microbiology laboratorians by providing the background and resources that demonstrate the laboratory's essential role in DxS. The heart of this review is to collate regulatory and accreditation requirements that, in essence, mandate DxS practices as a long-standing, core element of high-quality laboratory testing to deliver the best possible patient care. While examples of the clinical impact of DxS are plentiful in the literature, here, we focus on the operational and regulatory justification for the laboratory's role in stewardship activities.

Proceedings of the Clinical Microbiology Open 2023: discussions about pandemic preparedness.

The 4th Clinical Microbiology Open (CMO) took place in Carlsbad, California, on 10 and 11 February 2023. This event facilitated discussion between clinical and public health laboratory directors, government agencies, and industry representatives from the companies that make up ASM's Corporate Council. While many topics were discussed, much of the discussion focused on pandemic preparedness. There were four major questions addressed: (i) When is the perfect the enemy of good in pandemic testing? (ii) What other types of pathogens might cause another pandemic and how would this affect laboratory response? (iii) What research is needed to better understand the effectiveness of the pandemic response? (iv) What have we learned about the utility of self and at-home testing in future pandemics? This review serves as a summary of these discussions.

Pilot study to determine effect of an altruism intervention focusing on herd immunity to enhance influenza vaccination rates.

OBJECTIVE: A prospective, single-arm clinical trial was conducted to evaluate an altruism-tailored educational intervention to improve parental attitudes and vaccine uptake in vaccine-hesitant parents. METHODS: Vaccine-hesitant parents at two primary care sites, spanning two influenza seasons from 2020 to 2021 were provided an intervention (spoken and written communication) which highlighted altruistic benefits of accepting the seasonal influenza vaccine to optimize herd immunity to help protect pediatric cancer patients. Eligible parents included those with children eligible for the seasonal influenza vaccine, those who were proficient in English, and those with scores on the adjusted Vaccine Hesitancy Scale (aVHS) suggesting vaccine hesitancy (score ≥ 3). Enrollees completed a demographic questionnaire, underwent the educational intervention, and repeated the aVHS. Vaccination status at that visit was assessed. The primary outcome was change in aVHS scores obtained pre- and post-intervention. Influenza vaccine acceptance, along with demographic information, were also analyzed. RESULTS: We enrolled 510 parents of influenza vaccine eligible children and identified 73 vaccine-hesitant parents. There was an overall trend toward lower aVHS score, with a mean change in hesitancy score of -0.4 (P < 0.01). 43/73 (58.9 %) of the cohort experienced a positive effect toward a lower aVHS score, and 27/73 (37.0 %) of vaccine hesitant subjects became non-hesitant on the aVHS. Several demographic characteristics were associated with vaccine hesitancy in the screening population: educational level lower than bachelor's degree (p = 0.03), household income < 400 % of federal poverty level (p < 0.01), unmarried (p = 0.02), and identifying with a political affiliation other than Democrat (p < 0.01). However, no demographic characteristics were significantly associated with an individual becoming non-hesitant. Our altruism-tailored communication approach carried the largest positive impact on the altruism-specific question on the aVHS, decreasing the post-intervention response value by nearly 25 % (P < 0.01). CONCLUSIONS: Our altruism-tailored communication approach significantly improved attitudes regarding childhood influenza vaccine among vaccine-hesitant parents. CLINICALTRIALS: gov Identifier: NCT04568590.

Summary of the Fourth Annual American Sexually Transmitted Diseases Association Workshop on Improving Sexually Transmitted Infection Control Efforts Through Cross-Sector Collaboration.

ABSTRACT: The American Sexually Transmitted Diseases Association has, for several years, been conducting a cross-sector workshop to bring together a variety of stakeholders to develop ideas for collaboratively improving the sexually transmitted infection control efforts in the United States. In this summary, we share the content of discussions and ideas of the fourth annual workshop for future research and potential changes to practice with a focus on diagnostic capacity.

Rapid and Accurate Identification of SARS-CoV-2 Variants Using Real Time PCR Assays.

Background: Genomic surveillance efforts for SARS-CoV-2 are needed to understand the epidemiology of the COVID-19 pandemic. Viral variants may impact routine diagnostic testing, increase viral transmissibility, cause differences in disease severity, have decreased susceptibility to therapeutics, and/or confer the ability to evade host immunity. While viral whole-genome sequencing (WGS) has played a leading role in surveillance programs, many laboratories lack the expertise and resources for performing WGS. This study describes the performance of multiplexed real-time reverse transcription-PCR (RT-PCR) assays for identification of SARS-CoV-2 variants. Methods: SARS-CoV-2 specimens were tested for spike-gene variants using a combination of allele-specific primer and allele-specific detection technology (PlexPrime® and PlexZyme®). Targeted detection of spike gene mutations by RT-PCR was compared to variant detection in positive specimens by WGS, including the recently emerged SARS-CoV-2 Omicron variant. Results: A total of 398 SAR-CoV-2 RT-PCR positive and 39 negative specimens previously tested by WGS were re-tested by RT-PCR genotyping. PCR detection of spike gene mutations N501Y, E484K, and S982A correlated 100% with WGS for the 29 lineages represented, including Alpha (B.1.1.7), Beta (B.1.351), and Gamma (P.1). Incorporating the P681R spike gene mutation also allowed screening for the SARS-CoV-2 Delta variant (B.1.617.2 and AY sublineages). Further sampling of 664 specimens that were screened by WGS between June and August 2021 and then re-tested by RT-PCR showed strong agreement for Delta variant positivity: 34.5% for WGS vs 32.9% for RT-PCR in June; 100% vs 97.8% in August. In a blinded panel of 16 Omicron and 16 Delta specimens, results of RT-PCR were 100% concordant with WGS results. Conclusions: These data demonstrate that multiplexed real-time RT-PCR genotyping has strong agreement with WGS and may provide additional SARS-CoV-2 variant screening capabilities when WGS is unavailable or cost-prohibitive. RT-PCR genotyping assays may also supplement existing sequencing efforts while providing rapid results at or near the time of diagnosis to help guide patient management.

Characterization of Serum Samples With Discordant Results in 2 Herpes Simplex Virus Type 2 IgG Assays.

BACKGROUND: Our laboratory system tests sera for herpes simplex virus type 2 (HSV-2) IgG using the DiaSorin Liaison chemiluminescent immunoassay (CIA), with the option to confirm positive samples by a laboratory-developed HerpeSelect inhibition assay. As part of the confirmation process, the HerpeSelect HSV-2 IgG enzyme immunoassay (EIA) is performed. This study investigated the relationship between DiaSorin HSV-2 IgG CIA-positive indices and HerpeSelect HSV-2 IgG EIA results. METHODS: HerpeSelect HSV-2 IgG EIA results were compiled for a cohort of consecutive DiaSorin HSV-2 IgG CIA-positive (index ≥1.10) samples. To further characterize DiaSorin CIA-positive samples that were positive (concordant) or negative (discordant) by the HerpeSelect EIA, a separate composite reference study panel was constructed and also tested using the Biokit HSV-2 IgG assay and an HSV-2 IgG inhibition assay developed for the DiaSorin instrument. Samples were classified as DiaSorin HSV-2 IgG true positive or false positive based on a composite reference using HerpeSelect EIA, Biokit, and DiaSorin inhibition results. RESULTS: Of 2305 consecutive DiaSorin HSV-2 IgG CIA-positive samples, 411 (17.8%) were HerpeSelect HSV-2 IgG EIA negative; 343 of 411 (83%) had DiaSorin indices of 1.10 to 3.00. For the composite reference study panel (N = 120), 59 of 60 discordant samples were classified as DiaSorin HSV-2 IgG false positive based on the composite reference, whereas 58 of 60 concordant samples were classified as true positive. CONCLUSIONS: Nearly all DiaSorin HSV-2 IgG CIA-positive but HerpeSelect HSV-2 IgG EIA-negative sera are falsely positive in the DiaSorin CIA. Furthermore, most DiaSorin false-positive samples exhibit low-positive indices, suggesting that guidelines for confirmatory testing should include low-positive samples by CIA and EIA.

Transience of interference in an immunoassay measuring serum levels of beta-D-glucan.

Some sera tested for 1-3-beta-D-glucan to identify invasive fungal infections exhibit interference. To assess interference transience, we evaluated results for 426 patients with an interference sample followed by a later sample. Interference was transient for 73% of patients (later sample negative or positive); median time between samples was 8 days.

Trichomonas vaginalis Detection in Urogenital Specimens from Symptomatic and Asymptomatic Men and Women by Use of the cobas TV/MG Test.

Trichomonas vaginalis is a prevalent sexually transmitted infection (STI). Diagnosis has historically relied on either microscopic analysis or culture, the latter being the previous gold standard. However, these tests are not readily available for male diagnosis, generally only perform well for symptomatic women, and are not as sensitive as nucleic acid amplification tests (NAATs). Men are largely asymptomatic but carry the organism and transmit to their sexual partners. This multicenter, prospective study evaluated the performance of the cobas T. vaginalis/Mycoplasma genitalium (TV/MG) assay for detection of T. vaginalis DNA compared with patient infection status (PIS) defined by a combination of commercially available NAATs and culture using urogenital specimens. A total of 2,064 subjects (984 men and 1,080 women, 940 [45.5%] symptomatic, 1,124 [54.5%] asymptomatic) were evaluable. In women, sensitivity ranged from 99.4% (95% confidence interval [CI] 96.8 to 99.9%) using vaginal samples to 94.7% (95% CI 90.2 to 97.2%) in PreservCyt samples. Specificity ranged from 98.9 to 96.8% (95% CI 95.4 to 97.8%). In men, the cobas TV/MG assay was 100% sensitive for the detection of T. vaginalis in both male urine samples and meatal swabs, with specificity of 98.4% in urine samples and 92.5% in meatal swabs. The cobas TV/MG is a suitable diagnostic test for the detection of T. vaginalis, which could support public health efforts toward infection control and complement existing STI programs.

Impact of the COVID-19 Pandemic on Chlamydia and Gonorrhea Screening in the U.S.

INTRODUCTION: This study evaluates the impact of the COVID-19 pandemic on testing for common sexually transmitted infections. Specifically, changes are measured in chlamydia and gonorrhea testing and case detection among patients aged 14-49 years during the COVID-19 pandemic. METHODS: U.S. chlamydia and gonorrhea testing and positivity were analyzed on the basis of >18.6 million tests (13.6 million tests for female patients and 4.7 million tests for male patients) performed by a national reference clinical laboratory from January 2019 through June 2020. RESULTS: Chlamydia and gonorrhea testing reached a nadir in early April 2020, with decreases (relative to the baseline level) of 59% for female patients and 63% for male patients. Declines in testing were strongly associated with increases in weekly positivity rates for chlamydia (R2=0.96) and gonorrhea (R2=0.85). From March 2020 through June 2020, an expected 27,659 (26.4%) chlamydia and 5,577 (16.5%) gonorrhea cases were potentially missed. CONCLUSIONS: The COVID-19 pandemic impacted routine sexually transmitted infection services, suggesting an increase in syndromic sexually transmitted infection testing and missed asymptomatic cases. Follow-up analyses will be needed to assess the long-term implications of missed screening opportunities. These findings should serve as a warning for the potential sexual and reproductive health implications that can be expected from the overall decline in testing and potential missed cases.

Performance of Unobserved Self-Collected Nasal Swabs for Detection of SARS-CoV-2 by RT-PCR Utilizing a Remote Specimen Collection Strategy.

BACKGROUND: The use of a remote specimen collection strategy employing a kit designed for unobserved self-collection for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reverse transcription polymerase chain reaction (RT-PCR) can decrease the use of personal protective equipment (PPE) and exposure risk. To assess the impact of unobserved specimen self-collection on test performance, we examined results from a SARS-CoV-2 qualitative RT-PCR test for self-collected specimens from participants in a return-to-work screening program and assessed the impact of a pooled testing strategy in this cohort. METHODS: Self-collected anterior nasal swabs from employee return-to-work programs were tested using the Quest Diagnostics Emergency Use Authorization SARS-CoV-2 RT-PCR. The cycle threshold (Ct) values for the N1 and N3 N-gene targets and a human RNase P (RP) gene control target were tabulated. For comparison, we utilized Ct values from a cohort of health care provider-collected specimens from patients with and without coronavirus disease 2019 symptoms. RESULTS: Among 47 923 participants, 1.8% were positive. RP failed to amplify for 13/115 435 (0.011%) specimens. The median (interquartile range) Cts were 32.7 (25.0-35.7) for N1 and 31.3 (23.8-34.2) for N3. Median Ct values in the self-collected cohort were significantly higher than those of symptomatic but not asymptomatic patients. Based on Ct values, pooled testing with 4 specimens would have yielded inconclusive results in 67/1268 (5.2%) specimens but only a single false-negative result. CONCLUSIONS: Unobserved self-collection of nasal swabs provides adequate sampling for SARS-CoV-2 RT-PCR testing. These findings alleviate concerns of increased false negatives in this context. Specimen pooling could be used for this population, as the likelihood of false-negative results is very low when using a sensitive, dual-target methodology.

Evolution of Specimen Self-Collection in the COVID-19 Era: Implications for Population Health Management of Infectious Disease.

Laboratory testing is an important component in the diagnosis of respiratory tract infections such as with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). However, specimen collection not only risks exposure of health care workers and other patients to infection, but also necessitates use of personal protective equipment that may be in short supply during periods of heightened disease activity. Self-collection of nasal or oropharyngeal swabs offers an alternative to address these drawbacks. Although studies in the past decade have demonstrated the utility of this approach for respiratory infections, it has not been widely adopted in routine clinical practice. The rapid spread of coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, has focused attention on the need for safe, convenient, timely, and scalable methods for collecting upper respiratory specimens for testing. The goals of this article are to highlight the literature regarding self-collected nasal or oropharyngeal specimens for respiratory pathogen testing; discuss the role of self-collection in helping prevent the spread of the COVID-19 disease from infected patients and facilitating a shift toward "virtual" medicine or telemedicine; and describe the current and future state of self-collection for infectious agents, and the impacts these approaches can have on population health management and disease diagnosis and prevention.

Relationship between hepatitis C virus (HCV) IgG index values and quantitative HCV RNA results in HCV IgG-reactive serum samples.

Centers for Disease Control guidelines recommend hepatitis C virus (HCV) RNA testing of all HCV IgG-reactive samples, although earlier studies found that IgG-reactive samples with low indices were negative in qualitative RNA assays. To determine if previous study results could be confirmed using current real-time RT-PCR technology, we investigated the relationship between HCV IgG index (Ortho VITROS) and quantitative HCV RNA results (cobas HCV) for 2368 consecutive IgG-reactive sera. Results were segregated into Low (1.00-16.0), Medium (16.1-30.0), and High (>30.0) IgG index groups. Although median viral load (VL) of RNA-positive samples was similar in all 3 groups, the percentage with low VL (1.18-4.16 log IU/mL) was increased for the Low group. Further analysis of the Low group revealed that 23 of 370 (6%) samples with IgG indices ≤8.00 were RNA-positive, and 13/23 (57%) had low VL. Our analysis supports the Centers for Disease Control recommendation to test all HCV IgG-reactive sera for HCV RNA.

Relationship between DiaSorin Liaison Treponema pallidum antibody indices and confirmatory assay results in the reverse syphilis testing algorithm.

Published studies show that >99% of sera reactive in the reverse syphilis testing algorithm (RSTA) screening assay with an index above an assay-specific threshold confirm as reactive, with either a rapid plasma reagin-reactive (RPRR) or RPR-nonreactive/Treponema pallidum particle agglutination-reactive (TPPAR) result. However, the relationship between screen indices and confirmatory patterns has not been characterized. We thus assessed confirmatory testing results for 577 sera submitted for RSTA testing and a screen-reactive result in the DiaSorin Liaison assay. The median screen index was significantly higher for RPRR samples than TPPAR samples (55.6 versus 10.4), and the proportion with indices >28.3 (median for all 577 samples) was significantly higher for RPRR versus TPPAR samples (82% versus 26%). However, RPRR titers did not significantly correlate with screen indices (R2 = 0.02). These findings demonstrate a significant relationship between RSTA screen indices and confirmatory assay results. The clinical utility of this relationship requires further study.

Pooling of Upper Respiratory Specimens Using a SARS-CoV-2 Real-time RT-PCR Assay Authorized for Emergency Use in Low-Prevalence Populations for High-Throughput Testing.

BACKGROUND: Nucleic acid amplification testing is a critical tool for addressing the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Specimen pooling can increase throughput and conserve testing resources but requires validation to ensure that reduced sensitivity does not increase the false-negative rate. We evaluated the performance of a real-time reverse transcription polymerase chain reaction (RT-PCR) test authorized by the US Food and Drug Administration (FDA) for emergency use for pooled testing of upper respiratory specimens. METHODS: Positive specimens were selected from 3 prevalence groups, 1%-3%, >3%-6%, and >6%-10%. Positive percent agreement (PPA) was assessed by pooling single-positive specimens with 3 negative specimens; performance was assessed using Passing-Bablok regression. Additionally, we assessed the distributions of RT-PCR cycle threshold (Ct) values for 3091 positive specimens. RESULTS: PPA was 100% for the 101 pooled specimens. There was a linear relationship between Ct values for pooled and single-tested specimens (r = 0.96-0.99; slope ≈ 1). The mean pooled Ct shifts at 40 cycles were 2.38 and 1.90, respectively, for the N1 and N3 targets. The median Cts for 3091 positive specimens were 25.9 (N1) and 24.7 (N3). The percentage of positive specimens with Cts between 40 and the shifted Ct was 1.42% (N1) and 0.0% (N3). CONCLUSIONS: Pooled and individual testing of specimens positive for SARS-CoV-2 demonstrated 100% agreement, which demonstrates the viability of pooled specimens for SARS-COV-2 testing using a dual-target RT-PCR system. Pooled specimen testing can help increase testing capacity for SARS-CoV-2 with a low risk of false-negative results.

Detection of SARS-CoV-2 IgG Targeting Nucleocapsid or Spike Protein by Four High-Throughput Immunoassays Authorized for Emergency Use.

A total of 1,200 serum samples that were tested for SARS-CoV-2 IgG antibody using the Abbott Architect immunoassay targeting the nucleocapsid protein were run in 3 SARS-CoV-2 IgG immunoassays targeting spike proteins (DiaSorin Liaison, Ortho Vitros, and Euroimmun). Consensus-positive and consensus-negative interpretations were defined as qualitative agreement in at least 3 of the 4 assays. Agreement of the 4 individual assays with a consensus-negative interpretation (n = 610) ranged from 96.7% to 100%, and agreement with a consensus-positive interpretation (n = 584) ranged from 94.3% to 100%. Laboratory-developed inhibition assays were utilized to evaluate 49 consensus-negative samples that were positive in only one assay; true-positive reactivity was confirmed in only 2 of these 49 (4%) samples. These findings demonstrate very high levels of agreement among 4 SARS-CoV-2 IgG assays authorized for emergency use, regardless of antigen target or assay format. Although false-positive reactivity was identified, its occurrence was rare (no more than 1.7% of samples for a given assay).

Performance of the Roche cobas MTB Assay for the Molecular Diagnosis of Pulmonary Tuberculosis in a High HIV Burden Setting.

Increased access to and improved sensitivities of methods for diagnosing Mycobacterium tuberculosis infection and detecting rifampicin and isoniazid resistance are needed. Herein, the performance of the new cobas MTB assay for use on cobas 6800/8800 Systems (Roche) was assessed and compared with two other commercial assays: RealTime MTB (Abbott), and Xpert MTB/RIF (Cepheid). Molecular PCR-based assays were conducted on sputum specimens from individuals with presumptive and confirmed tuberculosis (n = 294) from two clinical facilities in South Africa between December 2016 and October 2017. Liquid mycobacterial culture was the reference. Test sensitivities were 94.7% (95% CI, 88%-98%), 92.6% (95% CI, 85%-97%), and 91.6% (95% CI, 84%-96%) for cobas MTB, RealTime MTB, and Xpert MTB/RIF assays, respectively. cobas MTB sensitivity was unaffected by HIV coinfection (95.7%; 95% CI, 88%-99%; n = 176) and sediment testing (94.7%; 95% CI, 88%-98%). Sensitivities were 81.8% (95% CI, 60%-95%), 72.7% (95% CI, 50%-89%), and 72.7% (95% CI, 50%-89%) among smear-negative, culture-positive individuals (n = 221) for cobas MTB, RealTime MTB, and Xpert MTB/RIF assays, respectively. cobas MTB specificity was 95.7% (95% CI, 89%-99%) and 99% (95% CI, 94%-100%) among HIV coinfected and uninfected individuals, respectively. The cobas 6800/8800 system is already implemented in South Africa for high-throughput HIV viral load testing, making it suitable for integrated HIV/tuberculosis diagnostics.