Specimen Source Research Articles

Antibiotic Overuse in Pediatric Patients Hospitalized with RSV, COVID-19, and Influenza

Abstract Background Children hospitalized with viral respiratory tract infections (RTIs) are often treated with antibiotics due to concern for bacterial co-infection, although most do not have concurrent bacterial infections. This practice leads to unnecessary antibiotic treatment, which can lead to development of resistant bacteria and adverse events. The extent of antibiotic overuse in hospitalized children with community-onset viral RTI has not been well-quantified in recent years and may have worsened during the COVID-19 pandemic. The objectives of this study were to quantify and compare the extent of antibiotic overtreatment and to determine predictors of antibiotic use post-COVID-19 pandemic in children hospitalized with RSV, COVID-19, and influenza. Methods We performed a single center retrospective study evaluating percentages of antibiotic use and bacterial co-infection among children and adolescents aged <19 years old, hospitalized with respiratory syncytial virus (RSV), influenza, or SARS-CoV-2 between April 2020 and May 2023. Bacterial infection was defined as growth of bacteria from any specimen source, including blood, urine, pleural fluid, cerebrospinal fluid, and respiratory secretions, excluding common contaminants like coagulase-negative Staphylococci. Predictors of antibiotic treatment were determined using multivariable logistic regression. Results During the study period 1,718 patients were included (1022 with RSV, 188 with influenza, 535 with COVID-19). RSV patients were younger and more likely to be in the ICU than influenza or COVID-19 patients. The overall proportion of patients with culture-confirmed bacterial co-infection was low at 8% and did not differ greatly by virus type (8% for influenza, 6.9% for RSV, 10.3% for COVID-19, p=0.03). The proportion of children receiving antibiotics was higher for influenza than for other viruses (60.6% for influenza, 41.2% for RSV, and 48.6% for COVID-19, p < 0.001). Among patients with the lowest severity of illness (with normal inflammatory markers [PCT <0.5 ug/mL or CRP <50 mg/L] and not admitted to the ICU), 7.3% had a culture-confirmed bacterial infection while 48.1% received antibiotics. Independent predictors for receiving antibiotics were elevated inflammatory markers (C-reactive protein or procalcitonin), having >3 comorbidities, mechanical ventilation, and influenza diagnosis. Conclusion In children hospitalized with community-onset viral RTIs, antibiotic treatment is substantially higher than the burden of culture-confirmed bacterial infection, suggesting antibiotic overuse. Antibiotic use was greater for patients with influenza than for those with RSV or COVID-19. Antibiotic overuse was estimated to occur in over 40% of patients with low severity of illness. Stewardship programs can use this data to design interventions to promote more judicious antibiotic use. Figure 1. Rates of bacterial co-infection and antibiotic utilization among patients hospitalized with influenza, RSV, and COVID. Blue, percentage of patients who had a culture-confirmed bacterial infection; orange, percentage of patients who received any antibiotics; green, percentage of patients who received >3 days of antibiotics.

Evaluation of Self-collected Saliva Samples Without Viral Transport Media for SARS-CoV-2 Testing via RT-PCR and Comparison of Amplicon Sequences Against Commonly Used Primers in Diagnostic Assays.

Mass screening for SARS-CoV-2 using nasopharyngeal swabs (NPS) is costly, uncomfortable for patients, and increases the chance of virus exposure to health care workers. Therefore, this study focused on determining if self-collected unpreserved saliva can be an effective alternative to NPS collection in COVID-19 surveillance. In this study, patients being tested for SARS-CoV-2 using NPS were asked to provide a saliva sample to compare their results. NPS samples were evaluated for SARS-CoV-2 using BioFire® FilmArray® Torch® or Cepheid® GeneXpert® systems while saliva samples were evaluated using an in-house developed reverse transcriptase polymerase chain reaction (RT-PCR) which targeted the Envelope (E) and Nucleocapsid (N) genes. Detection of SARS-CoV-2 using self-collected saliva was found to be only slightly less accurate (<5%) than testing using NPS. In addition, initial saliva RT-PCR identified 27 positive subjects, 18 of which provided amplicons sufficient for confirmatory sequencing. The sequencing data showed a genetic shift in the virus within our population sometime between 22 June and July 8, 2021 from Alpha to Delta variant. The saliva sample collection method identifies the E gene in SARS COVID-2 samples which provides an alternative specimen source to the NPS. This identifies the S gene and ORF1ab. Saliva collection is more convenient to the patient, yields comparable results to NPS collection, and potentially increases Covid-19 surveillance.

Factors associated with an inconclusive result from commercial homologous recombination deficiency testing in ovarian cancer.

Homologous recombination deficiency (HRD) testing is used to determine the appropriateness of poly ADP-ribose polymerase inhibitors for patients with epithelial ovarian cancer and no germline/somatic BRCA1/2 alterations. Myriad MyChoice CDx reports a genomic instability score (GIS) to quantify the level of HRD, with a positive score defined as ≥42. The authors sought to define factors associated with obtaining an inconclusive HRD test result. GIS was retrieved for patients at their institution with epithelial ovarian cancer without germline/somatic BRCA1/2 deleterious alterations who underwent HRD testing from April 2020-August 2023. Clinical data were abstracted from the medical record. Of 477 HRD test results identified, 57 (12%) were inconclusive. High-grade serous ovarian cancers had higher GIS than other histologic types (median 29 vs. 21, p<.001). Most HRD cases were of high-grade serous histology; no cases with clear cell or endometrioid histology were HRD-positive. On univariate analysis, interval versus primary cytoreductive surgery, other specimen sources versus surgical specimens, and chemotherapy exposure were risk factors for inconclusive HRD testing. On multivariable analysis, chemotherapy exposure, and tissue source were associated with an inconclusive test result, with surgical specimens more likely to yield a conclusive result than other sources (biopsy, cytology, other). Age, stage, self-reported race, and histology were not associated with an inconclusive result. Surgical tissue was more likely to yield a conclusive HRD test result versus other sources of epithelial ovarian cancer tissue acquisition. When feasible, laparoscopic biopsy before initiation of neoadjuvant chemotherapy may increase the likelihood of obtaining interpretable HRD test results.

Comparison of SARS-CoV-2 seroprevalence estimates between commercial lab serum specimens and blood donor specimens, United States, September-December 2021.

This study was the first systematic comparison between two nationwide severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) studies which estimated seroprevalence, or the proportion of the population with antibodies to the virus, using differing convenience sample populations. One study tested blood donor specimens; the other study tested specimens left over from clinical blood tests. The seroprevalence of anti-nucleocapsid and anti-spike antibodies was compared in the same states during the same months with statistical adjustments based on state demographics. Similar anti-nucleocapsid antibody seroprevalence estimates produced by two independent studies using differing convenience samples build confidence in the generalizability of their anti-nucleocapsid findings. Due to high blood donor vaccine rates, blood donor SARS-CoV-2 anti-spike antibody estimates might overestimate general population seroprevalence, an important consideration for interpreting national seroprevalence study results. Furthermore, because laboratory residuals and blood donations are two common sources of specimens for seroprevalence studies, study findings may be informative for other respiratory virus seroepidemiology studies.

Epidemiological Characteristics and Drug Resistance Analysis of 187 Strains of Carbapenem-Resistant Enterobacteriaceae in a General Hospital

Background: Carbapenem antibiotics are currently the most broad-spectrum and potent β-lactam antibiotics. Without effective intervention measures, Carbapenem-Resistant Enterobacteriaceae (CRE) may become widespread in all healthcare institutions within ten years, potentially developing into an endemic issue. Objectives: To understand the epidemiological characteristics and multilocus sequence typing (MLST) of CRE infections in hospitals, and to explore critical strategies for preventing and controlling CRE hospital infections. Methods: The study focused on hospitalized patients with CRE strains isolated by the Laboratory Department of the First Affiliated Hospital of Guizhou University of Traditional Chinese Medicine from 2020 to 2022. Patient data, bacterial isolation, culture, and drug sensitivity results were collected and analyzed to determine the epidemiological characteristics of CRE infections. Preserved Carbapenem-Resistant Klebsiella pneumoniae (CRKP) strains were reviewed, and the CRKP sequence type (ST) was obtained through MLST typing. Results: Over the past three years, 187 CRE strains were isolated in the hospital, with infections predominantly occurring in males and individuals over 60 years old. The departments with the highest detection rates were the ICU (35.29%), geriatric department (11.23%), and rehabilitation department (8.02%). The top three specimen sources were sputum (43.85%), clean midstream urine (32.09%), and secretions (13.90%). carbapenem-resistant Klebsiella pneumoniae accounted for the majority of CRE isolates (80.21%), followed by carbapenem-resistant Escherichia coli (14.44%). Carbapenem-resistant Enterobacteriaceae strains exhibited a high resistance rate to most commonly used antibiotics but were sensitive to tigecycline. Multilocus sequence typing results from 11 CRKP strains showed that 90.91% were of the ST11 type, while 9.09% were of the ST15 type. Conclusions: From 2020 to 2022, CRE infections at the hospital primarily affected elderly male patients over 60, with CRKP as the predominant pathogen and ST11 as the main sequence type. Carbapenem-resistant Enterobacteriaceae strains were highly resistant to most commonly used antimicrobial drugs. Hospitals should prioritize the prevention and control of CRE infections, emphasizing the management of antimicrobial drugs, patient identification, environmental hygiene, and infection control measures to prevent outbreaks caused by CRE.

Estimated Incidence of Hospitalizations Attributable to RSV Infection Among Adults in Ontario, Canada, Between 2013 and 2019.

Adult respiratory syncytial virus (RSV) burden is underestimated due to non-specific symptoms, limited standard-of-care and delayed testing, reduced diagnostic test sensitivity-particularly when using single diagnostic specimen-when compared to children, and variable test sensitivity based on the upper airway specimen source. We estimated RSV-attributable hospitalization incidence among adults aged ≥ 18 years in Ontario, Canada, using a retrospective time-series model-based approach. The Institute for Clinical Evaluative Sciences data repository provided weekly numbers of hospitalizations (from 2013 to 2019) for respiratory, cardiovascular, and cardiorespiratory disorders. The number of hospitalizations attributable to RSV was estimated using a quasi-Poisson regression model that considered probable overdispersion and was based on periodic and aperiodic time trends and viral activity. As proxies for viral activity, weekly counts of RSV and influenza hospitalizations in children under 2 years and adults aged 60 years and over, respectively, were employed. Models were stratified by age and risk group. In patients ≥ 60 years, RSV-attributable incidence rates were high for cardiorespiratory hospitalizations (range [mean] in 2013-2019: 186-246 [215] per 100,000 person-years, 3‒4% of all cardiorespiratory hospitalizations), and subgroups including respiratory hospitalizations (144-192 [167] per 100,000 person-years, 5‒7% of all respiratory hospitalizations) and cardiovascular hospitalizations (95-126 [110] per 100,000 person-years, 2‒3% of all cardiovascular hospitalizations). RSV-attributable cardiorespiratory hospitalization incidence increased with age, from 14-18 [17] hospitalizations per 100,000 person-years (18-49 years) to 317-411 [362] per 100,000 person-years (≥ 75 years). Estimated RSV-attributable respiratory hospitalization incidence among people ≥ 60 years in Ontario, Canada, is comparable to other incidence estimates from high-income countries, including model-based and pooled prospective estimates. Recently introduced RSV vaccines could have a substantial public health impact.

Resistance to Antifungals in Non-albicans Candida Species Isolates in the Southeast Region

Background: Antimicrobial resistance is a growing problem in Candida spp., leading to treatment challenges and increased morbidity and mortality. The World Health Organization (WHO) fungal priority pathogens list classifies C. glabrata, C. tropicalis, and C. parapsilosis as high priority and leading causes of candidemia with high fluconazole resistance. In the US, these organisms are the most frequently isolated non-albicans Candida species. In 2016, the Antibiotic Resistance Laboratory Network (ARLN) was created to monitor resistance threats, including in Candida spp. This study describes the proportion of resistance in C. glabrata, C. parapsilosis, and C. tropicalis isolates sent to the Southeast ARLN from 2017 to 2023. Methods: This study evaluated C. glabrata, C. parapsilosis, and C. tropicalis submitted to the Southeast ARLN from Alabama, Florida, Georgia, Louisiana, Mississippi, and Tennessee from February 2017- September 2023. Species identification was confirmed by Bruker Biotyper matrix assisted laser desorption-ionization time of flight (MALDI-TOF). Antifungal susceptibility testing (AFST) was performed using TREK frozen broth microdilution panels. Minimum inhibitory concentration values from the clinical instrument were used to determine susceptibility based on Clinical and Laboratory Standards Institute (CLSI) standard interpretations from the 2020 CLSI M60 guidelines. Data were extracted from the laboratory information management system. Analyses were conducted using SAS v9.4. Results: AFST testing was performed on 660 C. glabrata, 500 C. parapsilosis, and 233 C. tropicalis isolates from within the Southeast region. The predominant specimen sources by species were blood 25.30% C. glabrata; other/not specified 27.80% C. parapsilosis; and lower respiratory 36.91% C. tropicalis. Resistance to fluconazole is as follows: C. glabrata, 12.88%; C. parapsilosis, 3.41%; C. tropicalis, 36.64%. Resistance to voriconazole is as follows: C. parapsilosis, 1.00%; C. tropicalis 30.04%. Resistance to at least one echinocandin (Anidulafungin, Capsofungin, Micafungin) is as follows: C. glabrata, 1.67%; C. parapsilosis, 0.60%; C. tropicalis, 0.43%. Overall, there was a decreasing trend in resistance to fluconazole, and voriconazole in all three species between 2017 and 2023. Conclusions: Antifungal resistance in non-albicans Candida species represents an emerging public health threat, however, within the Southeast region, ARLN data has shown a decreasing trend of azole resistance. This may be due in part to changes in reporting requirements and submission criteria from within the region. Nevertheless, C. tropicalis showed high resistance to azoles within the Southeast region. These Candida species should be monitored to inform clinical decision making and identify resistance patterns in other US regions due to their increase in resistance worldwide.

Extended-Spectrum Beta-Lactamase Producing Enterobacterales Infections in the United States, 2012-2021

Background: The 2022 Special Report: COVID-19 U.S. Impact on Antimicrobial Resistance identified continued increases in the rate of extended- spectrum beta-lactamase producing (ESBL) infections in the United States from 2017 through 2020. Using similar data sources and methodology, we examined the trends of species-specific ESBL infections from 2012-2021. Methods: We identified a cohort of patients from the PINC AI and BD Research Insights databases with a clinical culture yielding a Klebsiella pneumoniae or Escherichia coli isolate with accompanying susceptibility testing. E. coli or K. pneumoniae isolates non-susceptible to ceftriaxone, cefotaxime, ceftazidime, or cefepime were considered suggestive of ESBL production. Isolates from patients with no culture yielding the same resistance phenotype of interest in the previous 14 days were counted as an incident case. Community-onset (CO) cultures were obtained ≤ day 3 of hospitalization; hospital-onset (HO) cultures were obtained ≥ day 4. We used a raking procedure to determine weights for extrapolating the number of discharges included in our sample to match the distribution of discharges, stratified by bed size, U.S. census division, urban/rural designation, and teaching status, for U.S. hospitals included in the American Hospital Association survey. We evaluated rates over time due to the changes in number of hospitalizations during the COVID-19 pandemic. Results were stratified by HO and CO, and sterile and non-sterile specimen sources. Results: In 2021, there were 48,936 ESBL K. pneumoniae and 153,112 ESBL E. coli infections among approximately 32 million discharges. Overall, most infections were CO and from non-sterile specimens. From 2012-2021, the rate of ESBL K. pneumoniae increased from 9.54 to 15.28 per 10,000 discharges. ESBL E. coli infections increased from 2012-2020 (30.18 to 51.32 per 10,000 discharges), then declined in 2021 (47.81 per 10,000 discharges) (Table 1, Figure 1). The proportion of non-sterile ESBL E. coli declined from 88% in 2012 to 83% in 2021, and the proportion of non-sterile ESBL K. pneumoniae was 85-87% over the study period (Figure 2). Conclusion: ESBL E. coli and K. pneumoniae infections increased from 2012-2021, although the CO ESBL E. coli rate decreased between 2020 and 2021. Understanding changes in culturing practices over time may provide insights into the increased proportion of ESBL E. coli from sterile sites. Additionally, further investigation into differences in organism trends, particularly in 2021, may inform prevention strategies.

Distribution and molecular characterization of carbapenemase-producing gram-negative bacteria in Henan, China

This study aimed to investigate the epidemiological characteristics and trends over time of carbapenemase-producing (e.g., KPC, NDM, VIM, IMP, and OXA-48) Gram-negative bacteria (CPGNB). Non-duplicated multi-drug resistant Gram-negative bacteria (MDRGNB) were collected from the First Affiliated Hospital of Zhengzhou University from April 2019 to February 2023. Species identification of each isolate was performed using the Vitek2 system and confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry according to the manufacturer's instructions. PCR detected carbapenem resistance genes in the strains, strains carrying carbapenem resistance genes were categorized as CPGNB strains after validation by carbapenem inactivation assay. A total of 5705 non-repetitive MDRGNB isolates belonging to 78 different species were collected during the study period, of which 1918 CPGNB were validated, with the respiratory tract being the primary source of specimens. Epidemiologic statistics showed a significant predominance of ICU-sourced strains compared to other departments. Klebsiella pneumoniae, Escherichia coli, Acinetobacter baumannii, and Pseudomonas aeruginosa were the significant CPGNB in Henan, and KPC and NDM were the predominant carbapenemases. Carbapenem-resistant infections in Henan Province showed an overall increasing trend, and the carriage of carbapenemase genes by CPGNB has become increasingly prevalent and complicated. The growing prevalence of CPGNB in the post-pandemic era poses a significant challenge to public safety.

Effects of coronavirus disease 2019 on the spread of respiratory-transmitted human-to-human bacteria

The coronavirus disease 2019 (COVID-19) pandemic has necessitated significant changes in medical systems, social behaviours, and non-pharmaceutical interventions (NPIs). We aimed to determine the effect of the COVID-19 pandemic on changes in the epidemiology of respiratory-transmitted bacteria that have been unexplored. We utilised a comprehensive national surveillance database from 2018 to 2021 to compare monthly number of patients with four respiratory-transmitted human-to-human bacteria (Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pyogenes) before and after the COVID-19 pandemic, stratified by specimen sources and age groups. The incidence of detected patients with S. pneumoniae, H. influenzae, and S. pyogenes from both respiratory and blood cultures significantly decreased from 2019 to 2020. In 2021, the incidence of detected patients with the respiratory-transmitted bacterial species, except for S. pyogenes, from respiratory cultures, increased again from April to July, primarily affecting the 0-4-year age group. Our comprehensive national surveillance data analysis demonstrates the dynamic changes and effects of NPIs on respiratory-transmitted bacteria during the COVID-19 pandemic, with variations observed among species, specimen sources, and age groups.

Molecular characteristics and drug susceptibility analysis of Streptococcus agalactiae from respiratory specimen sources

To study the carriage status of drug susceptibility, clonal complex groups, serotypes, surface proteins and virulence genes of Streptococcus agalactiae from respiratory specimen sources. A total of 35 strains of S.agalactiae meeting the criteria were collected from 3 hospitals in 2 locations, Tangshan and Jinan. The age span of the patients was 3 days-92 years, and the percentage of elderly patients≥60 years was 71.5%.The susceptibility to 9 antimicrobial drugs was measured and analyzed using the micro broth dilution method. The strains were 100.0% sensitive to penicillin, linezolid, vancomycin, and ceftriaxone; However, it exhibits high resistance rates to erythromycin, clindamycin and levofloxacin, at 97.1%, 85.7% and 82.9% respectively; and the resistance rates to tetracycline and chloramphenicol were 34.3% and 14.2%, respectively. Genome sequence determination and analysis showed that 16 resistance genes were detected in 35 strains, among which: macrolide and lincosamide resistance genes were mainly ermB, with a carrying rate of 74.2%; tetracycline resistance genes were mainly tetM, with a carrying rate of 25.7%; in addition, the mutation rates of the quinolone resistance determinants gyrA and parC were 88.5% and 85.7%, respectively. 35 strains belonged to 6 ST types and 4 clonal groups, with CC10/ST10 as the main one, accounting for 62.8%; they contained 4 serotypes of Ⅰb, Ⅱ, Ⅲ, and Ⅴ, as well as 1 untyped strain, with serotype Ⅰb as the main one, accounting for 65.7%. The strains carried three pilus types, PI1+PI2a, PI2a and PI2b types, respectively, and detected five surface proteins, alpha, alp1, rib, srr, and rdf_0594, and seven virulence factors, cba, cfb, cylE, fbsA, hylB, lmb, and pavA. Overall, S.agalactiae isolated from respiratory tract specimens is predominantly sourced from elderly patients, with CC10 strains being most prevalent. These strains harbor multiple drug-resistant and virulence genes, demonstrating elevated resistance rates to macrolides, lincosamides, and quinolones. This emphasizes the necessity for vigilant attention to the health threat posed by S. agalactiae from respiratory tract speciments of elderly patients.

Flushing fluid of post-bronchoscopic biopsy as a novel liquid biopsy specimen for genome profiling in advanced lung cancer.

e15038 Background: Tumour tissue is the primary substrate for molecular testing in advanced NSCLC. However, obtaining sufficient tissue samples from biopsy for molecular testing can often be challenging. Flushing fluid of post-bronchoscopic biopsy, which contains tumor-derived DNA, may serve as a supplement for genotyping. The aim of this study is to assess the efficacy and precision of detecting gene alterations in flushing fluid samples and evaluate their clinical potential. Methods: Flushing fluid, matched tissue (TIS), and whole blood were prospectively collected from 25 patients with stage III-IV NSCLC between June 2022 and December 2023. Ten milliliters of flushing fluid and whole blood were used to prepare flushing fluid supernatant (FFS) and plasma (PLA), respectively. Capture-based targeted sequencing of TIS, FFS and PLA samples was performed using a universal oncology panel. Results: In this cohort of 25 patients, there was a male predominance (22 out of 25) and the mean age was 60.2 years. The majority of patients were diagnosed with lung adenocarcinoma (23 out of 25), and smokers outnumbered non-smokers (16 out of 25). Furthermore, 18 out of 25 patients presented with metastatic disease at diagnosis. The performance of three sample types regarding key quality control indices was evaluated first. FFS exhibited a maximum allelic fraction similar to TIS, while PLA showed a significantly lower than TIS and FFS. Subsequently, we compared the positive detection rates, with TIS, FFS, and PLA achieving respective rates of 100%, 96%, and 80%. A total of 495 mutation events were detected from the three samples (FFP:209, FFS:196, PLA:90), Among these, 161 were concordant between TIS and FFS samples, and 72 were concordant between TIS and PLA samples. The sensitivity and positive predictive value (PPV) of detecting eight established driver alterations in NSCLC were compared using TIS as the gold standard with the two liquid biopsies. FFS demonstrated superior sensitivity and PPV compared to PLA (80.5% vs. 56.1%, 97% vs. 88.5%) and showed equivalent performance for EGFR mutation. Furthermore, estimates of tumor mutational burden (TMB) based on TIS and FFS were highly correlated (Pearson r= 0.97). Conclusions: Our study demonstrated that FFS performs comparably to matched TIS and superiorly to PLA for genotyping advanced NSCLC. FFS emerges as a valuable specimen source, especially in patients with insufficient tumor tissue.

Mycoplasmoides genitalium nucleic acid semi-quantitation and molecular macrolide resistance detection via automated assays: gender and specimen source considerations.

First-line macrolide treatment failure is of increasing concern with Mycoplasmoides genitalium in multiple settings. Recent sexually-transmitted infection treatment guidelines from the United States Centers for Disease Control and Prevention have predicated therapeutic approaches on the availability of a macrolide resistance/susceptibility result from a primary clinical specimen. In this report, we investigate potential correlation between macrolide resistance mutation detection rates (identified by a molecular amplified laboratory-developed test) and transcription-mediated amplification-based rRNA target semi-quantitation. Data reveal that rRNA semi-quantitation and laboratory-developed test detection rate differences exist as a function of gender and specimen source. These data can guide providers in proper specimen selection not only for the laboratory diagnosis of M. genitalium but also macrolide resistance mutation determination from primary clinical specimens.

Harpy eagle kill sample provides insights into the mandibular ontogenetic patterns of two-toed sloths (Xenarthra: Choloepus)

Abstract Skeletal ontogeny of xenarthrans is poorly known, especially because of the paucity of study specimens from distinct developmental stages. Here, we investigate morphometric aspects of the mandible ontogeny in the two-toed sloths, Choloepus spp. We examined mandibles of infant, juveniles and subadult sloths that were present in kill assemblages of harpy eagles, Harpia harpyja, and complemented our study with adult museum specimens. We carried out uni- and multivariate linear morphometric analyzes to assess the growth pattern of the mandible. Harpy eagles did not prey on adult two-toed sloths, preferring younger individuals. We found an overall strong correlation between the total length of the mandible and other mandibular measurements across age classes, with some of them scaling isometrically, and others presenting allometric growth. Also, morphometric data correlated with patterns of symphysial fusion across ontogenetic stages, rendering the latter a reliable indicator of the animal’s age category. Although it was necessary to complement our sample with museum material, individuals obtained from the harpy eagle kill assemblage proved to be a valuable complementary source of specimens to be studied.

Five previously undescribed thylacine (Thylacinus cynocephalus) specimens held in the museums of the University of Melbourne

ABSTRACT The last known captive thylacine (Thylacinus cynocephalus) died in Hobart's Beaumaris Zoo in 1936 and the species has since become an icon of extinction. In the 21st century, there is considerable interest in documenting, analysing, and rediscovering thylacine specimens in museum collections. Due to their age, location, and collecting practices, the teaching collections of Australia's oldest universities are a potential source of undocumented thylacine specimens. Within three departmental museum collections, the University of Melbourne holds five thylacine skulls. The skulls were visually inspected, measured, sexed, and described in detail for the first time. Archival research was undertaken to determine provenance and historical details. The five skulls were added to the University's collections between 1893 and 1932. They are derived from four adult males and one adult female. Specific locality data are available for three skulls, collected at Lake Saint Clair, south of Cressy, and Woolnorth. Holding five skulls, University of Melbourne's collection constitutes one of the top ten largest thylacine assemblages in Australasia, and the second largest held by an Australasian university.

Clinical characteristics and risk factors for mortality in Candida auris infections

AbstractBackgroundCandida auris infections pose a threat to public health, necessitating increased awareness in China. This study aimed to analyze the strains of C. auris, assess the infection status, and investigate clinical characteristics and risk factors for mortality.MethodsA retrospective analysis was conducted on 18 patients with Candida auris infection. We focused on evaluating basic characteristics, strain sources, and antibacterial susceptibility test results. Statistical methods were used to determine clinical features and identify risk factors for death.ResultsThe strain type, composition ratio, and specimen source of C. auris were not associated with mortality. Neither the infection index nor the length of hospitalization showed an association with the prognosis. However, significant risk factors for mortality included cerebral infarction, cardiac disease, renal dysfunction, hypoproteinemia, and anemia (all p &lt; 0.05).ConclusionsCerebral infarction, cardiac disease, renal dysfunction, hypoproteinemia, and anemia are significant risk factors for death in C. auris infections. These findings indicate the importance of recognizing and addressing these factors in the clinical management of C. auris infection.

Assessing Accuracy and Precision of Hemoglobin Determination in Venous, Capillary Pool, and Single-Drop Capillary Blood Specimens Using three Different HemoCue® Hb Models: The Multicountry Hemoglobin Measurement (HEME) Study

BackgroundAnemia prevalence estimates reported in population surveys can vary based on the blood specimen source (capillary or venous) and analytic device (hematology autoanalyzers or portable hemoglobinometers) used for hemoglobin (Hb) determination. ObjectivesThis study aimed to compare accuracy and precision of Hb measurement in three blood specimen types on three models of hemoglobinometers against the results from venous blood from the same individuals measured on automated analyzers (AAs). MethodsThis multisite (Cambodia, Ethiopia, Guatemala, Lebanon, Nigeria, and Tanzania) study assessed Hb measurements in paired venous and capillary blood specimens from apparently healthy women (aged 15–49 y) and children (aged 12–59 mo) using three HemoCue® Hb models (201+, 301, and 801). Measurements were compared against reference values: venous blood in hematology AA and adjusted via regression calibration or mean difference in HemoCue® Hb. Venous, capillary pool, and single-drop capillary blood specimens were assessed for accuracy and precision. ResultsVenous blood measured using HemoCue® Hb 301 exhibited a positive mean error, whereas responses in HemoCue® Hb 201+ and 801 were nondirectional compared with the reference. Adjustment with the reference harmonized mean errors for all devices across study sites to <1.0 g/L using venous blood. Precision was highest for venous blood (±5–16 g/L) in all sites, lowest for single-drop capillary (±9–37 g/L), and intermediate (±9–28 g/L) for capillary pool blood specimen. Imprecision differed across sites, especially with both capillary blood specimens, suggesting different levels of personnel skills. ConclusionsFindings suggest that venous blood is needed for accurate and precise Hb determination. Single-drop capillary blood use should be discouraged owing to high measurement variability. Further research should evaluate the viability and reliability of capillary pool blood for this purpose. Accuracy of HemoCue® Hb devices can be improved via standardization against results from venous blood assessed using AA.

Abstract 2500: Technical feasibility of CRISPR-based functional profiling for solid tumor precision medicine

Abstract Introduction: Next generation sequencing (NGS) has revolutionized precision oncology by enabling identification of cancer patients harboring actionable mutations who may benefit from corresponding targeted therapies. However, sequencing-based biomarkers are not detected for most patients and imperfectly predict response to therapy even when present. To address this unmet need, we have developed a CRISPR-based diagnostics platform for ‘personalized functional genomics’, allowing identification of patient-specific therapeutic vulnerabilities irrespective of mutation status. We have previously applied this approach to primary hematologic malignancies, and here report application to solid tumor-derived samples. Methods: Patient-derived solid tumor samples from several tumor types including breast, ovarian, colon and lung were obtained from 3 sources: fresh tissue (obtained from patients via core biopsy, surgical resection, or malignant fluid sampling), primary Patient-Derived Cell Lines (PDCLs, generated from surgical material and adapted for ex vivo culture), and commercially obtained viably frozen dissociated tumor cells (DTCs). Samples were dissociated to single cell suspensions and transduced with a lentivirus encoding Cas9 and an sgRNA library covering a custom targeted oncology gene panel. The panel consists of genes encoding targets of FDA-approved oncology drugs, as well as reference genes. After a short culture period, transduced cells were harvested and relative sgRNA distribution was measured by amplicon NGS. Changes in sgRNA abundance were collapsed into gene-level log2-fold changes, and significantly depleted genes scored as functional dependencies. For select PDCLs, cells were treated with cognate small molecule inhibitors over the course of 4 days. Cell viability was read out using CTG assay and a calculated IC50 lower than 500nM inferred as inhibitor sensitive. Results: In a double-blind study using primary PDCLs, functional dependencies identified by CRISPR perfectly discriminated alpelisib sensitive (2/2) from resistant samples (6/6). Using fresh primary samples from varying cancer types and specimen sources, we achieve a technical success rate &amp;gt;70%. Among gene dependencies identified, targets of FDA-approved oncology drugs and those under late-stage clinical investigation were both observed including BRAF, CDK4/6, VEGFR1/2 and PARP1/2. Conclusion: We demonstrate technical proof of concept of the FX platform for CRISPR-based personalized functional genomics. This approach is feasible when applied to primary patient samples from a variety of tumor types and tissue sources, and results are validated by comparison with orthogonal methods. CRISPR-based functional profiling represents a powerful adjunct to traditional genomic profiling, for the first time allowing drug target dependency mapping in a patient-specific and mutation-agnostic manner. Citation Format: Karl Hodel, Andreas Varkaris, Nicholas Chevalier, Maci Meyers, Allison Kehlmann, Stephane Corbel, Anastasia Stavridi, Peter Kilfeather, Leonardo Hagmann, Kathleen Tran, Yali Li, Christian Schmedt, Ioannis Sanidas, Srinath Sampath, Srihari Sampath, Dejan Juric. Technical feasibility of CRISPR-based functional profiling for solid tumor precision medicine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2500.

A novel hybridization capture method for direct whole genome sequencing of clinical specimens to inform Legionnaires' disease investigations.

The unprecedented precision and resolution of whole genome sequencing (WGS) can provide definitive identification of infectious agents for epidemiological outbreak tracking. WGS approaches, however, are frequently impeded by low pathogen DNA recovery from available primary specimens or unculturable samples. A cost-effective hybrid capture assay for Legionella pneumophila WGS analysis directly on primary specimens was developed. DNA from a diverse range of sputum and autopsy specimens PCR-positive for L. pneumophila serogroup 1 (LPSG1) was enriched with this method, and WGS was performed. All tested specimens were determined to be enriched for Legionella reads (up to 209,000-fold), significantly improving the discriminatory power to compare relatedness when no clinical isolate was available. We found the WGS data from some enriched specimens to differ by less than five single-nucleotide polymorphisms (SNPs) when compared to the WGS data of a matched culture isolate. This testing and analysis retrospectively provided previously unconfirmed links to environmental sources for clinical specimens of sputum and autopsy lung tissue. The latter provided the additional information needed to identify the source of these culture-negative cases associated with the South Bronx 2015 Legionnaires' disease (LD) investigation in New York City. This new method provides a proof of concept for future direct clinical specimen hybrid capture enrichment combined with WGS and bioinformatic analysis during outbreak investigations.IMPORTANCELegionnaires' disease (LD) is a severe and potentially fatal type of pneumonia primarily caused by inhalation of Legionella-contaminated aerosols from man-made water or cooling systems. LD remains extremely underdiagnosed as it is an uncommon form of pneumonia and relies on clinicians including it in the differential and requesting specialized testing. Additionally, it is challenging to obtain clinical lower respiratory specimens from cases with LD, and when available, culture requires specialized media and growth conditions, which are not available in all microbiology laboratories. In the current study, a method for Legionella pneumophila using hybrid capture by RNA baiting was developed, which allowed us to generate sufficient genome resolution from L. pneumophila serogroup 1 PCR-positive clinical specimens. This new approach offers an additional tool for surveillance of future LD outbreaks where isolation of Legionella is not possible and may help solve previously unanswered questions from past LD investigations.

Extensively Drug-Resistant Pseudomonas aeruginosa Outbreak Associated With Artificial Tears.

Carbapenemase-producing, carbapenem-resistant Pseudomonas aeruginosa (CP-CRPA) are extensively drug-resistant bacteria. We investigated the source of a multistate CP-CRPA outbreak. Cases were defined as a US patient's first isolation of P. aeruginosa sequence type 1203 with carbapenemase gene blaVIM-80 and cephalosporinase gene blaGES-9 from any specimen source collected and reported to the Centers for Disease Control and Prevention during 1 January 2022-15 May 2023. We conducted a 1:1 matched case-control study at the post-acute care facility with the most cases, assessed exposures associated with case status for all case-patients, and tested products for bacterial contamination. We identified 81 case-patients from 18 states, 27 of whom were identified through surveillance cultures. Four (7%) of 54 case-patients with clinical cultures died within 30 days of culture collection, and 4 (22%) of 18 with eye infections underwent enucleation. In the case-control study, case-patients had increased odds of receiving artificial tears versus controls (crude matched OR, 5.0; 95% CI, 1.1-22.8). Overall, artificial tears use was reported by 61 (87%) of 70 case-patients with information; 43 (77%) of 56 case-patients with brand information reported use of Brand A, an imported, preservative-free, over-the-counter (OTC) product. Bacteria isolated from opened and unopened bottles of Brand A were genetically related to patient isolates. Food and Drug Administration inspection of the manufacturing plant identified likely sources of contamination. A manufactured medical product serving as the vehicle for carbapenemase-producing organisms is unprecedented in the United States. The clinical impacts from this outbreak underscore the need for improved requirements for US OTC product importers.