Subset Of Specimens Research Articles

Truly the best of both worlds: Merging lineage‐specific and universal probe kits to maximize phylogenomic inference

AbstractPremiseHybridization capture kits are now commonly used for reduced representation approaches in genomic sequencing, with both universal and clade‐specific kits available. Here, we present a probe kit targeting 799 low‐copy genes for the plant family Annonaceae.MethodsThis new version of the kit combines the original 469 genes from the previous Annonaceae kit with 334 genes from the universal Angiosperms353 kit. We also compare the results obtained using the original Angiosperms353 kit with our custom approach using a subset of specimens. Parsimony‐informative sites and the results of maximum likelihood phylogenetic inference were assessed for combined matrices using the genera Asimina and Deeringothamnus.ResultsThe Annonaceae799 genes derived from the Angiosperms353 kit have extremely high recovery rates. Off‐target reads were also detected. When evaluating size, the proportion of on‐ and off‐target regions, and the number of parsimony‐informative sites, the genes incorporated from the Angiosperms353 panel generally outperformed the genes from the original Annonaceae probe kit.DiscussionWe demonstrated that the new sequences from the Angiosperms353 probe set are variable and relevant for future studies on species‐level phylogenomics and within‐species studies in the Annonaceae. The integration of kits also establishes a connection between projects and makes new genes available for phylogenetic and population studies.

Articular Surface Damage Following Headless Intramedullary Nail Fixation of Proximal Phalanx Fractures

Offering the benefits of rigid fixation while minimizing soft tissue dissection, intramedullary implants have become a popular choice among hand surgeons. Their placement often requires traversing or passing in proximity to joint surfaces. This study aimed to assess the damage to the articular cartilage of the base of the proximal phalanx resulting from antegrade placement of threaded headless intramedullary nails. A cadaveric study comparing two techniques for antegrade placement of threaded headless intramedullary nails was conducted in 56 digits. The first entailed a single 2.1 mm intramedullary nail placed via the dorsal base of the proximal phalanx, whereas the second used two 1.8 mm intramedullary nails inserted via the collateral recesses of the phalangeal base. All specimens were analyzed for articular surface damage with the cartilage defect measured as a percentage of total joint surface area. Damage to the extensor tendons was also assessed in a subset of specimens. No significant difference in the percentage of articular surface damage was observed, with an average 3.21% ± 2.34% defect in the single 2.1 mm nail group and a 2.71% ± 3.42% mean defect in the two 1.8 mm nails group. There was no articular surface injury in 18% of digits in each group. Damage to extensor tendons was seen in three (9.4%) specimens and in all cases involved either the extensor indicis proprius or extensor digiti minimi. Hardware insertion using either the dorsal base of the proximal phalanx or the collateral recesses of the phalangeal base both demonstrated minimal articular cartilage damage and infrequent injury to the extensor tendons. With proper technique for antegrade insertion into the proximal phalanx, the cartilage defect observed often encompasses only a small percentage of the overall joint surface area.

An integrative framework for dark taxa biodiversity assessment at scale: A case study using Megaselia (Diptera, Phoridae)

Abstract Species extinctions increase at a global scale; therefore, rapid inventorying of our planet's biodiversity is becoming more and more important. As insects represent the highest portion of the fauna and play key ecological roles, it is a pressing need to investigate their biodiversity and accelerate species discovery, especially for understudied insect groups, also known as “dark taxa.” Phoridae (Diptera) are a great example of a “dark taxon,” in particular the genus Megaselia Rondani. The use of integrative methodologies is the best approach to face up to the task of describing hyperdiverse and dark taxa, as morphology alone can be imprecise and slow, and molecular methods alone are often insufficient and lead to errors. Here, we used the Large‐Scale Integrative Taxonomy (LIT) approach to sort 9000 Megaselia into 277 putative species based on DNA barcodes. Each cluster passed through an evaluation of the predictors for incongruence indices between clusters and morphology (maximum p‐distance, stability index), and a subset of specimens were morphologically examined. We provided species estimates with Chao1, and our results suggest a 15% increase in species richness on our dataset. As this estimate was mostly based on samples from southern Germany, the species count will likely increase with expanded geographic sampling. This is a step forward in the study of this taxon, despite the fact that the German insect fauna is one of the best known in Europe and boasts more than one hundred years of study on phorids.

Prevalence of nasopharyngeal myiasis in roe deer (Capreolus capreolus) from an area with high sympatry between wild and domestic ungulates in Central Spain

Infestation by the sheep bot fly Oestrus ovis was firstly reported in a single roe deer from Central Spain in 2022. For assessing the current situation of nasal myiases in this ungulate in this area, the nasopharyngeal cavities of 184 roe deer hunted in Central Spain between January-June 2023 were examined. All larvae were recovered and morphologically identified; in addition, species identification was molecularly confirmed in a subset of specimens. Forty-four roe deer (23.9%; CI 95 17.95–30.74) were positive for different Oestrinae larval stages. Twenty-six animals (14.1%; CI 95 9.44–20.02%) were infested by the roe deer nasal bot fly (Cephenemyia stimulator) with a mean intensity of 35.2 (SD 49.71) larvae/infested animal, and eighteen (9.8%; CI 95 5.90-15.02%) roe deer harboured the sheep bot fly (O. ovis), with a mean intensity of 2.0 (SD 1.33) larvae/infested animal. No mixed infestations by both Oestrinae were found in a single animal. All larval instars (L1, L2 and L3) of both species were identified. Most C. stimulator specimens were located at the nasal turbinates, and a small percentage (3.2%) at the pharynx; all O. ovis larvae were found at the nasal turbinates. Since O. ovis is highly prevalent in sheep and goat flocks from Central Spain, the high sympatry between roe deer and small ruminant populations in the studied area may have increased the risk of cross-infection. Moreover, the finding of mature L3 of O. ovis suggests that this species can complete its life cycle in roe deer. Therefore, monitoring bot flies in sheep and goat flocks as well as in sympatric wild ruminants is strongly recommended for achieving an optimum control of nasal myiases.

Detection of a Human Adenovirus Outbreak, Including Some Critical Infections, Using Multipathogen Testing at a Large University, September 2022-January 2023.

Human adenoviruses (HAdVs) can cause outbreaks of flu-like illness in university settings. Most infections in healthy young adults are mild; severe illnesses rarely occur. In Fall 2022, an adenovirus outbreak was identified in university students. HAdV cases were defined as university students 17-26 years old who presented to the University Health Service or nearby emergency department with flu-like symptoms (eg, fever, cough, headache, myalgia, nausea) and had confirmed adenovirus infections by polymerase chain reaction (PCR). Demographic and clinical characteristics were abstracted from electronic medical records; clinical severity was categorized as mild, moderate, severe, or critical. We performed contact investigations among critical cases. A subset of specimens was sequenced to confirm the HAdV type. From 28 September 2022 to 30 January 2023, 90 PCR-confirmed cases were identified (51% female; mean age, 19.6 years). Most cases (88.9%) had mild illness. Seven cases required hospitalization, including 2 critical cases that required intensive care. Contact investigation identified 44 close contacts; 6 (14%) were confirmed HAdV cases and 8 (18%) reported symptoms but never sought care. All typed HAdV-positive specimens (n = 36) were type 4. While most students with confirmed HAdV had mild illness, 7 otherwise healthy students had severe or critical illness. Between the relatively high number of hospitalizations and proportion of close contacts with symptoms who did not seek care, the true number of HAdV cases was likely higher. Our findings illustrate the need to consider a wide range of pathogens, even when other viruses are known to be circulating.

A Rare Fish Amphistome Revisited: The Phylogenetic Position of Kalitrema kalitrema (Trematoda: Cladorchiidae) Found in Hypostomus spp. (Siluriformes: Loricariidae) from Brazil

Despite recent advances in the molecular knowledge of amphistome trematodes, most genera known from fish remain to be genetically characterized. This is the case for Kalitrema, a genus of the speciose family Cladorchiidae and the type of Kalitrematinae. The type and only species of this genus, Kalitrema kalitrema Travassos, 1933, was originally proposed based on two specimens found in an armored suckermouth catfish from Brazil, and its phylogenetic position has not been evaluated. In this study, paramphistomes found in Hypostomus alatus (2/9; 22.2%) and Hypostomus francisci (4/143; 2.8%) from the Paraobepa River (São Francisco River basin), Minas Gerais, Brazil, between December 2019 and November 2021, were subjected to morphological study. The parasites were identified in low intensity of infection [1.2 (1–2)] and redescribed as K. kalitrema. This species exhibits unique features such as a linguiform body with a circular ridge near the anterior end and a deep, median notch present at the posterior extremity of the body, apparently dividing the body into two lobes. A subset of specimens was further subject to phylogenetic analyses based on the most densely sampled markers, the nuclear ribosomal RNA (28S and ITS2) and mitochondrial cox1, which revealed the inclusion of K. kalitrema in a Neotropical clade of fish paramphistomes. The most comprehensive phylogenetic tree, based on the 28S dataset, confirmed K. kalitrema as an independent, early diverging lineage among Neotropical fish cladorchiids. However, the monophyly of Kalitrematinae was not sustained, given that species of the other kalitrematine genera Pseudocladorchis and Iquitostrema included in the phylogenetic analysis fell in a distinct clade with other fish cladorchiids. As a result, we propose here a narrower concept for Kalitrematinae sensu stricto, accommodating only Kalitrema (type genus) until a more natural subfamilial or familial classification is provided.

Phylogenetic insight into the Lecidea atrobrunnea complex – evidence of narrow geographic endemics and the pressing need for integrative taxonomic revisions

AbstractSpecies of lichen-forming fungi (LFF) display an array of geographical distribution patterns. Among the broadly distributed lichen-forming fungal species, the degree of reproductive isolation and genetic substructure among populations varies widely, in some cases masking unrecognized diversity or meaningful biogeographical patterns. Lecidea atrobrunnea (Raymond ex Lam. & DC.) Schaer. s. lat. (Lecideaceae) is a widespread species complex that has been studied for over two centuries since its initial description. The diversity of the L. atrobrunnea group is highest in western North America, where a dizzying array of morphologies and chemistry can occur at local scales. Here we investigate whether the assumed cosmopolitan distribution of L. atrobrunnea s. lat. is an artifact of taxonomic limitations and masks biogeographical patterns in this species complex. To address these questions, we compiled sequence data from the standard fungal barcoding marker (ITS) for over 100 specimens within this complex, in addition to genome-scale data from a subset of these representing over 1600 single-copy nuclear genes spanning over 3 Mb of the genome. Our study corroborates the perspective that the morphologically and chemically variable Lecidea atrobrunnea group reflects a complex of distinct species-level lineages, with 42–83 candidate species inferred from the ITS region and high levels of diversity inferred from a subset of specimens using genome-scale data. However, both phenotype- and molecular-based species boundaries remained unsettled, with the most common nominal taxa recovered as highly polyphyletic and with conflict among different molecular species delimitation approaches. Our study also highlights the potential for geographically restricted species, with fascinating biogeographical patterns, challenging, in part, the assumed cosmopolitan distribution of L. atrobrunnea s. lat. This study provides valuable direction for future research that will be crucial in understanding diversification and establishing a robust taxonomy for this well-known species complex.

Hook Effect in Semiquantitative SARS-CoV-2 Antispike Total Antibody Assay.

The hook effect is a common preanalytical error that results in falsely decreased analyte concentrations in immunoassays. We present here an example in a semiquantitative SARS-CoV-2 antispike total antibody assay and report the incidence of this error at our institution. All specimens with initial results within the reportable range of the assay were diluted. Results with higher results upon dilution were determined to have the hook effect. In a subset of specimens, these results were also confirmed as elevated on an alternative SARS-CoV-2 antibody assay. Over 1 month, 12 (9.1%) of 132 results were within the analytical measuring range of the assay. Of these, 11 showed the hook effect and required dilution to obtain accurate results. These represented 8.3% of our total testing volume. The hook effect was detected in a semiquantitative SARS-CoV-2 antispike total antibody assay at a high incidence. This error results in observed concentrations much lower than is accurate. Laboratories should be aware of this issue and consider manually diluting specimens within the reportable range of the assay to detect this issue.

New York State Tick Blitz: harnessing community-based science to understand range expansion of ticks.

Ticks and tick-borne diseases are an immense public health burden in New York State (NYS), United States. Tick species and associated pathogens are expanding into new areas, shifting threats to human, and animal health across the state. For example, the invasive tick, Haemaphysalis longicornis Neumann (Acari: Ixodidae), was first detected in the United States in 2017 and has since been identified in 17 states, including NYS. In addition, Amblyomma americanum (L.) (Acari: Ixodidae) is a native tick thought to be reestablishing historical populations in NYS. We implemented a community-based science project called the "NYS Tick Blitz" to determine the distribution of A. americanum and H. longicornis in NYS. Community volunteers were recruited, provided with education, training, and materials to conduct active tick sampling during a 2-wk period in June 2021. Fifty-nine volunteers sampled 164 sites across 15 counties, for a total of 179 separate collection events and 3,759 ticks collected. The most frequently collected species was H. longicornis, followed by Dermacentor variabilis Say (Acari: Ixodidae), Ixodes scapularis Say (Acari: Ixodidae), and A. americanum, respectively. Through the NYS Tick Blitz collections, H. longicornis was identified for the first time in Putnam County. We conducted pooled pathogen testing on a subset of specimens, with the highest rates of infection detected for pathogens transmitted by I. scapularis, including Borrelia burgdorferi, Anaplasma phagocytophilum, and Babesia microti. Most participants who completed a follow-up survey (n = 23, 71.9%) were promoters of the NYS Tick Blitz and 50% (n = 15) reported that they enjoyed participating in meaningful science.

MP50-12 CLINICOPATHOLOGIC ANALYSIS AND DIGITAL EVALUATION OF ORCHIECTOMY SPECIMENS IN PATIENTS WITH GENDER DYSPHORIA

You have accessJournal of UrologyCME1 Apr 2023MP50-12 CLINICOPATHOLOGIC ANALYSIS AND DIGITAL EVALUATION OF ORCHIECTOMY SPECIMENS IN PATIENTS WITH GENDER DYSPHORIA Ivan De La Riva, Ruoji Zhou, Alcino Gama, Bonnie Choy, Bogdan Isaila, Diana Bowen, Robert Brannigan, and Ximing Yang Ivan De La RivaIvan De La Riva More articles by this author , Ruoji ZhouRuoji Zhou More articles by this author , Alcino GamaAlcino Gama More articles by this author , Bonnie ChoyBonnie Choy More articles by this author , Bogdan IsailaBogdan Isaila More articles by this author , Diana BowenDiana Bowen More articles by this author , Robert BranniganRobert Brannigan More articles by this author , and Ximing YangXiming Yang More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003298.12AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Gender-affirmation therapy for transgender individuals is an area of increasing importance in genitourinary pathology. These patients undergo various forms of hormonal replacement therapy (HRT) followed by bilateral orchiectomy. To date, there are very limited studies on the histological and biochemical alterations in these testes. METHODS: We conducted a retrospective search of the database for gender-affirming orchiectomies performed at our institution from 2018 to 2022. Clinical features, including age, length and types of hormonal treatment, and pathologic features, were reviewed and summarized. 19 cases were subjected to whole slide imaging scanning for digital analysis using the Leica system. Immunohistochemistry for OCT4 and CD117 was performed on selected cases. RESULTS: Atrophic changes and decreased spermatogenesis were found in all the gender-affirming orchiectomy specimens examined, with decreased mean tubule diameter (102.9 μm, 202 tubules examined) compared to the tubular size of germ cell neoplasia in situ (GCNIS) and normal testis (139.3 μm and 143.7 μm, 326 and 185 tubules examined, respectively). All cases showed significant maturation arrest, with 55 testes showing aspermatogenesis and the remaining 14 testes showing hypospermatogenesis. Various degrees of basement membrane thickening were observed in all testes, and 55.1% of the testes were found to have focal tubule hyalinization/obliteration. The number of Leydig cells was reduced, with marked cytoplasmic vacuolization and degenerative changes. In a subset of specimens, scattered germ cells displayed prominent nucleomegaly. Using digital analysis, the average nuclear size of these abnormal germ cells was 14.4 μm in diameter (35 cells counted), approaching or exceeding the size of the neoplastic nucleus in GCNIS (mean diameter=12.1 μm, 33 cells counted). However, the enlarged nuclei of these germ cells demonstrated a smooth nuclear membrane, small nucleoli, and fine granular and smudged chromatin, significantly different from the nuclei in GCNIS. Furthermore, immunohistochemistry staining of the germ cells with nucleomegaly in tested cases demonstrated negative staining for both OCT4 and CD117, militating again the possibility of GCNIS. CONCLUSIONS: All the testes from gender-affirming orchiectomies in our study exhibit significantly reduced spermatogenesis, including maturation arrest and atrophic changes. In addition, the presence of scattered germ cells with nucleomegaly in these specimens can mimic GCNIS. Source of Funding: NA © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e689 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Ivan De La Riva More articles by this author Ruoji Zhou More articles by this author Alcino Gama More articles by this author Bonnie Choy More articles by this author Bogdan Isaila More articles by this author Diana Bowen More articles by this author Robert Brannigan More articles by this author Ximing Yang More articles by this author Expand All Advertisement PDF downloadLoading ...

Vector mapping and bloodmeal metabarcoding demonstrate risk of urban Chagas disease transmission in Caracas, Venezuela.

Chagas disease is a significant public health risk in rural and semi-rural areas of Venezuela. Triatomine infection by the aetiological agent Trypanosoma cruzi is also observed in the Metropolitan District of Caracas (MDC), where foodborne T. cruzi outbreaks occasionally occur but active vector-to-human transmission (infection during triatomine bloodmeal) is considered absent. Citizen science-based domiciliary triatomine collection carried out between 2007 and 2013 in the MDC has advanced understanding of urban T. cruzi prevalence patterns and represents an important public awareness-building tool. The present study reports on the extension of this triatomine collection program from 2014 to 2019 and uses mitochondrial metabarcoding to assess feeding behavior in a subset of specimens. The combined, thirteen-year dataset (n = 4872) shows a high rate of T. cruzi infection (75.2%) and a predominance of Panstrongylus geniculatus (99.01%) among triatomines collected in domiciliary areas by MDC inhabitants. Collection also involved nymphal stages of P. geniculatus in 18 of 32 MDC parishes. Other collected species included Triatoma nigromaculata, Triatoma maculata, Rhodnius prolixus, and Panstrongylus rufotuberculatus. Liquid intestinal content indicative of bloodmeal was observed in 53.4% of analyzed specimens. Dissection pools representing 108 such visually blooded P. geniculatus specimens predominantly tested positive for human cytochrome b DNA (22 of 24 pools). Additional bloodmeal sources detected via metabarcoding analysis included key sylvatic T. cruzi reservoirs (opossum and armadillo), rodents, and various other synanthropic and domesticated animals. Results suggest a porous sylvatic-domiciliary transmission interface and ongoing adaptation of P. geniculatus to the urban ecotope. Although P. geniculatus defecation traits greatly limit the possibility of active T. cruzi transmission for any individual biting event, the cumulation of this low risk across a vast metropolitan population warrants further investigation. Efforts to prevent triatomine contact with human food sources also clearly require greater attention to protect Venezuela's capital from Chagas disease.

Combining high‐resolution ex vivo MRI and histopathology to identify medial temporal lobe atrophy patterns specific to tau pathology in Alzheimer’s Disease

AbstractBackgroundTau pathology in the medial temporal lobe (MTL) is closely linked to neurodegeneration, and is an early pathological change associated with Alzheimer’s disease (AD). MRI measures of MTL neurodegeneration have proven to be sensitive to change during preclinical AD. Current measures are confounded by the presence of non‐AD pathologies (e.g., TDP‐43, ageing). Here, we combine ex vivo imaging with histopathological ratings of tau and TDP‐43 to identify fine‐grained MTL atrophy patterns specific to tau. Such an analysis could be used to define MTL “hotspots” where in vivo measures of neurodegeneration are expected to be strongly associated with tau, potentially enabling the development of biomarkers that are more effective during early AD clinical trials.MethodEx vivo MRI scans (0.2x0.2x0.2mm3, 9.4T) of human MTL specimens were combined using a customized registration approach to construct a 3D atlas. Using serial histology available in a subset of specimens (n = 11), MTL subregions in the atlas were labelled based on cytoarchitecture (Ravikumar et al., 2021) (Figure 1A). To perform thickness analysis, 29 specimens containing a primary diagnosis of AD or primary age‐related tauopathy were registered to the atlas (13 specimens were registered to the atlas during atlas construction, and 16 were registered to the atlas after it was constructed) (Figure 1B, Table 1). Using histopathology measures of tau and TDP‐43 pathology (based on contralateral sampling), we investigated the association between tau and thickness by fitting a linear model (with age/TDP‐43 as covariates) at each point along the MTL and SRLM surface.ResultPointwise thickness analysis reveals significant atrophy patterns in the transentorhinal region and SRLM. When excluding age from the model, stronger tau associations are observed in the SRLM, entorhinal cortex, and extending further towards Brodmann Area 35 (Figure 2).ConclusionOur findings are consistent with early Braak stages but suggest that covarying for age may be obscuring some associations due to tau since age and tau burden have a highly correlated relationship (Figure 3). In future work, quantitative maps of NFT burden will be mapped from serial pathology images into atlas space, allowing us to characterize NFT distribution in 3D (Yushkevich et al., 2021).

Evaluation of Dried Blood and Cerebrospinal Fluid Filter Paper Spots for Storing and Transporting Clinical Material for the Molecular Diagnosis of Invasive Meningococcal Disease.

To improve the storage and transport of clinical specimens for the diagnosis of Neisseria meningitidis (Nm) infections in resource-limited settings, we have evaluated the performance of dried blood spot (DBS) and dried cerebrospinal fluid spot (DCS) assays. DBS and DCS were prepared on filter paper from liquid specimens previously tested for Nm in the United Kingdom. Nm was detected and genogrouped by real-time PCR performed on crude genomic DNA extracted from the DBS (n = 226) and DCS (n = 226) specimens. Targeted whole-genome sequencing was performed on a subset of specimens, DBS (n = 4) and DCS (n = 6). The overall agreement between the analysis of liquid and dried specimens was (94.2%; 95% CI 90.8–96.7) for blood and (96.4%; 95% CI 93.5–98.0) for cerebrospinal fluid. Relative to liquid specimens as the reference, the DBS and DCS assays had sensitivities of (89.1%; 95% CI 82.7–93.8) and (94.2%; 95% CI 88.9–97.5), respectively, and both assays had specificities above 98%. A genogroup was identified by dried specimen analysis for 81.9% of the confirmed meningococcal infections. Near full-length Nm genome sequences (>86%) were obtained for all ten specimens tested which allowed determination of the sequence type, clonal complex, presence of antimicrobial resistance and other meningococcal genotyping. Dried blood and CSF filter spot assays offer a practical alternative to liquid specimens for the molecular and genomic characterisation of invasive meningococcal diseases in low-resource settings.

Expanded geographic distribution and host preference of Anopheles gibbinsi (Anopheles species 6) in northern Zambia

BackgroundNchelenge District in northern Zambia suffers from holoendemic malaria transmission despite a decade of yearly indoor residual spraying (IRS) and insecticide-treated net (ITN) distributions. One hypothesis for this lack of impact is that some vectors in the area may forage in the early evening or outdoors. Anopheles gibbinsi specimens were identified in early evening mosquito collections performed in this study area, and further insight was gleaned into this taxon, including characterizing its genetic identity, feeding preferences, and potential role as a malaria vector.MethodsMosquitoes were collected in July and August 2019 by CDC light traps in Nchelenge District in indoor sitting rooms, outdoor gathering spaces, and animal pens from 16:00–22:00. Host detection by PCR, COI and ITS2 PCR, and circumsporozoite (CSP) ELISA were performed on all samples morphologically identified as An. gibbinsi, and a subset of specimens were selected for COI and ITS2 sequencing. To determine risk factors for increased abundance of An. gibbinsi, a negative binomial generalized linear mixed-effects model was performed with household-level variables of interest.ResultsComparison of COI and ITS2 An. gibbinsi reference sequences to the NCBI database revealed > 99% identity to “Anopheles sp. 6” from Kenya. More than 97% of specimens were morphologically and molecularly consistent with An. gibbinsi. Specimens were primarily collected in animal pen traps (59.2%), followed by traps outdoors near where humans gather (24.3%), and traps set indoors (16.5%). Host DNA detection revealed a high propensity for goats, but 5% of specimens with detected host DNA had fed on humans. No specimens were positive for Plasmodium falciparum sporozoites. Animal pens and inland households > 3 km from Lake Mweru were both associated with increased An. gibbinsi abundance.ConclusionsThis is the first report of An. gibbinsi in Nchelenge District, Zambia. This study provided a species identity for unknown “An. sp. 6” in the NCBI database, which has been implicated in malaria transmission in Kenya. Composite data suggest that this species is largely zoophilic and exophilic, but comes into contact with humans and the malaria parasites they carry. This species should continue to be monitored in Zambia and neighbouring countries as a potential malaria vector.

Anti-phospholipid antibodies are elevated and functionally active in chronic rhinosinusitis with nasal polyps.

BackgroundPolyps from patients with chronic rhinosinusitis with nasal polyps (CRSwNP) contain increased levels of autoreactive antibodies, B cells and fibrin deposition. Anti‐phospholipid antibodies (APA) are autoantibodies known to cause thrombosis but have not been implicated in chronic rhinosinusitis (CRS).ObjectiveTo compare APA levels (anti‐cardiolipin, anti‐phosphatidylethanolamine (anti‐PE), and anti‐β2‐glycoprotein (anti‐B2GP)) in nasal polyp (NP) tissue with tissue from control and CRS without nasal polyp (CRSsNP) patients, we tested whether NP antibodies affect coagulation, and correlate APAs with anti‐dsDNA IgG and markers of coagulation.MethodsPatient specimens were assayed for APA IgG, anti‐dsDNA IgG and thrombin‐anti‐thrombin (TaT) complex by ELISA. Antibodies from a subset of specimens were tested for modified activated partial thromboplastin time (aPTT) measured on an optical‐mechanical coagulometer.ResultsAnti‐cardiolipin IgG in NP was 5‐fold higher than control tissue (p < .0001). NP antibodies prolonged aPTT compared to control tissue antibodies at 400 µg/mL (36.7 s vs. 33.8 s, p = .024) and 600 µg/mL (40.9 s vs. 34.7 s, p = .0037). Anti‐PE IgG antibodies were increased in NP (p = .027), but anti‐B2GP IgG was not significantly higher (p = .084). All APAs correlated with anti‐dsDNA IgG levels, which were also elevated (R = .77, .71 and .54, respectively, for anti‐cardiolipin, anti‐PE, and anti‐B2GP; all p < .001), but only anti‐cardiolipin (R = .50, p = .0185) and anti‐PE (R = 0.45, p = .037) correlated with TaT complex levels.ConclusionsAPA IgG antibodies are increased in NP and correlate with autoreactive tissue antibodies. NP antibodies have in vitro anti‐coagulant activity similar to those observed in anti‐phospholipid syndrome, suggesting that they may have pro‐coagulant effects in polyp tissue.

Genomic analysis of group A Streptococcus isolated during a correctional facility outbreak of MRSA in 2004

In 2004-2005, an outbreak of impetigo occurred at a correctional facility during a sentinel outbreak of methicillin- resistant Staphylococcus aureus (MRSA) in Alberta, Canada. Next-generation sequencing (NGS) was used to characterize the group A Streptococcus (GAS) isolates and evaluate whether genomic biomarkers could distinguish between those recovered alone and those co-isolated with S. aureus. Superficial wound swabs collected from all adults with impetigo during this outbreak were cultured using standard methods. NGS was used to characterize and compare all of the GAS and S. aureus genomes. Fifty-three adults were culture positive for GAS, with a subset of specimens also positive for MRSA (n = 5) or methicillin-sensitive S. aureus (n = 3). Seventeen additional MRSA isolates from this facility from the same time frame (no GAS co-isolates) were also included. All 78 bacterial genomes were analyzed for the presence of known virulence factors, plasmids, and antimicrobial resistance (AMR) genes. Among the GAS isolates were 12 emm types, the most common being 41.2 (n = 27; 51%). GAS genomes were phylogenetically compared with local and public datasets of invasive and non-invasive isolates. GAS genomes had diverse profiles for virulence factors, plasmids, and AMR genes. Pangenome analysis did not identify horizontally transferred genes in the co-infection versus single infections. GAS recovered from invasive and non-invasive sources were not genetically distinguishable. Virulence factors, plasmids, and AMR profiles grouped by emm type, and no genetic changes were identified that predict co-infection or horizontal gene transfer between GAS and S. aureus.

Alvocidib Enhances Venetoclax Sensitivity in Unfavorable-Risk Multiple Myeloma Cells In Vitro and In Vivo

▪Abstract:Background: Multiple myeloma (MM) is a lymphoproliferative neoplasm involving mature plasma cells and remains incurable. New and more effective treatment options are clearly needed for higher-risk MM groups. The BCL-2-specific BH3-mimetic venetoclax (ABT-199) has been reported to be active in patients with the t(11;14) translocation, an intermediate risk group in response to immunomodulatory drugs (IMiDS) and proteasome inhibitors (PIs), prompting efforts to extend venetoclax activity to include more resistant, higher-risk MM subsets.Methods:Effects of the CDK9 inhibitor alvocidib (flavopiridol; FP) on responses to the BCL-2-specific BH3-mimetic venetoclax (ABT-199) were examined in multiple myeloma (MM) cells, including higher-risk sub-types exhibiting low BCL-2/MCL-1 expression profiles and limited sensitivity to venetoclax alone. Cell death and protein expression were evaluated by western blot, and immunofluorescence. Xenograft models, including an orthotopic murine model (NOD/SCID-γ mice) and an immune-competent model (C57BL/KaLwR1 mice) were used to characterize combination effects in vivo.Results:Alvocidib (100-200 nM; 24 h) synergistically increased ABT-199 (250-750 nM) lethality in both ABT-199-sensitive and insensitive MM cells, and these interactions were synergistic by Median Dose Effect analysis with CI values < 1.0 in both t(4;14) H929 and OPM-2 cells. Synergistic interactions or markedly enhanced lethality were also observed in multiple other intermediate or high-risk models (e.g., KMS11(t(4;14)), OPM2(t(4;14)), RPMI8226 (t(14;16) and t(8;22)), and KMS28-PE (t(4;14))). The alvocidib/venetoclax regimen was effective in various drug-resistant MM lines e.g., bortezomib, melphalan, and revlimid. Alvocidib blocked CDK9 activation and P-TEFb phosphorylation, down-regulated MCL-1, increased BCL-2/MCL-1 ratios, and up-regulated BIM. MCL-1 ectopic expression or knock-down in MM cells significantly diminished or increased ABT-199 sensitivity respectively. CDK9 shRNA knock-down triggered MCL-1 down-regulation and increased ABT-199 activity, while BIM knock-down significantly reduced alvocidib/venetoclax lethality. HS-5 cell co-culture failed to protect MM cells from the FP/ABT-199 regimen, suggesting circumvention of microenvironmental signals. The effects of venetoclax +/- alvocidib were investigated in a larger number of primary specimens (N =23). Combined treatment very significantly reduced the survival of cells compared to single agent treatment (24 h; P < 0.0001 and 0.0016). In the sub-set of specimens in which primitive progenitor cells could be identified (CD138-/CD19+/CD20+/CD127+; N=18), significant reductions in survival following combined treatment were also observed (P < 0.0074 and 0.0001). In contrast, alvocidib did not increase ABT199 lethality in normal CD34+ cord blood cells. To assess the in vivo relevance of these findings, NOD/SCID-γ mice were inoculated with fluorescently-labled bortezomib-resistant PS-R cells. Combined treatment sharply reduced tumor burden and significantly prolonged survival compared to single-agent treatment (P < 0.0001). Median survival was 79 days for animals in the combined treatment group, versus 62, 63, and 71 days for those in vehicle, venetoclax, or alvocidib groups, respectively. In addition, combined treatment was not associated with significant increases in weight loss or other forms of toxicity e.g., behavioral changes, fur loss etc. Finally, parallel studies were performed in an immune-competent model. C57BL/KaLwR1 mice were injected intravenously with murine MM 5TGM1 cells stably expressing luciferase. Combined administration suppressed tumor growth, manifested by clearly diminished intensity and significantly prolonged survival compared to single agents (P < 0.001), but did not lead to increases in toxicity e.g., weight loss. Specifically, median survival was 69 days for animals in the combined treatment group, versus 39, 49, and 53 days for those in vehicle, venetoclax, or alvocidib groups, respectively.Conclusions:These findings argue that CDK9 inhibitors e.g., FP may increase the anti-myeloma activity of ABT-199, including in unfavorable-risk MM minimally responsive to ABT-199 alone. DisclosuresOrlowski:BioTheryX: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Potential Familial Predisposition to Hematologic Neoplasm Detected Incidentally in Adult Patients Presenting with Myeloid Neoplasms

Introduction: Germline mutations in CEBPA, ETV6, RUNX1, and GATA2 have been reported to be associated with predisposition to hematologic malignancies. Next Generation Sequencing (NGS), used to identify somatic mutations to aid in targeted therapies, may incidentally uncover inherited germline mutations. Recognizing familial predisposition is important as it may impact management and surveillance. We assessed the prevalence of potentially clinically significant germline mutations detected incidentally by NGS in samples from patients investigated for myeloid disorders.Methods: NGS was performed to identify somatic mutations in 54 genes, on 6100 consecutive bone marrow aspirates or peripheral blood specimens between January 2014 and mid-2017. The average depth of sequencing was 10,000x. DNA was extracted using the QIAamp DNA Mini Kit and subjected to the TruSight Myeloid panel (Illumina, San Diego, CA). Most (92%) of the common dbSNP variants (>10% allele frequency in dbSNP) were found between 45-55% variant allele frequency (VAF) or 98-100% VAF for hetero- and homozygous states. This suggests that common SNPs are mostly found between those frequencies; hence, alleles with these frequencies were defined as candidate germline variants. However, to further enrich for germline variants, we only analyzed those cases with at least one other somatic variant with VAF of 5-40%; the rational being that it is unlikely that a sample would have two true independent somatic mutation events in different subclones. Following this enrichment process, 2951 specimens were eligible for further analysis for potential germline mutations. The ClinVar and dbSNP databases were used to assess allele frequencies and clinical significance.Results: In 2951 of 6100 specimens, we reported at least one somatic mutation with a variant allele frequency in the range of 5-40%. A total of 5322 variants were identified in this subset of specimens. Among the candidate germline mutations in 4 genes implicated in myeloid neoplasm predisposition, we identified 93 unique putative loss of function variants that met our germline criteria. Nine of these mutations were identified in more than 3 cases and more than half of these were represented in ClinVar as germline loss of function variants. One of them, RUNX1 : p.Arg201Gln, identified in 5 patients, is described as a germline pathogenic variant. In contrast, the other 84 variants identified in the 4 genes were rare (observed in 1 or 2 cases) and few (7/84) had been characterized in dbSNP. Most loss of function germline variants are unique and rare with few hotspots, which is consistent with our data.Analysis of germline mutations in genes, which have not yet been implicated in predisposition to myeloid neoplasm, revealed 4858 candidate mutations in 23 genes: 15.4% (750/4858) were unique, 13.6% (102/750) were more common (> 3 cases), while most (86.4%; 648/750) were found in only 1 or 2 cases. Some of the more common variants were identified in ClinVar as pathogenic germline variants, including IDH2 (p.Arg140Gln; MIM: 613657), identified in 84 cases, and a TP53 loss of function mutation (p.Pro72Arg) identified in 424 cases.Conclusion: Germline mutations, GATA2, RUNX1, CEBPA, and ETV6, known to predispose to myeloid malignancies, may be identified incidentally during testing for somatic mutations in myeloid malignancies.In addition, if the NGS panel includes genes other than those known to predispose towards myeloid malignancies, pathogenic variants of wide ranging clinical implication may be uncovered, as observed in the cases with mutation in IDH2 or TP53 . Although the specific mutation observed in IDH2 has been identified most often as a somatic mutation, it has been reported to be inherited in one instance. Inheriting this allele can confer D-2-hydroxyglutaric aciduria-2 (D2HGA2). The TP53 mutation observed is known to be involved in other hereditary cancers (e.g. Li-Fraumeni syndrome) and could affect the recommended therapy of antineoplastic agents.Rare incidentally identified potential germline mutations remain a challenge as their clinical significance may be unknown. These mutations require additional investigation to determine if they could contribute to clinical management. DisclosuresFunari:NeoGenomics: Employment. Thangavelu:NeoGenomics: Employment. Ma:NeoGenomics: Employment. De Dios:NeoGenomics: Employment. Agersborg:NeoGenomics: Employment. Blocker:NeoGenomics: Employment. Albitar:NeoGenomics: Employment.

Supplementation of sputum cultures with culture filtrate to detect tuberculosis in a cross-sectional study of HIV-infected individuals

While some healthcare systems have shifted to molecular diagnostics, culture still remains the gold standard for tuberculosis diagnosis, but it is limited by its long duration to a positive result. Methods to reduce time to culture positivity (TTP) are urgently required. We determined if growth factor supplementation in the mycobacterial growth indicator tube (MGIT) culture system reduces TTP. MGITs were supplemented with fresh culture filtrate (CF) as a source of growth stimulatory molecules from axenic Mycobacterium tuberculosis culture. Different volumes of CF and media components were tested. The performance of these modified MGITs was assessed with sputum from HIV-TB co-infected individuals. Reducing the volume of MGIT cultures and removal of detergent from cultures grown to generate CF had a marginal but significant benefit on reducing TTP. In a subset of specimens, CF inhibited growth. Following optimization of methods, a reduced TTP occurred in specimens with low bacillary load as measured by GeneXpert, smear microscopy and colony forming units. Three specimens that were negative under standard conditions flagged positive following CF supplementation. Our data provide preliminary evidence that addition of CF to MGIT cultures can enhance detection of M. tuberculosis in HIV-TB co-infected patients with low sputum bacillary loads.

Unheeded biodiversity: perspectives of DNA barcoding of Trichoptera in Bosnia and Herzegovina

Bosnia and Herzegovina has valuable natural resources with a high percentage of endemic and autochthonous species (Kučinić et al. 2008, Đug and Drešković 2012). The freshwater fauna of Trichoptera in this area is under-investigated, with a lack of morphological description of different life stages and DNA barcode data. Public data show 58,993 barcode entries for Trichoptera in the Barcode of Life Data Systems (BOLD) submitted from 92 countries, and none from Bosnia and Herzegovina (B&amp;amp;H) (BOLD 2021). Previous research in Bosnia and Herzegovina has provided the first DNA barcode for the endemic species Rhyacophila bosnica, stored in GeneBank, under accession number MK211322 by a domestic institution (Kalamujić Stroil et al. 2018). A few DNA barcodes of adult individuals of Trichoptera from Bosnia and Herzegovina were found in BOLD. However, these specimens were collected on B&amp;amp;H territory, but analyzed, processed, and stored by foreign institutions. To change the current state of DNA barcoding of Trichoptera in Bosnia and Herzegovina, we aimed to employ this approach in investigating caddisflies in selected habitats in the Sarajevo Canton. Our fieldwork was done in all five protected areas (spring of the Bosna River, Bijambare, Trebević, Skakavac, and Bentbaša) in which larvae samples were collected according to the AQEM sampling methodology. The standard animal DNA barcode was successfully obtained using degenerated primers LCO1490- JJ and HCO2198-JJ (Astrin and Stüben 2008). Out of 684 collected individuals (313 Trebević, 130 spring of the Bosna River, 117 Bijambare, 71 Bentbaša, 53 Skakavac), a subset of specimens were sequenced. We uncovered 14 different taxa, 11 genera and six families (Limnephilidae, Glossosomatidae, Rhyacophilidae, Goeridae, Hydropsychidae, Polycentropodidae). The preliminary data of Trichoptera composition in the Sarajevo Canton indicated species richness. Based on our sequential data, a new subspecies was discovered in two investigated areas (Valladolid et al. 2020), proving that Trichoptera species diversity in our country is far from entirely uncovered. The benefit and power of the DNA barcoding approach are that it can pinpoint the areas of vast and unknown species diversity more economically, both financially and temporarily, than the morphological approach. Therefore, we believe that it is critical to support the development of DNA barcoding for the bioassessment of freshwater ecosystems in Bosnia and Herzegovina. Several problems prevented us from exploiting sequential data to the fullest. Despite a general notion among scientists that European Trichoptera species are well covered in the BOLD database, most of the sequences we obtained were absent from the database. Secondly, we recognized that morphological data about the larval developmental stage of B&amp;amp;H Trichoptera species are largely missing. The unified, updated, and complete data on this order of insects is urgently needed. However, insufficient financial support by governmental institutions and lack of systematic approach to barcoding the wildlife of Bosnia and Herzegovina hampers this process. Further attempts to collaborate with the stakeholders can be crucial with profound and substantial implications for biomonitoring of aquatic macroinvertebrates in general. New approaches, such as novel DNA barcoding-based methodology can fill an important gap in our knowledge of Balkan caddisflies haplotypes, lineages, and their diversification and distribution patterns.