From e-voucher to genomic data: Preserving archive specimens as demonstrated with medically important mosquitoes (Diptera: Culicidae) and kissing bugs (Hemiptera: Reduviidae)

Scientific collections such as the U.S. National Museum (USNM) are critical to filling knowledge gaps in molecular systematics studies. The global taxonomic impediment has resulted in a reduction of expert taxonomists generating new collections of rare or understudied taxa and these large historic collections may be the only reliable source of material for some taxa. Integrated systematics studies using both morphological examinations and DNA sequencing are often required for resolving many taxonomic issues but as DNA methods often require partial or complete destruction of a sample, there are many factors to consider before implementing destructive sampling of specimens within scientific collections. We present a methodology for the use of archive specimens that includes two crucial phases: 1) thoroughly documenting specimens destined for destructive sampling—a process called electronic vouchering, and 2) the pipeline used for whole genome sequencing of archived specimens, from extraction of genomic DNA to assembly of putative genomes with basic annotation. The process is presented for eleven specimens from two different insect subfamilies of medical importance to humans: Anophelinae (Diptera: Culicidae)—mosquitoes and Triatominae (Hemiptera: Reduviidae)—kissing bugs. Assembly of whole mitochondrial genome sequences of all 11 specimens along with the results of an ortholog search and BLAST against the NCBI nucleotide database are also presented.

Evaluation of Merkel Cell Polyomavirus DNA in Tissue Samples from Italian Patients with Diagnosis of MCC

Because the incidence of Merkel cell carcinoma (MCC) has increased significantly during the last 10 years and it is recognized that Merkel cell polyomavirus (MCPyV) and ultraviolet (UV) radiation represent two different etiological inputs sharing clinical, histopathological, and prognostic similar features, although with different prognosis, this study investigated the detection of MCPyV in skin and lymph nodes with histological diagnosis of MCC. Formalin-fixed paraffin-embedded tissue (FFPE) were retrieved from archived specimens and MCPyV non-coding control region (NCCR) and viral capsid protein 1 (VP1) sequences were amplified and sequenced. Results provide an interesting observation concerning the discrepancy between the MCPyV DNA status in primary and metastatic sites: in fact, in all cases in which primary and metastatic lesions were investigated, MCPyV DNA was detected only in the primary lesions. Our data further support the “hit-and-run” theory, also proposed by other authors, and may lead to speculation that in some MCCs the virus is only necessary for the process of tumor initiation and that further mutations may render the tumor independent from the virus. Few point mutations were detected in the NCCR and only silent mutations were observed in the VP1 sequence compared to the MCPyV MCC350 isolate. To unequivocally establish a role of MCPyV in malignancies, additional well-controlled investigations are required, and larger cohorts should be examined.

Multicenter Evaluation of the Unyvero Platform for Testing Bronchoalveolar Lavage Fluid

Bronchoalveolar lavage (BAL) culture is a standard, though time consuming, approach for identifying microorganisms in severe lower respiratory tract infections. Sensitivity of BAL culture is relatively low and prior antimicrobial therapy decreases the sensitivity further, leading to overuse of empiric antibiotics. The Unyvero LRT BAL Application (Curetis GmbH, Germany) is a multiplex molecular panel that detects 19 bacteria, 10 antibiotic resistance markers and a fungus, Pneumocystis jirovecii, in BAL fluid in ∼4.5 hours. Performance was evaluated using 1,016 prospectively collected and 392 archived specimens from 11 clinical trial sites in the United States. Overall positive and negative percent agreement with culture for identification of bacteria that grow in routine cultures were 93.4% and 98.3%, respectively, with additional potential pathogens identified by Unyvero in 21.7% of prospectively collected specimens. For detection of P. jirovecii, positive percent agreement with standard testing was 87.5%. Antibiotic resistance marker results were compared to standard antibiotic susceptibility testing to determine positive predictive values (PPVs). PPVs ranged from 80-100%, based on the microorganism and specific resistance marker(s). The Unyvero LRT BAL Application provides accurate detection of common agents of bacterial pneumonia and of P. jirovecii. The sensitivity and rapidity of this panel suggests significant clinical value for choosing appropriate antibiotics and for antibiotic stewardship.

BIOM-54. A RAPID GENOTYPING PANEL FOR SENSITIVE AND SPECIFIC SEGREGATION OF CNS PATHOLOGIES

Primary central nervous system lymphoma (PCNSL) remains challenging to diagnose due to nonspecific clinical and radiologic features and low diagnostic yields of cerebrospinal fluid (CSF) studies. We sought to characterize the diagnostic approach of suspected PCNSL, in order to improve clinical workflow. We first reviewed 1,007 new brain lesions of unknown etiology that included PCNSL in the radiologic differential diagnosis. The most common final diagnoses included high-grade glioma (28.2%) and PCNSL (14.6%). Diagnostic biopsies were frequently performed for high-grade glioma (100%) and PCNSL (94.4%), while CSF was frequently sampled for PCNSL (78.7%). We next identified 159 patients with an established new diagnosis of PCNSL. CSF studies were non-diagnostic in 86.7% of cases, whereas biopsy was positive in 93%. However, intraoperative histopathology was inconclusive for PNCSL in 54.5%, likely contributing to 22% of patients undergoing surgical resection. These challenges resulted in 12 days median time to treatment initiation, and readmission for further workup or treatment initiation in 27% of patients. These results indicated the need for a rapid, sensitive and specific platform to segregate PCNSL and glioma using CSF and tissue samples. We developed a qPCR-based assay to genotype the MYD88 L265P hotspot mutation from CSF and plasma within 80 minutes of sample acquisition. Results were concordant with orthogonal DNA sequencing in extracts from 87 archived specimens, with detection limits of 490pg of input genomic DNA and 0.15% mutant allele frequency. When performed simultaneously with assays for TERT promoter, IDH1/2, H3F3A and BRAF point mutations, the resulting panel accurately segregated PCNSL and adult diffuse glioma molecular diagnoses in 87 archived specimens and 19 prospective liquid biopsies, including cases of lymphoma and glioma. We propose that inclusion of targeted analysis of these mutually exclusive recurrent molecular alterations characterizing gliomas and PCNSL will facilitate rapid, sensitive diagnosis from solid and liquid biopsies.

Specimens in a Broader Context: The National Ecological Observatory Network and the extended specimen

Community innovations in both specimen digitization (e.g., Morphbank; SlideAtlas; Inselect, Hudson et al. 2015) and data standards (e.g., the National Science Foundation initative "Advancing Digitization of Biodiversity Collections", Page et al. 2015, Nelson and Shari 2019; Darwin Core (Darwin Core Task Group 2009)), have resulted in digitized specimens with rich contextual metadata and the capacity to share such specimen information widely. These extended specimens have allowed for the exploration of cross-scale research questions that traverse multiple taxonomic, spatial and temporal scales. As a relatively new collection organization, the National Ecological Observatory Network (NEON; Keller et al. 2008) has curated and archived >200,000 specimens to date and is projected to archive between 80,000 and 120,000 specimens annually through its 30-year, continental-scale environmental monitoring program. NEON has embraced the Extended Specimen paradigm (introduced by Webster 2017; NEON's implementation described in Lendemer et al. 2020), and each sample is physically and digitally curated from the point of collection enabling sample discoverability that maximizes specimen Findability, Accessibility, Interoperability, and Reusability (the FAIR standard; Wilkinson et al. 2016). All archived specimens are associated with precise spatial and temporal information and (where available/applicable) NEON also integrates specimen images, morphometrics, genetic sequences and taxonomic data with the specimen records within a Symbiota platform. Any additional analyses or derived specimens created by the research community are also linked in the specimen record. NEON has benefited substantially from community development of tools and standards, but the process of data integration has not been without problems. Here, we will discuss challenges NEON has faced in the implementation of the extended specimen as well as solutions.

Look before diving into pooling of SARS-CoV-2 samples on high throughput analyzers

Given the unprecedented demand for SARS-CoV-2 testing during the COVID-19 pandemic, the benefits of specimen pooling have recently been explored. As previous studies were limited to mathematical modeling or testing on low throughput PCR instruments, this study aimed to assess pooling on high throughput analyzers. To assess the impact of pooling, SARS-CoV-2 dilutions were performed at varying pool depths (i.e. 1:2, 1:4, and 1:8) into test-negative nasopharyngeal or oropharynx/anterior nares swabs matrix. Testing was evaluated on the automated Roche Cobas 6800 system, or the Roche MagNApure LC 2.0 or MagNAPure 96 instruments paired with a laboratory-developed test using a 96-well PCR format. The frequency of detection in specimens with low viral loads was evaluated using archived specimens collected throughout the first pandemic wave. The proportion of detectable results per pool depths was used to estimate the potential impact. In addition, workflow at the analytical stage, and pre-and post-stages of testing were also considered. The current study estimated that pool depths of 1:2, 1:4, and 1:8 would have allowed the detection of 98.3%, 96.0%, and 92.6% of positive SARS-CoV-2 results identified in the first wave of the pandemic in Nova Scotia. Overall, this study demonstrated that pooling on high throughput instrumentation can dramatically increase the overall testing capacity to meet increased demands, with little compromising to sensitivity at low pool depths. However, the human resources required at the pre-analytical stage of testing is a particular challenging to achieve.

Pathology Associated With Streptococcus spp. Infection in Baboons (Papio spp.)

Streptococcus spp. are a source of morbidity and mortality in captive nonhuman primate populations. However, little is known about the lesions associated with naturally occurring streptococcal infections in baboons ( Papio spp.). The pathology database of the Southwest National Primate Research Center was searched for all baboon autopsies from 1988 to 2018 in which Streptococcus spp. were cultured. Baboons on experimental protocol were excluded. The gross autopsy and histopathology reports were reviewed. Archived specimens were retrieved and reviewed as needed for confirmation or clarification. Fifty-six cultures were positive for Streptococcus spp. in 54 baboons with evidence of bacterial infection. Associated gross lesions included purulent exudate, fibrinous to fibrous adhesions, hemorrhage, mucosal thickening, organomegaly, and abscessation. Histologic lesions included suppurative inflammation, abscessation, necrosis, hemorrhage, fibrin accumulation, and thrombosis. Lungs and pleura ( n = 31) were the most commonly infected organ followed by the central nervous system ( n = 16), spleen ( n = 15), soft tissues ( n = 12), air sacs, liver, peritoneum, adrenal glands, heart, lymph nodes, uterus, kidneys, biliary system, bones, ears, umbilical structures, mammary glands, pancreas, placenta, and salivary glands. Infections by non-β-hemolytic Streptococcus spp. predominated in the lungs and air sacs; the most common isolate was Streptococcus pneumoniae. Infections by β-hemolytic Streptococcus spp. predominated in the soft tissues and reproductive tract. Naturally occurring β-hemolytic and non-β-hemolytic Streptococcus spp. infections cause morbidity and mortality in captive baboon populations. The lesions associated with streptococcal infection are similar to those reported in human infection. Thus, the baboon may represent an underutilized model for studying Streptococcus spp. as pathogens.