Proof of concept: Detection of cell free RNA from EDTA plasma in patients with lung cancer and non-cancer patients.

INTRODUCTION: Nucleic acid sequencing technologies have advanced significantly in recent years, thereby allowing for the development of liquid biopsies as new means to detect cancer biomarkers and cancer heterogenicity. Most of the assays available, clinically, focus on cell free DNA (cfDNA), however, cell free RNA (cfRNA) is also present. cfRNA has the potential to complement and improve cancer detection especially in cancers like lung cancer, which are usually only diagnosed at late stages and therefore have poor long-term survival outcomes. METHODS: Remnant EDTA plasma was collected from lung cancer patients and non-cancer individuals at the University of Maryland Medical Center. RNA was extracted and processed for next generation sequencing with a tagmentation-based library preparation approach. RESULTS: cfRNA was successfully extracted and sequenced from 52 EDTA-treated plasma samples with volumes as low as 1.5 mL. This quantity was sufficient to prepare libraries with the length of libraries averaging from 264 bp to 381 bp and resulted in over 2.2 to 3.6 million total sequence reads respectively. Sequential dilution of cfRNA samples from healthy individuals indicated that the starting cfRNA concentration influenced the detection of differentially expressed genes. CONCLUSIONS: This proof-of-concept study provides a framework for screening cfRNA for identifying biomarkers for early detection of lung cancer (and other cancers), using minimal amounts of samples (1.5 mL) from standard EDTA 3-mL collection tubes routinely used for patient care. Further studies in large populations are required to establish limit of detection and other parameters including precision, accuracy, sensitivity, and specificity, to standardize this method.

Kinetics of SARS-CoV-2 antibody responses pre-COVID-19 and post-COVID-19 convalescent plasma transfusion in patients with severe respiratory failure: an observational case-control study.

AIMS: While the SARS-CoV-2 pandemic may be contained through vaccination, transfusion of convalescent plasma (CCP) from individuals who recovered from COVID-19 (CCP) is considered an alternative treatment. We investigate if CCP transfusion in patients with severe respiratory failure increases plasma titres of SARS-CoV-2 antibodies and improves clinical outcomes. METHODS: Patients with COVID-19 (n=34) were consented for CCP transfusion and serial blood draws pretransfusion and post-transfusion. Plasma SARS-CoV-2 antireceptor binding domain (RBD) IgG and IgM titres were measured by ELISA serially, and compared with serial plasma titre levels from control patients (n=68). The primary outcome was survival at 30 days, and secondary outcomes were length of ventilator and/or extracorporeal membrane oxygenation (ECMO) support, length of stay (LOS) in the hospital and in the intensive care unit (ICU). Outcomes were compared with matched control patients (n=34). Kinetics of antibodies and clinical outcomes were compared using LOess regression and ORs, respectively. RESULTS: Prior to CCP transfusion, 74% of patients were anti-RBD seropositive for IgG (median 1:3200), and 81% were anti-RBD IgM seropositive (median 1:320), while 16% were seronegative. The kinetics of antibody titres in CCP recipients were similar to controls. CCP recipients presented with similar survival, duration on ventilatory and/or ECMO support, as well as ICU and hospital LOS compared with controls. CONCLUSIONS: CCP transfusion did not increase the kinetics of SARS-CoV2 antibodies and did not result in improved clinical outcomes in patients with COVID-19 with severe respiratory failure, suggesting that CCP may not be indicated in this category of patients.

Renin Kinetics Are Superior to Lactate Kinetics for Predicting In-Hospital Mortality in Hypotensive Critically Ill Patients.

OBJECTIVES: Whole blood lactate concentration is widely used in shock states to assess perfusion. We aimed to determine if the change in plasma renin concentration over time would be superior to the change in lactate concentration for predicting in-hospital mortality in hypotensive patients on vasopressors. DESIGN: Prospective, observational cohort study. SETTING: Tertiary academic ICU. PATIENTS: Adult patients on vasopressors for greater than 6 hours to maintain a mean arterial pressure greater than or equal to 65 mm Hg during January 2020. INTERVENTIONS: Plasma renin concentrations were measured at enrollment and at 24, 48, and 72 hours. Whole blood lactate measurements were performed according to normal standard of care. Logistic regression was performed to evaluate whether the change in renin or lactate concentration could predict in-hospital mortality. Generalized estimating equations were used to analyze the association between renin and lactate concentration and in-hospital mortality. The area under the receiver operating characteristics curve was performed to measure the discriminative ability of initial and peak renin and lactate concentration to predict mortality. The association between renin and lactate concentration above the upper limit of normal at each timepoint with in-hospital mortality was also examined. MEASUREMENTS AND MAIN RESULTS: The study included 197 renin and 148 lactate samples obtained from 53 patients. The slope of the natural log (ln) of renin concentration was independently associated with mortality (adjusted odds ratio, 10.35; 95% CI, 1.40-76.34; p = 0.022), but the slope of ln-lactate concentration was not (adjusted odds ratio, 4.78; 95% CI, 0.03-772.64; p = 0.55). The generalized estimating equation models found that both ln-renin (adjusted odds ratio, 1.18; 95% CI, 1.02-1.37; p = 0.025) and ln-lactate (adjusted odds ratio, 2.38; 95% CI, 1.05-5.37; p = 0.037) were associated with mortality. Area under the receiver operating characteristics curve analysis demonstrated that initial renin could predict in-hospital mortality with fair discrimination (area under the receiver operating characteristics curve, 0.682; 95% CI, 0.503-0.836; p = 0.05), but initial lactate could not (area under the receiver operating characteristics curve, 0.615; 95% CI, 0.413-0.803; p = 0.27). Peak renin (area under the receiver operating characteristics curve, 0.728; 95% CI, 0.547-0.888; p = 0.01) and peak lactate (area under the receiver operating characteristics curve, 0.746; 95% CI, 0.584-0.876; p = 0.01) demonstrated moderate discrimination. There was no significant difference in discriminative ability between initial or peak renin and lactate concentration. At each study time point, a higher proportion of renin values exceeded the threshold of normal (40 pg/mL) in nonsurvivors than in survivors, but this association was not significant for lactate. CONCLUSIONS: Although there was no significant difference in the performance of renin and lactate when examining the absolute values of each laboratory, a positive rate of change in renin concentration, but not lactate concentration, over 72 hours was associated with in-hospital mortality. For each one-unit increase in the slope of ln-renin, the odds of mortality increased 10-fold. Renin levels greater than 40 pg/mL, but not lactate levels greater than 2 mmol/L, were associated with in-hospital mortality. These findings suggest that plasma renin kinetics may be superior to lactate kinetics in predicting mortality of hypotensive, critically ill patients.

Performance of 4 Automated SARS-CoV-2 Serology Assay Platforms in a Large Cohort Including Susceptible COVID-19-Negative and COVID-19-Positive Patients.

BACKGROUND: Anti-SARS-CoV-2 serological responses may have a vital role in controlling the spread of the disease. However, the comparative performance of automated serological assays has not been determined in susceptible patients with significant comorbidities. METHODS: In this study, we used large numbers of samples from patients who were negative (n = 2030) or positive (n = 112) for COVID-19 to compare the performance of 4 serological assay platforms: Siemens Healthineers Atellica IM Analyzer, Siemens Healthineers Dimension EXL Systems, Abbott ARCHITECT, and Roche cobas. RESULTS: All 4 serology assay platforms exhibited comparable negative percentage of agreement with negative COVID-19 status ranging from 99.2% to 99.7% and positive percentage of agreement from 84.8% to 87.5% with positive real-time reverse transcriptase PCR results. Of the 2142 total samples, only 38 samples (1.8%) yielded discordant results on one or more platforms. However, only 1.1% (23/2030) of results from the COVID-19-negative cohort were discordant. whereas discordance was 10-fold higher for the COVID-19-positive cohort, at 11.3% (15/112). Of the total 38 discordant results, 34 were discordant on only one platform. CONCLUSIONS: Serology assay performance was comparable across the 4 platforms assessed in a large population of patients who were COVID-19 negative with relevant comorbidities. The pattern of discordance shows that samples were discordant on a single assay platform, and the discordance rate was 10-fold higher in the population that was COVID-19 positive.

Validation of COVID-19 serologic tests and large scale screening of asymptomatic healthcare workers.

OBJECTIVES: Serologic testing for SARS-CoV-2 is an important element in the fight to slow the COVID-19 pandemic. This study aimed to validate two serologic tests for total (IgM, IgG, IgA) SARS-CoV-2 antibodies, (i) the Ortho-Clinical Diagnostics Anti-SARS-CoV-2 Total Antibody assay for the Vitros 5600 analyzers and (ii) a manual laboratory developed ELISA (FDA EUA pending), for use in parallel orthogonal testing of asymptomatic healthcare workers and affiliates of the University of Maryland Medical System. DESIGN AND METHODS: Validation and verification of the two tests was performed using samples from hospitalized patients that were found to be PCR positive for SARS-CoV-2, samples pre-COVID-19, and samples from individuals with current/previous infections with other viruses. Healthcare workers and affiliates from across the University of Maryland Health System were provided testing free of charge and their results were reported as reactive or non-reactive if the two tests were concordance, or indeterminate if the results were discordant. RESULTS: Validation testing found the Ortho Vitros test to be 100% (73/73) sensitive, and 99.3% (152/153) specific, while the UMMC ELISA was found to be 97.6% (204/209) sensitive and 100% (288/288) specific. Real world testing among 8399 healthcare workers found that 2.9% (247/8399) of healthcare workers were positive for anti- SARS-CoV-2 antibodies by both tests. An indeterminate rate of 1.1% (91/8399), in which one test reported reactive results, and one as non-reactive was also seen. CONCLUSIONS: Parallel orthogonal testing improves the positive and negative predictive value of serologic testing in populations with low prevalence. The use of an indeterminate result from parallel orthogonal testing allows for the follow-up and re-testing, which helps resolve discrepancies between assays.

Optimal Detection of Acute Myocardial Injury and Infarction with Cardiac Troponin: Beyond the 99th Percentile, into the High-Sensitivity Era.

PURPOSE OF REVIEW: Cardiac troponin (cTn) is the biomarker of choice for the diagnosis of acute myocardial infarction (MI); use of this biomarker has centered around the 99th percentile upper reference limit (URL) for healthy populations. Recent development and regulatory approval of high-sensitivity cardiac troponin (hs-cTn) assays have required a fresh look at utilization of the 99th percentile URL. This review covers issues regarding the 99th percentile URL and approaches for use of cTn as biomarker for detecting cardiac injury and diagnosis of acute myocardial infarction. RECENT FINDINGS: Development of hs-cTn assays has allowed determination and use of sex-specific 99th percentile URLs for assessing cardiac injury and increased the utility of cTn values below the 99th percentile URL. This improved analytical performance for hs-cTn assays has allowed for development of accelerated diagnostic protocols (ADPs) for rapid assessment and disposition of patients based on serial sampling of cTn for use in acute MI diagnosis as soon as 0-1 h after clinical presentation. The 99th percentile URLs of cTn is essential for detecting cardiac injury; however, use of the 99th percentile URLs in the era of hs-cTn results may be modified. ADPs have the potential to substantially decrease the time many patients spend under evaluation for acute MI, thereby potentiating improvement in patient satisfaction, decreased healthcare costs, and reducing the burden on emergency departments.

Analytical and clinical characterization of a novel high-sensitivity cardiac troponin assay in a United States population.

BACKGROUND: Cardiac troponin (cTn) is the keystone for diagnosis of acute myocardial infarction (AMI). We examined the analytical and clinical diagnostic characteristics of the ACCESS hsTnI assay in a United States (US) population. METHODS: All measurements and studies were conducted using a lithium heparin matrix. Sex-specific 99th percentile upper reference limits (URLs) were determined for 1089 healthy women (54.6%) and men using non-parametric statistics. High-sensitivity (hs) performance was assessed to determine if the total CV was ≤10% at sex-specific URLs, and if ≥50% of cTnI values for each sex exceeded the assay's limit of detection (LoD). Precision, analytical measurement range, high-dose hook effect, and endogenous/exogenous interferences were examined with CLSI guidance. Clinical characterization included serial sampling of 1854 suspected AMI subjects presenting to 14 US Emergency Departments. AMI was adjudicated by a panel of expert cardiologists. The study's only exclusion was end stage renal disease. RESULTS: 99th percentile URLs were 11.6-, 19.8- and 17.5-ng/L for respective female, male and all-subject populations. Total %CV was <8% from 6.8 to 19,000 ng/L, and <6% at sex-specific 99th percentiles; ≥99% of ACCESS hsTnI values for each sex exceeded the LoD. No high-dose hook effect or endogenous/exogenous interferences were identified. A comparison of Baseline samples collected at ≤1 h and any-time after presentation, found 4% lower sensitivity for AMI than with earlier sampling. For 1-9 h post presentation, the sensitivity was >90%, specificity >85%; and negative and positive predictive value were ≥99% and >60%, respectively. CONCLUSION: Analytical and clinical performance of the ACCESS hsTnI assay meets the definition of a hs cTn method. The ACCESS hsTnI assay has good precision over a wide range, no significant interferences, and sensitivity >90% and NPV ≥99%. Performance is appropriate for aiding in AMI diagnosis.

Rickettsial Infections among Cats and Cat Fleas in Riverside County, California.

Presently, few studies have investigated the role of domestic cats (Felis catus) in the recrudescence of flea-borne rickettsioses in California and the southern United States. In this study, we aimed to investigate the presence of Rickettsia typhi or Rickettisa felis in domestic cats (F. catus) and the fleas (primarily Ctenocephalides felis, the cat flea) associated with these cats in Riverside County, California. Thirty cats and 64 pools of fleas collected from these cats were investigated for rickettsial infections. Three cats and 17 flea pools (from 10 cats) tested positive for rickettsial infections. polymerase chain reaction and DNA sequencing indicated that one of the cats was positive for R. felis infections, whereas two were positive for Candidatus Rickettsia senegalensis infection. In addition, 12 of the flea pools were positive for R. felis, whereas five were positive for Ca. R. senegalensis. By contrast, no cats or their associated fleas tested positive for R. typhi. Finally, eight sera from these cats contained spotted fever group Rickettsia (SFGR) antibodies. The detection of R. felis and SFGR antibodies and the lack of R. typhi and TGR antibodies support R. felis as the main rickettsial species infecting cat fleas. The detection of Ca. R. senegalensis in both fleas and cats also provides additional evidence that cats and their associated fleas are infected with other R. felis-like organisms highlighting the potential risk for human infections with R. felis or R. felis-like organisms.

Whole-Genome Analysis of Bartonella ancashensis, a Novel Pathogen Causing Verruga Peruana, Rural Ancash Region, Peru.

The genus Bartonella contains >40 species, and an increasing number of these Bartonella species are being implicated in human disease. One such pathogen is Bartonella ancashensis, which was isolated in blood samples from 2 patients living in Caraz, Peru, during a clinical trial of treatment for bartonellosis. Three B. ancashensis strains were analyzed by using whole-genome restriction mapping and high-throughput pyrosequencing. Genome-wide comparative analysis of Bartonella species showed that B. ancashensis has features seen in modern and ancient lineages of Bartonella species and is more related to B. bacilliformis. The divergence between B. ancashensis and B. bacilliformis is much greater than what is seen between known Bartonella genetic lineages. In addition, B. ancashensis contains type IV secretion system proteins, which are not present in B. bacilliformis. Whole-genome analysis indicates that B. ancashensis might represent a distinct Bartonella lineage phylogenetically related to B. bacilliformis.

Complete Genome Sequence of Bartonella ancashensis Strain 20.00, Isolated from the Blood of a Patient with Verruga Peruana.

Here we present the complete genome sequence of Bartonella ancashensis strain 20.00, isolated from the blood of a Peruvian patient with verruga peruana, known as Carrion's disease. Bartonella ancashensis is a Gram-negative bacillus, phylogenetically most similar to Bartonella bacilliformis, the causative agent of Oroya fever and verruga peruana.

Description of Bartonella ancashensis sp. nov., isolated from the blood of two patients with verruga peruana.

Three novel isolates of the genus Bartonella were recovered from the blood of two patients enrolled in a clinical trial for the treatment of chronic stage Bartonella bacilliformis infection (verruga peruana) in Caraz, Ancash, Peru. The isolates were initially characterized by sequencing a fragment of the gltA gene, and found to be disparate from B. bacilliformis. The isolates were further characterized using phenotypic and genotypic methods, and found to be genetically identical to each other for the genes assessed, but distinct from any known species of the genus Bartonella, including the closest relative B. bacilliformis. Other characteristics of the isolates, including their morphology, microscopic and biochemical properties, and growth patterns, were consistent with members of the genus Bartonella. Based on these results, we conclude that these three isolates are members of a novel species of the genus Bartonella for which we propose the name Bartonella ancashensis sp. nov. (type strain 20.00T = ATCC BAA-2694T = DSM 29364T).

Molecular typing of "Candidatus Bartonella ancashi," a new human pathogen causing verruga peruana.

A recently described clinical isolate, "Candidatus Bartonella ancashi," was obtained from a blood sample of a patient presenting with verruga peruana in the Ancash region of Peru. This sample and a second isolate obtained 60 days later from the same patient were molecularly typed using multilocus sequence typing (MLST) and multispacer sequence typing (MST). The isolates were 100% indistinguishable from each other but phylogenetically distant from Bartonella bacilliformis and considerably divergent from other known Bartonella species, confirming their novelty.