The clinical impact of early detection of ESBL-producing Enterobacterales with PCR-based blood culture assays.

BACKGROUND: Starting January 4, 2021, our health system core microbiology laboratory changed blood culture identification (BCID) platforms to ePlex BCID from BioFire BCID1 with the additional capability to detect the blaCTX-M-Type gene of ESBL-producing organisms. Clinical outcomes of ESBL bloodstream infections (BSI) after implementing ePlex BCID were unknown. METHODS: Patients with ESBL BSI were compared pre and postimplementation of ePlex BCID in this 11-hospital retrospective analysis (BioFire BCID1 in 2019 vs ePlex BCID in 2021). The primary outcome was time from the Gram stain result to escalation to a carbapenem. Secondary outcomes included in-hospital mortality, 30-day readmission rate, length of stay (LOS), and the duration of antimicrobial therapy. RESULTS: A total of 275 patients were analyzed. The median time of Gram stain result to escalation to carbapenem was reduced from 44.5 hours with BioFire BCID1 to 7.9 hours with ePlex BCID (P < .001). There were no significant differences in mortality, 30-day readmission, or LOS. The duration of antimicrobial therapy for ESBL BSI was lower in the ePlex BCID group (from 14.4 days to 12.7 days, P = .014). CONCLUSIONS: Timely detection of the blaCTX-M-Type gene by BCID provides valuable information for the early initiation of appropriate and effective antimicrobial therapy. Although it was not associated with lower mortality, 30-day readmission, or LOS, it may have benefits such as decreasing antimicrobial exposure to patients.

Identification of basal complex protein that is essential for maturation of transmission-stage malaria parasites.

Malaria remains a global driver of morbidity and mortality. To generate new antimalarials, one must elucidate the fundamental cell biology of Plasmodium falciparum, the parasite responsible for the deadliest cases of malaria. A membranous and proteinaceous scaffold called the inner membrane complex (IMC) supports the parasite during morphological changes, including segmentation of daughter cells during asexual replication and formation of transmission-stage gametocytes. The basal complex lines the edge of the IMC during segmentation and likely facilitates IMC expansion. It is unknown, however, what drives IMC expansion during gametocytogenesis. We describe the discovery of a basal complex protein, PfBLEB, which we find to be essential for gametocytogenesis. Parasites lacking PfBLEB harbor defects in IMC expansion and are unable to form mature gametocytes. This article demonstrates a role for a basal complex protein outside of asexual division, and, importantly, highlights a potential molecular target for the ablation of malaria transmission.

Evaluation of the automated LIAISON® SARS-CoV-2 TrimericS IgG assay for the detection of circulating antibodies.

OBJECTIVES: COVID-19 has brought about tests from many manufacturers. While molecular and rapid antigen tests are targeted for early diagnosis, immunoassays have a larger role in epidemiological studies, understanding longitudinal immunity, and in vaccine development and response. METHODS: The performance of the LIAISON® SARS-CoV-2 TrimericS IgG assay was evaluated against the Beckman ACCESS SARS-CoV-2 IgG assay in New Mexico, and against the Siemens ADVIA Centaur COV2G assay in New York. Discordant samples were parsed using a microneutralization assay. RESULTS: A SARS-CoV-2 antibody positivity rate of 23.8% was observed in the samples tested in New York (September 2020), while in the same month the positivity rate was 1.5% in New Mexico. Positive and negative agreement were 67.6% (95% CI 49.5-82.6%) and 99.8% (95% CI 99.5-99.9%), respectively, with the Beckman test, and 98.0% (95% CI 95.7-99.3%) and 94.8% (95% CI 93.4-96.0%), respectively, with the Siemens test. Receiver operating characteristic analysis for the detection of SARS-CoV-2 antibodies discloses an AUC, area under the curve, of 0.996 (95% CI 0.992-0.999) for the LIAISON® SARS-CoV-2 TrimericS IgG assay. The criterion associated to the Youden Index was determined to be >12.9 kAU/L with a sensitivity of 99.44% and a specificity of 99.82%. CONCLUSIONS: The LIAISON® SARS-CoV-2 TrimericS IgG assay is highly sensitive and specific. The balance of these parameters, without emphasis on high specificity alone, is particularly important when applied to high prevalence populations, where a highly sensitive assay will result in reporting a lower number of false negative subjects.

SARS-CoV-2 IgG antibody responses in New York City.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a strain of coronavirus that causes coronavirus disease 2019 (Covid-19) and has been declared a global pandemic by the World Health Organization. Total cases of SARS-CoV-2 worldwide exceed 10.2 million, with over 503,000 deaths recorded. Little is known about the body's immune response to SARS-CoV-2 infection. In this paper, we describe SARS-CoV-2 IgG antibody responses in 28,523 patients from the New York City metropolitan area and report a SARS-CoV-2 IgG positivity rate of 44%, indicating the widespread nature of the pandemic in the city and state of New York. Additionally, for a subset of patients, we report on the correlation between SARS-CoV-2 patient symptom severity and level of SARS-CoV-2 IgG antibody found in the patient sample.

A Novel Antiparasitic Compound Kills Ring-Stage Plasmodium falciparum and Retains Activity Against Artemisinin-Resistant Parasites.

Spreading antimalarial resistance threatens effective treatment of malaria, an infectious disease caused by Plasmodium parasites. We identified a compound, BCH070, that inhibits asexual growth of multiple antimalarial-resistant strains of Plasmodium falciparum (half maximal inhibitory concentration [IC50] = 1-2 µM), suggesting that BCH070 acts via a novel mechanism of action. BCH070 preferentially kills early ring-form trophozoites, and, importantly, equally inhibits ring-stage survival of wild-type and artemisinin-resistant parasites harboring the PfKelch13:C580Y mutation. Metabolomic analysis demonstrates that BCH070 likely targets multiple pathways in the parasite. BCH070 is a promising lead compound for development of new antimalarial combination therapy that retains activity against artemisinin-resistant parasites.

An essential contractile ring protein controls cell division in Plasmodium falciparum.

During the blood stage of human malaria, Plasmodium falciparum parasites divide by schizogony-a process wherein components for several daughter cells are produced within a common cytoplasm and then segmentation, a synchronized cytokinesis, produces individual invasive daughters. The basal complex is hypothesized to be required for segmentation, acting as a contractile ring to establish daughter cell boundaries. Here we identify an essential component of the basal complex which we name PfCINCH. Using three-dimensional reconstructions of parasites at electron microscopy resolution, we show that while parasite organelles form and divide normally, PfCINCH-deficient parasites develop inviable conjoined daughters that contain components for multiple cells. Through biochemical evaluation of the PfCINCH-containing complex, we discover multiple previously undescribed basal complex proteins. Therefore, this work provides genetic evidence that the basal complex is required for precise segmentation and lays the groundwork for a mechanistic understanding of how the parasite contractile ring drives cell division.

Transcription coupled repair and biased insertion of human retrotransposon L1 in transcribed genes.

BACKGROUND: L1 retrotransposons inserted within genes in the human genome show a strong bias against sense orientation with respect to the gene. One suggested explanation for this observation was the possibility that L1 inserted randomly, but that there was negative selection against sense-oriented insertions. However, multiple studies have now found that de novo and polymorphic L1 insertions, which have little opportunity for selection to act, also show the same bias. RESULTS: Here we show that the transcription-coupled sub-pathway of nucleotide excision repair does not affect the overall rate of insertion of L1 elements, which is in contrast with the regulation by the global sub-pathway of nucleotide excision repair. The transcription-coupled subpathway does cause a strong bias against insertion in the sense orientation relative to genes. CONCLUSIONS: This suggests that a major portion of the L1 orientation bias might be generated during the process of insertion through the action of transcription-coupled nucleotide excision repair.

The Malaria Parasite Cyclin H Homolog PfCyc1 Is Required for Efficient Cytokinesis in Blood-Stage Plasmodium falciparum.

All well-studied eukaryotic cell cycles are driven by cyclins, which activate cyclin-dependent kinases (CDKs), and these protein kinase complexes are viable drug targets. The regulatory control of the Plasmodium falciparum cell division cycle remains poorly understood, and the roles of the various CDKs and cyclins remain unclear. The P. falciparum genome contains multiple CDKs, but surprisingly, it does not contain any sequence-identifiable G1-, S-, or M-phase cyclins. We demonstrate that P. falciparum Cyc1 (PfCyc1) complements a G1 cyclin-depleted Saccharomyces cerevisiae strain and confirm that other identified malaria parasite cyclins do not complement this strain. PfCyc1, which has the highest sequence similarity to the conserved cyclin H, cannot complement a temperature-sensitive yeast cyclin H mutant. Coimmunoprecipitation of PfCyc1 from P. falciparum parasites identifies PfMAT1 and PfMRK as specific interaction partners and does not identify PfPK5 or other CDKs. We then generate an endogenous conditional allele of PfCyc1 in blood-stage P. falciparum using a destabilization domain (DD) approach and find that PfCyc1 is essential for blood-stage proliferation. PfCyc1 knockdown does not impede nuclear division, but it prevents proper cytokinesis. Thus, we demonstrate that PfCyc1 has a functional divergence from bioinformatic predictions, suggesting that the malaria parasite cell division cycle has evolved to use evolutionarily conserved proteins in functionally novel ways.IMPORTANCE Human infection by the eukaryotic parasite Plasmodium falciparum causes malaria. Most well-studied eukaryotic cell cycles are driven by cyclins, which activate cyclin-dependent kinases (CDKs) to promote essential cell division processes. Remarkably, there are no identifiable cyclins that are predicted to control the cell cycle in the malaria parasite genome. Thus, our knowledge regarding the basic mechanisms of the malaria parasite cell cycle remains unsatisfactory. We demonstrate that P. falciparum Cyc1 (PfCyc1), a transcriptional cyclin homolog, complements a cell cycle cyclin-deficient yeast strain but not a transcriptional cyclin-deficient strain. We show that PfCyc1 forms a complex in the parasite with PfMRK and the P. falciparum MAT1 homolog. PfCyc1 is essential and nonredundant in blood-stage P. falciparum PfCyc1 knockdown causes a stage-specific arrest after nuclear division, demonstrating morphologically aberrant cytokinesis. This work demonstrates a conserved PfCyc1/PfMAT1/PfMRK complex in malaria and suggests that it functions as a schizont stage-specific regulator of the P. falciparum life cycle.

The Nucleotide Excision Repair Pathway Limits L1 Retrotransposition.

Long interspersed elements 1 (L1) are active mobile elements that constitute almost 17% of the human genome. They amplify through a "copy-and-paste" mechanism termed retrotransposition, and de novo insertions related to these elements have been reported to cause 0.2% of genetic diseases. Our previous data demonstrated that the endonuclease complex ERCC1-XPF, which cleaves a 3' DNA flap structure, limits L1 retrotransposition. Although the ERCC1-XPF endonuclease participates in several different DNA repair pathways, such as single-strand annealing, or in telomere maintenance, its recruitment to DNA lesions is best characterized in the nucleotide excision repair (NER) pathway. To determine if the NER pathway prevents the insertion of retroelements in the genome, we monitored the retrotransposition efficiencies of engineered L1 elements in NER-deficient cells and in their complemented versions. Core proteins of the NER pathway, XPD and XPA, and the lesion binding protein, XPC, are involved in limiting L1 retrotransposition. In addition, sequence analysis of recovered de novo L1 inserts and their genomic locations in NER-deficient cells demonstrated the presence of abnormally large duplications at the site of insertion, suggesting that NER proteins may also play a role in the normal L1 insertion process. Here, we propose new functions for the NER pathway in the maintenance of genome integrity: limitation of insertional mutations caused by retrotransposons and the prevention of potentially mutagenic large genomic duplications at the site of retrotransposon insertion events.

Receptor for Activated C-Kinase 1 (PfRACK1) is required for Plasmodium falciparum intra-erythrocytic proliferation.

Emerging resistance to current anti-malarials necessitates a more detailed understanding of the biological processes of Plasmodium falciparum proliferation, thus allowing identification of new drug targets. The well-conserved protein Receptor for Activated C-Kinase 1 (RACK1) was originally identified in mammalian cells as an anchoring protein for protein kinase C (PKC) and has since been shown to be important for cell migration, cytokinesis, transcription, epigenetics, and protein translation. The P. falciparum ortholog, PfRACK1, is expressed in blood stages of the parasite and is diffusely localized in the parasite cytoplasm. Using a destabilizing domain to allow inducible knockdown of the endogenous protein level, we evaluated the requirement for PfRACK1 during blood-stage replication. Following destabilization, the parasites demonstrate a nearly complete growth arrest at the trophozoite stage. The essential nature of PfRACK1 suggests that the protein itself or the pathways regulated by the protein are potential targets for novel anti-malarial therapeutics.

Sequencing, identification and mapping of primed L1 elements (SIMPLE) reveals significant variation in full length L1 elements between individuals.

BACKGROUND: There are over a half a million copies of L1 retroelements in the human genome which are responsible for as much as 0.5% of new human genetic diseases. Most new L1 inserts arise from young source elements that are polymorphic in the human genome. Highly active polymorphic "hot" L1 source elements have been shown to be capable of extremely high levels of mobilization and result in numerous instances of disease. Additionally, hot polymorphic L1s have been described to be highly active within numerous cancer genomes. These hot L1s result in mutagenesis by insertion of new L1 copies elsewhere in the genome, but also have been shown to generate additional full length L1 insertions which are also hot and able to further retrotranspose. Through this mechanism, hot L1s may amplify within a tumor and result in a continued cycle of mutagenesis. RESULTS AND CONCLUSIONS: We have developed a method to detect full-length, polymorphic L1 elements using a targeted next generation sequencing approach, Sequencing Identification and Mapping of Primed L1 Elements (SIMPLE). SIMPLE has 94% sensitivity and detects nearly all full-length L1 elements in a genome. SIMPLE will allow researchers to identify hot mutagenic full-length L1s as potential drivers of genome instability. Using SIMPLE we find that the typical individual has approximately 100 non-reference, polymorphic L1 elements in their genome. These elements are at relatively low population frequencies relative to previously identified polymorphic L1 elements and demonstrate the tremendous diversity in potentially active L1 elements in the human population.

The contribution of alu elements to mutagenic DNA double-strand break repair.

Alu elements make up the largest family of human mobile elements, numbering 1.1 million copies and comprising 11% of the human genome. As a consequence of evolution and genetic drift, Alu elements of various sequence divergence exist throughout the human genome. Alu/Alu recombination has been shown to cause approximately 0.5% of new human genetic diseases and contribute to extensive genomic structural variation. To begin understanding the molecular mechanisms leading to these rearrangements in mammalian cells, we constructed Alu/Alu recombination reporter cell lines containing Alu elements ranging in sequence divergence from 0%-30% that allow detection of both Alu/Alu recombination and large non-homologous end joining (NHEJ) deletions that range from 1.0 to 1.9 kb in size. Introduction of as little as 0.7% sequence divergence between Alu elements resulted in a significant reduction in recombination, which indicates even small degrees of sequence divergence reduce the efficiency of homology-directed DNA double-strand break (DSB) repair. Further reduction in recombination was observed in a sequence divergence-dependent manner for diverged Alu/Alu recombination constructs with up to 10% sequence divergence. With greater levels of sequence divergence (15%-30%), we observed a significant increase in DSB repair due to a shift from Alu/Alu recombination to variable-length NHEJ which removes sequence between the two Alu elements. This increase in NHEJ deletions depends on the presence of Alu sequence homeology (similar but not identical sequences). Analysis of recombination products revealed that Alu/Alu recombination junctions occur more frequently in the first 100 bp of the Alu element within our reporter assay, just as they do in genomic Alu/Alu recombination events. This is the first extensive study characterizing the influence of Alu element sequence divergence on DNA repair, which will inform predictions regarding the effect of Alu element sequence divergence on both the rate and nature of DNA repair events.

Regulation of L1 expression and retrotransposition by melatonin and its receptor: implications for cancer risk associated with light exposure at night.

Expression of long interspersed element-1 (L1) is upregulated in many human malignancies. L1 can introduce genomic instability via insertional mutagenesis and DNA double-strand breaks, both of which may promote cancer. Light exposure at night, a recently recognized carcinogen, is associated with an increased risk of cancer in shift workers. We report that melatonin receptor 1 inhibits mobilization of L1 in cultured cells through downregulation of L1 mRNA and ORF1 protein. The addition of melatonin receptor antagonists abolishes the MT1 effect on retrotransposition in a dose-dependent manner. Furthermore, melatonin-rich, but not melatonin-poor, human blood collected at different times during the circadian cycle suppresses endogenous L1 mRNA during in situ perfusion of tissue-isolated xenografts of human cancer. Supplementation of human blood with exogenous melatonin or melatonin receptor antagonist during the in situ perfusion establishes a receptor-mediated action of melatonin on L1 expression. Combined tissue culture and in vivo data support that environmental light exposure of the host regulates expression of L1 elements in tumors. Our data imply that light-induced suppression of melatonin production in shift workers may increase L1-induced genomic instability in their genomes and suggest a possible connection between L1 activity and increased incidence of cancer associated with circadian disruption.

A droplet digital PCR detection method for rare L1 insertions in tumors.

BACKGROUND: The active human mobile element, long interspersed element 1 (L1) currently populates human genomes in excess of 500,000 copies per haploid genome. Through its mobility via a process called target primed reverse transcription (TPRT), L1 mobilization has resulted in over 100 de novo cases of human disease and has recently been associated with various cancer types. Large advances in high-throughput sequencing (HTS) technology have allowed for an increased understanding of the role of L1 in human cancer; however, researchers are still limited by the ability to validate potentially rare L1 insertion events detected by HTS that may occur in only a small fraction of tumor cells. Additionally, HTS detection of rare events varies greatly as a function of read depth, and new tools for de novo element discovery are needed to fill in gaps created by HTS. RESULTS: We have employed droplet digital PCR (ddPCR) to detect rare L1 loci in mosaic human genomes. Our assay allows for the detection of L1 insertions as rare as one cell in every 10,000. CONCLUSIONS: ddPCR represents a robust method to be used alongside HTS techniques for detecting, validating and quantitating rare L1 insertion events in tumors and other tissues.

LINE-1 and Alu retrotransposition exhibit clonal variation.

BACKGROUND: The non-long terminal repeat (non-LTR) retrotransposons, long interspersed element-1 (LINE-1) and Alu are currently active retroelements in humans. We, and others, have observed that different populations of HeLa cells from different laboratories support retrotransposition of LINE-1 and Alu to varying degrees. We therefore tested whether individual cell clones of HeLa and HCT116 cell lines supported different levels of LINE-1 and Alu retrotransposition, and whether these variations were stable upon re-cloning. FINDINGS: Standard retrotransposition tissue culture assays were used to measure a cell's ability to support LINE-1 and Alu retrotransposition in clonal HeLa and HCT116 cell lines. We observed that both LINE-1 and Alu retrotransposition exhibited clonal variation in HeLa cells, with certain HeLa cell clones supporting high levels of LINE-1 and Alu retrotransposition and other cell clones being essentially retrotransposition-dead. This clonal variation was similarly observed in HCT116 cells, although possibly not to the same extent. These patterns of clonal variation are relatively consistent upon re-cloning. CONCLUSIONS: Observations of the variability of LINE-1 and Alu retrotransposition in different populations of the same cell line are supported by our results that indicate in some cell types, individual cell clones can have dramatically differing capacity for retrotransposition. The mixed populations of cells commonly used in laboratories have often been passaged for many generations and accumulated significant genetic and epigenetic diversity. Our results suggest that the clonal variability observed by our cloning experiments may lead to a homogenization of retrotransposition capacity, with the resulting mixed population of cells being composed of individual variants having either increased or decreased retrotransposition potential compared to the starting population.