Defining the value of medical microbiology consultation.

Medical microbiologists, defined as doctoral-level laboratory directors with subspecialty training in medical microbiology, lead the clinical laboratory operations through activities such as clinical consultations, oversight of diagnostic testing menu, institutional leadership, education, and scholastic activities. However, unlike their clinical colleagues, medical microbiologists are largely unable to bill for clinical consultations performed within the hospital and, therefore, unable to generate relative value units or a similar quantifiable metric. As hospital budgets tighten and justification of staffing becomes a necessity, this may present a challenge to the medical microbiologist attempting to prove their value to the organization. To aid in providing tangible data, the Personnel Standards and Workforce subcommittee of the American Society for Microbiology conducted a multi-center study across seven medical centers to document clinical consultations and their impact. Consults were generated equally from internal (laboratory-based) and external (hospital-based) parties, with the majority directly impacting patient management. Near universal acceptance of the medical microbiologist's recommendation highlights the worth derived from their expertise. External consults required more time commitment from the medical microbiologist than internal consults, although both presented ample opportunity for secondary value, including impact through stewardship, education, clinical guidance, and cost reduction. This study is a description of the content and impact of consultations that underscore the importance of the medical microbiologist as a key member of the healthcare team. IMPORTANCE: Medical microbiologists are invaluable to the clinical microbiology laboratory and the healthcare system as a whole. However, as medical microbiologists do not regularly generate relative value units, capturing and quantifying the value provided is challenging. As hospital budgets tighten, justification of staffing becomes a necessity. To aid in providing tangible data, the Personnel Standards and Workforce subcommittee of the American Society for Microbiology conducted a multi-center study across seven medical centers to document clinical consultations and their impact. To our knowledge, this is the first study to provide detailed evaluation of the consultative value provided by medical microbiologists.

Status check: next-generation sequencing for infectious-disease diagnostics.

Next-generation sequencing (NGS) applications for the diagnostics of infectious diseases has demonstrated great potential with three distinct approaches: whole-genome sequencing (WGS), targeted NGS (tNGS), and metagenomic NGS (mNGS, also known as clinical metagenomics). These approaches provide several advantages over traditional microbiologic methods, though challenges still exist.

SARS-CoV-2 evolution during prolonged infection in immunocompromised patients.

Prolonged infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in immunocompromised patients provides an opportunity for viral evolution, potentially leading to the generation of new pathogenic variants. To investigate the pathways of viral evolution, we carried out a study on five patients experiencing prolonged SARS-CoV-2 infection (quantitative polymerase chain reaction-positive for 79-203 days) who were immunocompromised due to treatment for lymphoma or solid organ transplantation. For each timepoint analyzed, we generated at least two independent viral genome sequences to assess the heterogeneity and control for sequencing error. Four of the five patients likely had prolonged infection; the fifth apparently experienced a reinfection. The rates of accumulation of substitutions in the viral genome per day were higher in hospitalized patients with prolonged infection than those estimated for the community background. The spike coding region accumulated a significantly greater number of unique mutations than other viral coding regions, and the mutation density was higher. Two patients were treated with monoclonal antibodies (bebtelovimab and sotrovimab); by the next sampled timepoint, each virus population showed substitutions associated with monoclonal antibody resistance as the dominant forms (spike K444N and spike E340D). All patients received remdesivir, but remdesivir-resistant substitutions were not detected. These data thus help elucidate the trends of emergence, evolution, and selection of mutational variants within long-term infected immunocompromised individuals. IMPORTANCE: SARS-CoV-2 is responsible for a global pandemic, driven in part by the emergence of new viral variants. Where do these new variants come from? One model is that long-term viral persistence in infected individuals allows for viral evolution in response to host pressures, resulting in viruses more likely to replicate efficiently in humans. In this study, we characterize replication in several hospitalized and long-term infected individuals, documenting efficient pathways of viral evolution.

Detection and quantification of Babesia species intraerythrocytic parasites by flow cytometry.

OBJECTIVES: Recent work has demonstrated that automated fluorescence flow cytometry (FLC) is a potential alternative for the detection and quantification of Plasmodium parasites. The objective of this study was to apply this novel FLC method to detect and quantify Babesia parasites in venous blood and compare results to light microscopy and polymerase chain reaction methods. METHODS: An automated hematology/malaria analyzer (XN-31; Sysmex) was used to detect and quantify B microti-infected red blood cells from residual venous blood samples (n = 250: Babesia positive, n = 170; Babesia negative, n = 80). As no instrument software currently exists for Babesia, qualitative and quantitative machine learning (ML) algorithms were developed to facilitate analysis. RESULTS: Performance of the ML models was verified against the XN-31 software using P falciparum-infected samples. When applied to Babesia-infected samples, the qualitative ML model demonstrated an area under the curve (AUC) of 0.956 (sensitivity, 95.9%; specificity, 83.3%) relative to polymerase chain reaction. For valid scattergrams, the qualitive model achieved an AUC of 1.0 (sensitivity and specificity, 100%), while the quantitative model demonstrated an AUC of 0.986 (sensitivity, 94.4%; specificity, 100%). CONCLUSIONS: This investigation demonstrates that Babesia parasites can be detected and quantified directly from venous blood using FLC. Although promising, opportunities remain to improve the general applicability of the method.

Development of a scoring system to identify high-yield specimens for bacterial broad-range 16S rRNA gene PCR with sequencing at a tertiary care medical center.

OBJECTIVES: Broad-range bacterial polymerase chain reaction with sequencing (BRBPS) provides valuable diagnostic data, especially in cases of culture-negative infections. However, as BRBPS testing demonstrates generally low positivity, cost per impactful result can be high and commonly involves longer turnaround times compared with other methods, targeting use of this assay to high-yield situations remains a challenging goal. Diagnostic stewardship can help alleviate these challenges and increase clinical utility, yet not all laboratories have a dedicated stewardship team, and little formal guidance exists on identifying high-yield samples outside of specific clinical syndromes. In this study, we performed a retrospective review of 86 BRBPS orders from a tertiary care medical center, with a focus on identifying high-yield cases using an infectious markers scoring system, visualized inflammation or organism (VIO) score, to predict return of actionable diagnostic data. METHODS: Using chart review, we evaluated how results from high VIO score or low VIO score specimens contributed to clinical management. RESULTS: Testing low VIO score samples identified an organism in only 10% of samples, and of these positive results, 33% were considered to represent contamination. Despite negative routine workup and no identified pathogen via BRBPS, broad antimicrobial treatment was continued in 85% of cases with a low VIO score. In contrast, specimens with high VIO scores were more predictably positive by BRBPS, identified organisms that were universally considered pathogens, and provided opportunities to target or de-escalate antimicrobial therapy. CONCLUSIONS: This study describes the VIO scoring system to guide the identification of high-yield samples and steward the appropriate use of BRBPS testing.

When to Think About Other Borreliae:: Hard Tick Relapsing Fever (Borrelia miyamotoi), Borrelia mayonii, and Beyond.

In North America, several hard tick-transmitted Borrelia species other than Borrelia burgdorferi cause human disease, including Borrelia miyamotoi, Borrelia mayonii, and possibly Borrelia bissettii. Due to overlapping clinical syndromes, nonspecific tickborne disease (TBD) testing strategies, and shared treatment approaches, infections with these lesser known Borrelia are likely under-reported. In this article, we describe the epidemiology, clinical manifestations, diagnosis, and treatment of these less common Borrelia pathogens.

Multiple Introductions of SARS-CoV-2 Alpha and Delta Variants into White-Tailed Deer in Pennsylvania.

The SARS-CoV-2 pandemic began by viral spillover from animals to humans; today multiple animal species are known to be susceptible to infection. White-tailed deer, Odocoileus virginianus, are infected in North America at substantial levels, and genomic data suggests that a variant in deer may have spilled back to humans. Here, we characterize SARS-CoV-2 in deer from Pennsylvania (PA) sampled during fall and winter 2021. Of 123 nasal swab samples analyzed by RT-qPCR, 20 (16.3%) were positive for SARS-CoV-2. Seven whole genome sequences were obtained, together with six more partial spike gene sequences. These annotated as alpha and delta variants, the first reported observations of these lineages in deer, documenting multiple new jumps from humans to deer. The alpha lineage persisted in deer after its displacement by delta in humans, and deer-derived alpha variants diverged significantly from those in humans, consistent with a distinctive evolutionary trajectory in deer. IMPORTANCE Coronaviruses have been documented to replicate in numerous species of vertebrates, and multiple spillovers of coronaviruses from animals into humans have founded human epidemics. The COVID-19 epidemic likely derived from a spillover of SARS-CoV-2 from bats into humans, possibly via an intermediate host. There are now several examples of SARS-CoV-2 jumping from humans into other mammals, including mink and deer, creating the potential for new animal reservoirs from which spillback into humans could occur. For this reason, data on formation of new animal reservoirs is of great importance for understanding possible sources of future infection. Here, we identify extensive infection in white-tailed deer in Pennsylvania, including what appear to be multiple independent transmissions. Data further suggests possible transmission among deer. These data thus help identify a potential new animal reservoir and provide background information relevant to its management.

Novel Assays for Molecular Detection of Severe Acute Respiratory Syndrome Coronavirus 2.

From the onset of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)/COVID-19 pandemic, there has been a major emphasis on molecular laboratory tests for the virus. Shortages in various testing supplies, the desire to increase testing capacity, and a push to make point-of-care or home-based testing available have fostered considerable innovation for SARS-CoV-2 molecular diagnostics, advancements likely to be applicable to other diagnostic uses. The authors attempt to cover some of the most compelling novel types of molecular assays or novel approaches in adapting established molecular methodologies for SARS-CoV-2 detection or characterization.

Partial ORF1ab Gene Target Failure with Omicron BA.2.12.1.

Mutations in the genome of SARS-CoV-2 can affect the performance of molecular diagnostic assays. In some cases, such as S-gene target failure, the impact can serve as a unique indicator of a particular SARS-CoV-2 variant and provide a method for rapid detection. Here, we describe partial ORF1ab gene target failure (pOGTF) on the cobas SARS-CoV-2 assays, defined by a ≥2-thermocycle delay in detection of the ORF1ab gene compared to that of the E-gene. We demonstrate that pOGTF is 98.6% sensitive and 99.9% specific for SARS-CoV-2 lineage BA.2.12.1, an emerging variant in the United States with spike L452Q and S704L mutations that may affect transmission, infectivity, and/or immune evasion. Increasing rates of pOGTF closely mirrored rates of BA.2.12.1 sequences uploaded to public databases, and, importantly, increasing local rates of pOGTF also mirrored increasing overall test positivity. Use of pOGTF as a proxy for BA.2.12.1 provides faster tracking of the variant than whole-genome sequencing and can benefit laboratories without sequencing capabilities.

Partial ORF1ab Gene Target Failure with Omicron BA.2.12.1.

Mutations in the viral genome of SARS-CoV-2 can impact the performance of molecular diagnostic assays. In some cases, such as S gene target failure, the impact can serve as a unique indicator of a particular SARS-CoV-2 variant and provide a method for rapid detection. Here we describe partial ORF1ab gene target failure (pOGTF) on the cobas ® SARS-CoV-2 assays, defined by a ≥2 thermocycles delay in detection of the ORF1ab gene compared to the E gene. We demonstrate that pOGTF is 97% sensitive and 99% specific for SARS-CoV-2 lineage BA.2.12.1, an emerging variant in the United States with spike L452Q and S704L mutations that may impact transmission, infectivity, and/or immune evasion. Increasing rates of pOGTF closely mirrored rates of BA.2.12.1 sequences uploaded to public databases, and, importantly increasing local rates of pOGTF also mirrored increasing overall test positivity. Use of pOGTF as a proxy for BA.2.12.1 provides faster tracking of the variant than whole-genome sequencing and can benefit laboratories without sequencing capabilities.

SARS-CoV-2 Delta Variant (AY.3) in the Feces of a Domestic Cat.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections have spilled over from humans to companion and wild animals since the inception of the global COVID-19 pandemic. However, whole genome sequencing data of the viral genomes that infect non-human animal species have been scant. Here, we detected and sequenced a SARS-CoV-2 delta variant (AY.3) in fecal samples from an 11-year-old domestic house cat previously exposed to an owner who tested positive for SARS-CoV-2. Molecular testing of two fecal samples collected 7 days apart yielded relatively high levels of viral RNA. Sequencing of the feline-derived viral genomes showed the two to be identical, and differing by between 4 and 14 single nucleotide polymorphisms in pairwise comparisons to human-derived lineage AY.3 sequences collected in the same geographic area and time period. However, several mutations unique to the feline samples reveal their divergence from this cohort on phylogenetic analysis. These results demonstrate continued spillover infections of emerging SARS-CoV-2 variants that threaten human and animal health, as well as highlight the importance of collecting fecal samples when testing for SARS-CoV-2 in animals. To the authors' knowledge, this is the first published case of a SARS-CoV-2 delta variant in a domestic cat in the United States.

Detection and Interspecies Comparison of SARS-CoV-2 Delta Variant (AY.3) in Feces from a Domestic Cat and Human Samples.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections have spilled over from humans to companion and wild animals since the inception of the global COVID-19 pandemic. However, whole genome sequencing data of the viral genomes that infect non-human animal species has been scant. Here, we detected and sequenced a SARS-CoV-2 delta variant (AY.3) in fecal samples from an 11-year-old domestic house cat previously exposed to an owner who tested positive for SARS-CoV-2. Molecular testing of two fecal samples collected 7 days apart yielded relatively high levels of viral RNA. Sequencing of the feline-derived viral genomes showed the two to be identical, and differing by between 4 and 14 single nucleotide polymorphisms in pairwise comparisons to human-derived lineage AY.3 sequences collected in the same geographic area and time period. However, several mutations unique to the feline samples reveal their divergence from this cohort on phylogenetic analysis. These results demonstrate continued spillover infections of emerging SARS-CoV-2 variants that threaten human and animal health, as well as highlight the importance of collecting fecal samples when testing for SARS-CoV-2 in animals. To the authors' knowledge, this is the first published case of a SARS-CoV-2 delta variant in a domestic cat in the United States.

Collaboration between Clinical and Academic Laboratories for Sequencing SARS-CoV-2 Genomes.

Genomic sequencing of severe acute respiratory syndrome coronavirus 2 (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 genome sequences have been deposited in Global Initiative on Sharing All Influenza Data (GISAID), but contributions from the United States, particularly through 2020, lagged the global effort. The primary goal of clinical microbiology laboratories is seldom rooted in epidemiologic or public health testing, and many laboratories 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 laboratories, outline best practices, describe two divergent strategies in accomplishing a similar goal, and discuss the challenges with implementing and maintaining such programs.

Novel Applications of Metagenomics for Detection of Tickborne Pathogens.

BACKGROUND: Tick populations have expanded in many parts of the globe, bringing with them an enhanced appreciation and discovery of novel tickborne pathogens, as well an increased in reported human cases of tickborne disease. Targeted and unbiased (shotgun) clinical metagenomic sequencing tests are increasingly used for detection of known and emerging infectious agents and have recently been employed for detection of tickborne pathogens. CONTENT: This review describes the types of metagenomic sequencing assays used for detection of emerging tickborne pathogens and reviews the recent literature on this topic. Important diagnostic and interpretative challenges are also covered. SUMMARY: Metagenomic analysis has emerged as a powerful tool for detection, discovery, characterization, and classification of tickborne pathogens. Shotgun metagenomics is especially promising because it allows for detection of all tickborne bacteria, viruses, and parasites in a single specimen. Despite the potential advantages, there are several important challenges, including high cost, complexity of testing and interpretation, and slow turnaround time. No doubt, these challenges will diminish with increased use and advances in the field.

SARS-CoV-2 variants associated with vaccine breakthrough in the Delaware Valley through summer 2021.

The severe acute respiratory coronavirus-2 (SARS-CoV-2) is the cause of the global outbreak of COVID-19. Evidence suggests that the virus is evolving to allow efficient spread through the human population, including vaccinated individuals. Here we report a study of viral variants from surveillance of the Delaware Valley, including the city of Philadelphia, and variants infecting vaccinated subjects. We sequenced and analyzed complete viral genomes from 2621 surveillance samples from March 2020 to September 2021 and compared them to genome sequences from 159 vaccine breakthroughs. In the early spring of 2020, all detected variants were of the B.1 and closely related lineages. A mixture of lineages followed, notably including B.1.243 followed by B.1.1.7 (alpha), with other lineages present at lower levels. Later isolations were dominated by B.1.617.2 (delta) and other delta lineages; delta was the exclusive variant present by the last time sampled. To investigate whether any variants appeared preferentially in vaccine breakthroughs, we devised a model based on Bayesian autoregressive moving average logistic multinomial regression to allow rigorous comparison. This revealed that B.1.617.2 (delta) showed three-fold enrichment in vaccine breakthrough cases (odds ratio of 3; 95% credible interval 0.89-11). Viral point substitutions could also be associated with vaccine breakthroughs, notably the N501Y substitution found in the alpha, beta and gamma variants (odds ratio 2.04; 95% credible interval of 1.25-3.18). This study thus provides a detailed picture of viral evolution in the Delaware Valley and a geographically matched analysis of vaccine breakthroughs; it also introduces a rigorous statistical approach to interrogating enrichment of viral variants.

Detection of Blastomyces dermatitidis Antigen in Urine Using a Commercially Available Quantitative Enzyme Immunoassay.

Laboratory diagnosis of blastomycosis relies on a combination of methods, including antigen detection. We assessed the performance of analyte-specific reagents from Gotham Biotech (Portland, ME) for quantitative detection of Blastomyces dermatitidis galactomannan (GM) in urine using an enzyme immunoassay (EIA) compared to the Blastomyces quantitative EIA from MiraVista Diagnostics (Indianapolis, IN). Residual urine from 232 unique patients previously tested by the MiraVista assay was evaluated using the Gotham EIA, which showed 97.4% (74/76), 100% (156/156), and 99.1% (230/232) positive, negative, and overall agreement, respectively. Correlation between the quantitative B. dermatitidis antigen levels by the Gotham and MiraVista EIAs was low (R2 = 0.20). Medical records were available for 36 of the 232 patients, among whom four had confirmed blastomycosis and both the Gotham and MiraVista EIAs were positive. Nine of these patients had histoplasmosis, and the Gotham and MiraVista EIAs yielded negative results in 44.4% (4/9) and 22.2% (2/9) of cases, respectively. Both assays were negative in the remaining 23 patients. After laboratory implementation of the Gotham EIA, chart reviews were performed on the first 50 unique patients (51 samples) tested by the assay in our hospital. Among these, 3/50 (6%) samples were positive by the Gotham EIA, including two samples from a patient with culture-confirmed blastomycosis and one from a patient with histoplasmosis (also positive by the MiraVista Blastomyces EIA). All remaining patients were negative by the Gotham EIA and had alternative diagnoses. Our findings show comparable performance between the Gotham and MiraVista quantitative EIAs for detection of B. dermatitidis GM in urine.

Femtomolar SARS-CoV-2 Antigen Detection Using the Microbubbling Digital Assay with Smartphone Readout Enables Antigen Burden Quantitation and Tracking.

BACKGROUND: High-sensitivity severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen assays are desirable to mitigate false negative results. Limited data are available to quantify and track SARS-CoV-2 antigen burden in respiratory samples from different populations. METHODS: We developed the Microbubbling SARS-CoV-2 Antigen Assay (MSAA) with smartphone readout, with a limit of detection of 0.5 pg/mL (10.6 fmol/L) nucleocapsid antigen or 4000 copies/mL inactivated SARS-CoV-2 virus in nasopharyngeal (NP) swabs. We developed a computer vision and machine learning-based automatic microbubble image classifier to accurately identify positives and negatives and quantified and tracked antigen dynamics in intensive care unit coronavirus disease 2019 (COVID-19) inpatients and immunocompromised COVID-19 patients. RESULTS: Compared to qualitative reverse transcription-polymerase chain reaction methods, the MSAA demonstrated a positive percentage agreement of 97% (95% CI 92%-99%) and a negative percentage agreement of 97% (95% CI 94%-100%) in a clinical validation study with 372 residual clinical NP swabs. In immunocompetent individuals, the antigen positivity rate in swabs decreased as days-after-symptom-onset increased, despite persistent nucleic acid positivity. Antigen was detected for longer and variable periods of time in immunocompromised patients with hematologic malignancies. Total microbubble volume, a quantitative marker of antigen burden, correlated inversely with cycle threshold values and days-after-symptom-onset. Viral sequence variations were detected in patients with long duration of high antigen burden. CONCLUSIONS: The MSAA enables sensitive and specific detection of acute infections and quantification and tracking of antigen burden and may serve as a screening method in longitudinal studies to identify patients who are likely experiencing active rounds of ongoing replication and warrant close viral sequence monitoring.