Detection of an emerging pathogen: A real time qualitative pcr assay targeting Haematospirillum jordaniae for EDTA whole blood and plasma clinical specimens.

Haematospirillum jordaniae is a gram-negative bacterium that has been identified in the blood of septic patients. The environmental source or potential zoonotic host of this bacterium, recently described as a human bacterial pathogen is unknown. An increasing number of H. jordaniae clinical infections identified by our laboratory suggested the need for an assay to detect this organism in order to aid clinical teams and practitioners with faster identification and treatment thus improving patient prognosis. Described here is a real-time qualitative PCR assay designed using gene targets identified from the analysis of 14 H. jordaniae genomes sequenced by the Center for Disease Control and Prevention's (CDC) Special Bacterial Reference Laboratory (SBRL) culture collection. The assay was validated on clinical EDTA whole blood samples as well as on plasma and determined to be effective at detecting as few as 10 copies per microliter (10,000 copies per mL, 4 log/mL) for whole blood samples and 1 copy per microliter (1,000 copies per mL, 3 log mL) for plasma samples.

Human plasma-like medium (HPLM) induces Cryptococcus neoformans in vivo cell morphologies.

The fungal pathogen Cryptococcus neoformans (C. neoformans) forms yeast cells of different sizes and morphological characteristics during infection. These features are usually not seen in standard laboratory in vitro conditions. Here, we describe in vivo cell morphologies when C. neoformans is grown in human plasma-like medium at 37°C, 5% CO2. We observed mixed-size populations of cells less than 1 µm up to 16.8 µm in cell diameter, increased capsule size, high chitin, and DNA content in larger cells. Our findings show that serum is not required for human plasma-like medium (HPLM)-induced C. neoformans cellular heterogeneity. Thus, this new method offers an opportunity to investigate factors of C. neoformans that mediate pathogenesis or host-pathogen interactions in a physiologically relevant setting.IMPORTANCEWe provide a description of new in vitro culture condition using the human plasma-like medium that supports the formation of the full range of in vivo cell morphologies of C. neoformans.

Cell-Free Microbial DNA Analysis: Effects of Blood Plasma and Serum Quantity, Biobanking Protocols, and Isolation Kits.

Recent studies highlight the presence of bacterial sequences in the human blood, suggesting potential clinical significance for circulating microbial signatures. These sequences could presumably serve in the diagnosis, prediction, or monitoring of various health conditions. Ensuring the similarity of samples before bacterial analysis is crucial, especially when combining samples from different biobanks prepared under varying conditions (such as different DNA extraction kits, centrifugation conditions, blood collection tubes, etc.). In this study, we aimed to analyze the impact of different sample collection and nucleic acid extraction criteria (blood collection tube, centrifugation, input volume, and DNA extraction kit) on circulating bacterial composition. Blood samples from four healthy individuals were collected into three different sample collection tubes: K2EDTA plasma tube, sodium citrate plasma tube, and gel tube for blood serum. Tubes were centrifugated at standard and double centrifugation conditions. DNA extraction was performed using 100, 200, and 500 µL plasma/serum input volumes. DNA extraction was performed using three different isolation kits: Norgen plasma/serum cell-free circulating DNA purification micro kit, Applied Biosystems MagMAX cell-free DNA isolation kit, and Qiagen QIAamp MinElute cell-free circulating DNA mini kit. All samples were subjected to 16S rRNA V1-V2 library preparation and sequencing. In total, 216 DNA and 18 water control samples were included in the study. According to PERMANOVA, PCoA, Mann-Whitney, and FDR tests the effect of the DNA extraction kit on the microbiota composition was the greatest, whereas the type of blood collection tube, centrifugation type, and sample input volume for the extraction had minor effects. Samples extracted with the Norgen DNA extraction kit were enriched with Gram-negative bacteria, whereas samples extracted with the Qiagen and MagMAX kits were enriched with Gram-positive bacteria. Bacterial profiles of samples prepared with the Qiagen and MagMAX DNA extraction kits were more similar, whereas samples prepared with the Norgen DNA extraction kit were significantly different from other groups.

[Metagenomic next-generation sequencing of plasma for the identification of bloodstream infectious pathogens in severe aplastic anemia].

Objective: To analyze the diagnostic value of cell-free plasma metagenomic next-generation sequencing (mNGS) pathogen identification for severe aplastic anemia (SAA) bloodstream infection. Methods: From February 2021 to February 2022, mNGS and conventional detection methods (blood culture, etc.) were used to detect 33 samples from 29 consecutive AA patients admitted to the Anemia Diagnosis and Treatment Center of the Hematology Hospital of the Chinese Academy of Medical Sciences to assess the diagnostic consistency of mNGS and conventional detection, as well as the impact on clinical treatment benefits and clinical accuracy. Results: ①Among the 33 samples evaluated by mNGS and conventional detection methods, 25 cases (75.76%) carried potential pathogenic microorganisms. A total of 72 pathogenic microorganisms were identified from all cases, of which 65 (90.28%) were detected only by mNGS. ②All 33 cases were evaluated for diagnostic consistency, of which 2 cases (6.06%) were Composite, 18 cases (54.55%) were mNGS only, 2 cases (6.06%) were Conventional method only, 1 case (3.03%) was both common compliances (mNGS/Conventional testing) , and 10 cases (30.3%) were completely non-conforming (None) . ③All 33 cases were evaluated for clinical treatment benefit. Among them, 8 cases (24.24%) received Initiation of targeted treatment, 1 case (3.03%) received Treatment de-escalation, 13 cases (39.39%) received Confirmation, and the remaining 11 cases (33.33%) received No clinical benefit. ④ The sensitivity of 80.77%, specificity of 70.00%, positive predictive value of 63.64%, negative predictive value of 84.85%, positive likelihood ratio of 2.692, and negative likelihood ratio of 0.275 distinguished mNGS from conventional detection methods (21/12 vs 5/28, P<0.001) . Conclusion: mNGS can not only contribute to accurately diagnosing bloodstream infection in patients with aplastic anemia, but can also help to guide accurate anti-infection treatment, and the clinical accuracy is high.

Evaluation of the effect of extended refrigerated storage of serum and plasma specimens on syphilis serologic test results.

The effect of extended refrigerated storage of 14 serum and plasma specimens on 5 syphilis serologic tests was evaluated for 16 weeks. Higher stability of nontreponemal and treponemal antibodies in serum was recorded compared to plasma. Described work may provide insights on refrigerated specimens' stability and suitability for syphilis tests.

Plasma adipocytokines distinguish tuberculous lymphadenitis from pulmonary tuberculosis.

Adipocytokines are the major secretory products of adipose tissue and potential markers of metabolism and inflammation. However, their association in host immune response against tuberculous lymphadenitis (TBL) disease is not known. Thus, we measured the systemic levels of adipocytokines in TBL (n = 44) and compared to pulmonary tuberculosis (PTB, n = 44) and healthy control (HC, n = 44) individuals. We also examined the pre and post-treatment adipocytokine levels in TBL individuals upon completion of standard anti-tuberculosis treatment (ATT). The receiver operating characteristics (ROC) were performed between TBL, PTB and HCs to find the potential discriminatory markers. Finally, principal component (PCA) analysis was performed to reveal the expression patterns of adipocytokines among study groups. Our results demonstrate that TBL is associated with significantly higher systemic levels of adipocytokines (except resistin) when compared with PTB and significantly lower levels when compared with HC (except adiponectin) individuals. Upon completion of ATT, the systemic levels of adiponectin and resistin were significantly decreased when compared to pre-treatment levels. Upon ROC analysis, all the three adipocytokines discriminated TBL from PTB but not with HCs, respectively. Similarly, adipocytokines were differentially clustered in TBL in comparison to PTB in PCA analysis. Therefore, adipocytokines are a distinguishing feature in TBL compared to PTB individuals.

Quantitative flow chamber system for evaluating in vitro biofilms and the kinetics of S. aureus biofilm formation in human plasma media.

BACKGROUND: It has been well established that biofilm formation on orthopaedic implants is a critical event in the pathogenesis of orthopaedic infections, yet the natural history of this process with respect to bacterial adhesion, proliferation, and glycocalyx matrix production remains poorly understood. Moreover, there are no quantitative methods yet available to assess the differences in biofilm formation between different bacterial strains or implant materials. Consequently, this study aimed to investigate the natural history of S. aureus in in vitro biofilm formation in human plasma media using a flow chamber system. Bioluminescent S. aureus strains were used to better understand the bacterial growth and biofilm formation on orthopaedic materials. Also, the effects of human plasma media were assessed by loading the chamber with Tryptic Soy Broth with 10% human plasma (TSB + HP). RESULTS: Scanning electron microscopy (SEM) was utilized to assess the morphological appearance of the biofilms, revealing that S. aureus inoculation was required for biofilm formation, and that the phenotypes of biofilm production after 24 h inoculation with three tested strains (SH1000, UAMS-1, and USA300) were markedly different depending on the culture medium. Time course study of the bioluminescence intensity (BLI) and biofilm production on the implants due to the UAMS-1 and USA300 strains revealed different characteristics, whereby UAMS-1 showed increasing BLI and biofilm growth until peaking at 9 h, while USA300 showed a rapid increase in BLI and biofilm formation at 6 h. The kinetics of biofilm formation for both UAMS-1 and USA300 were also supported and confirmed by qRT-PCR analysis of the 16S rRNA gene. Biofilms grown in our flow chamber in the plasma media were also demonstrated to involve an upregulation of the biofilm-forming-related genes icaA, fnbA, and alt. The BLI and SEM results from K-wire experiments revealed that the in vitro growth and biofilm formation by UAMS-1 and USA300 on stainless-steel and titanium surfaces were virtually identical. CONCLUSION: We demonstrated a novel in vitro model for S. aureus biofilm formation with quantitative BLI and SEM outcome measures, and then used this model to demonstrate the presence of strain-specific phenotypes and its potential use to evaluate anti-microbial surfaces.

Temporal dynamics of the plasma microbiome in recipients at early post-liver transplantation: a retrospective study.

BACKGROUND: Immunosuppression during liver transplantation (LT) enables the prevention and treatment of organ rejection but poses a risk for severe infectious diseases. Immune modulation and antimicrobials affect the plasma microbiome. Thus, determining the impact of immunosuppression on the microbiome may be important to understand immunocompetence, elucidate the source of infection, and predict the risk of infection in LT recipients. We characterized the plasma microbiome of LT recipients at early post-LT and assessed the association between the microbiome and clinical events. RESULTS: In this study, 51 patients who received LT at Nagoya University Hospital from 2016 to 2018 were enrolled. Plasma samples were retrospectively collected at the following time points: 1) within a week after LT; 2) 4 ± 1 weeks after LT; 3) 8 ± 1 weeks after LT; and 4) within 2 days after a positive blood culture. A total of 111 plasma samples were analyzed using shotgun next-generation sequencing (NGS) with the PATHDET pipeline. Relative abundance of Anelloviridae, Nocardiaceae, Microbacteriaceae, and Enterobacteriaceae significantly changed during the postoperative period. Microbiome diversity was higher within a week after LT than that at 8 weeks after LT. Antimicrobials were significantly associated with the microbiome of LT recipients. In addition, the proportion of Enterobacteriaceae was significantly increased and the plasma microbiome diversity was significantly lower in patients with acute cellular rejection (ACR) than non-ACR patients. Sequencing reads of bacteria isolated from blood cultures were predominantly identified by NGS in 8 of 16 samples, and human herpesvirus 6 was detected as a causative pathogen in one recipient with severe clinical condition. CONCLUSIONS: The metagenomic NGS technique has great potential in revealing the plasma microbiome and is useful as a comprehensive diagnostic procedure in clinical settings. Temporal dynamics of specific microorganisms may be used as indirect markers for the determination of immunocompetence and ACR in LT recipients.

Plasma pooling in combination with amotosalen/UVA pathogen inactivation to increase standardisation and safety of therapeutic plasma units.

OBJECTIVES: Assessment of the impact of pooling five single-donor plasma (SDP) units to obtain six pathogen-reduced therapeutic plasma (PTP) units on standardisation and the retention of labile coagulation factors. BACKGROUND: SDP shows a high inter-donor variability with potential implications for the clinical treatment outcome. Additionally, there is still an existing risk for window-period transmissions of blood borne pathogens including newly emerging pathogens. METHODS/MATERIALS: Five ABO-identical SDP units were pooled, treated with the INTERTCEPT™ Blood System (Cerus Corporation, U.S.A.) and split into six PTP units which were frozen and thawed after 30 days. The variability in volume, labile coagulation factor retention and activity was assessed. RESULTS: The variability of volumes between the PTP units was reduced by 46% compared to SDP units. The variability in coagulation factor content between the PTP units was reduced by 63% compared to SDP units. Moderate, but significant losses of coagulation factors (except for vWF) were observed in PTPs compared to SDPs. CONCLUSION: The pooling of five SDP units to obtain six PTP units significantly increases product standardisation with potential implications for safety, economics as well as transfusion-transmitted pathogen safety, making it an interesting alternative to quarantine SDP (qSDP) and pathogen-reduced SDP.

Use of Plasma Metagenomic Next-generation Sequencing for Pathogen Identification in Pediatric Endocarditis.

Pediatric infective endocarditis incurs significant morbidity and generally occurs among children with underlying heart disease. Identification of a pathogen is critical in determining appropriate therapy. However, standard diagnostic testing has limited sensitivity. We describe a case series of children with infective endocarditis in whom plasma next-generation sequencing (Karius, Redwood, CA) identified an organism in 8 of 10 cases.

Fetal mitochondrial DNA in maternal plasma in surrogate pregnancies: Detection and topology.

OBJECTIVES: Due to the maternally-inherited nature of mitochondrial DNA (mtDNA), there is a lack of information regarding fetal mtDNA in the plasma of pregnant women. We aim to explore the presence and topologic forms of circulating fetal and maternal mtDNA molecules in surrogate pregnancies. METHODS: Genotypic differences between fetal and surrogate maternal mtDNA were used to identify the fetal and maternal mtDNA molecules in plasma. Plasma samples were obtained from the surrogate pregnant mothers. Using cleavage-end signatures of BfaI restriction enzyme, linear and circular mtDNA molecules in maternal plasma could be differentiated. RESULTS: Fetal-derived mtDNA molecules were mainly linear (median: 88%; range: 80%-96%), whereas approximately half of the maternal-derived mtDNA molecules were circular (median: 51%; range: 42%-60%). The fetal DNA fraction of linear mtDNA was lower (median absolute difference: 9.8%; range: 1.1%-27%) than that of nuclear DNA (median: 20%; range: 9.7%-35%). The fetal-derived linear mtDNA molecules were shorter than the maternal-derived ones. CONCLUSION: Fetal mtDNA is present in maternal plasma, and consists mainly of linear molecules. Surrogate pregnancies represent a valuable clinical scenario for exploring the biology and potential clinical applications of circulating mtDNA, for example, for pregnancies conceived following mitochondrial replacement therapy.

Candida albicans adhesion to central venous catheters: Impact of blood plasma-driven germ tube formation and pathogen-derived adhesins.

Candida albicans-related bloodstream infections are often associated with infected central venous catheters (CVC) triggered by microbial adhesion and biofilm formation. We utilized single-cell force spectroscopy (SCFS) and flow chamber models to investigate the adhesion behavior of C. albicans yeast cells and germinated cells to naïve and human blood plasma (HBP)-coated CVC tubing. Germinated cells demonstrated up to 56.8-fold increased adhesion forces to CVC surfaces when compared to yeast cells. Coating of CVCs with HBP significantly increased the adhesion of 60-min germinated cells but not of yeast cells and 30-min germinated cells. Under flow conditions comparable to those in major human veins, germinated cells displayed a flow directional-orientated adhesion pattern to HBP-coated CVC material, suggesting the germ tip to serve as the major adhesive region. None of the above-reported phenotypes were observed with germinated cells of an als3Δ deletion mutant, which displayed similar adhesion forces to CVC surfaces as the isogenic yeast cells. Germinated cells of the als3Δ mutant also lacked a clear flow directional-orientated adhesion pattern on HBP-coated CVC material, indicating a central role for Als3 in the adhesion of germinated C. albicans cells to blood exposed CVC surfaces. In the common model of C. albicans, biofilm formation is thought to be mediated primarily by yeast cells, followed by surface-triggered the formation of hyphae. We suggest an extension of this model in which C. albicans germ tubes promote the initial adhesion to blood-exposed implanted medical devices via the germ tube-associated adhesion protein Als3.

The effect of pathogen inactivation on cryoprecipitate: a functional and quantitative evaluation.

BACKGROUND: As a pooled donor blood product, cryoprecipitate (cryo) carries risks of pathogen transmission. Pathogen inactivation (PI) improves the safety of cryoprecipitate, but its effects on haemostatic properties remain unclear. This study investigated protein expression in samples of pathogen inactivated cryoprecipitate (PI-cryo) using non-targeted quantitative proteomics and in vitro haemostatic capacity of PI-cryo. MATERIALS AND METHODS: Whole blood (WB)- and apheresis (APH)-derived plasma was subject to PI with INTERCEPT® Blood System (Cerus Corporation, Concord, CA, USA) and cryo was prepared from treated plasma. Protein levels in PI-cryo and paired controls were quantified using liquid chromatography-tandem mass spectrometry. Functional haemostatic properties of PI-cryo were assessed using a microparticle (MP) prothrombinase assay, thrombin generation assay, and an in vitro coagulopathy model subjected to thromboelastometry. RESULTS: Over 300 proteins were quantified across paired PI-cryo and controls. PI did not alter the expression of coagulation factors, but levels of platelet-derived proteins and platelet-derived MPs were markedly lower in the WB PI-cryo group. Compared to controls, WB (but not APH) cryo samples demonstrated significantly lower MP prothrombinase activity, prolonged clotting time, and lower clot firmness on thromboelastometry after PI. However, PI did not affect overall thrombin generation variables in either group. DISCUSSION: Data from this study suggest that PI via INTERCEPT® Blood System does not significantly impact the coagulation factor content or function of cryo but reduces the higher MP content in WB-derived cryo. PI-cryo products may confer benefits in reducing pathogen transmission without affecting haemostatic function, but further in vivo assessment is warranted.

Plasma of Acute Lymphoblastic Leukemia Patients React to the Culture of a Mycovirus Containing Aspergillus flavus.

Acute lymphoblastic leukemia (ALL) is the most common cancer in children and is also seen in adults. Currently, no plasma-based test for the detection of ALL is available. We have cultured the home of a patient with ALL and isolated a mycovirus containing Aspergillus flavus. This culture was subjected to electron microscopy, purification, and mass spectrometry. Using enzyme-linked immunosorbent assay technique, plasma of patients with ALL and long-term survivors of this disease were tested for antibodies, utilizing supernatant of the culture of this organism. The results were compared with 3 groups of controls, including healthy individuals, patients with sickle cell disease, and solid tumors. Using electron microscopy, the isolated A. flavus contained mycovirus particles. In chemical analysis, this organism did not produce any aflatoxin. Using an enzyme-linked immunosorbent assay technique, the supernatant of the culture of the mycovirus containing A. flavus could differentiate ALL patients from each group of controls (P<0.001). These studies provide a new technique for the detection of ALL and may add information for future research regarding leukemogenesis.

Microbiome analyses of blood and tissues suggest cancer diagnostic approach.

Systematic characterization of the cancer microbiome provides the opportunity to develop techniques that exploit non-human, microorganism-derived molecules in the diagnosis of a major human disease. Following recent demonstrations that some types of cancer show substantial microbial contributions1-10, we re-examined whole-genome and whole-transcriptome sequencing studies in The Cancer Genome Atlas11 (TCGA) of 33 types of cancer from treatment-naive patients (a total of 18,116 samples) for microbial reads, and found unique microbial signatures in tissue and blood within and between most major types of cancer. These TCGA blood signatures remained predictive when applied to patients with stage Ia-IIc cancer and cancers lacking any genomic alterations currently measured on two commercial-grade cell-free tumour DNA platforms, despite the use of very stringent decontamination analyses that discarded up to 92.3% of total sequence data. In addition, we could discriminate among samples from healthy, cancer-free individuals (n = 69) and those from patients with multiple types of cancer (prostate, lung, and melanoma; 100 samples in total) solely using plasma-derived, cell-free microbial nucleic acids. This potential microbiome-based oncology diagnostic tool warrants further exploration.

Efficacy of antiseptics in a novel 3-dimensional human plasma biofilm model (hpBIOM).

The increasing incidence of non-healing wounds constitutes a pivotal socio-economic burden. 60-80% of chronic wounds are colonized by pathogenic microorganisms within a protective extracellular polymeric substance, bearing a great challenge in wound management. Human plasma was used to prepare the biofilm model (hpBIOM), adding pathogens to the plasma and forming Coagula-like discs with integrated pathogens were produced. The antiseptics Octenisept and Lavasorb were tested regarding their antibacterial properties on clinically relevant biofilm-growing bacteria (MRSA, P. aeruginosa) in the hpBIOM. Biofilm-typical glycocalyx-formation was confirmed using immunohistochemical staining. Treatment of a 12 h-maturated biofilm with Octenisept resulted in complete eradication of P. aeruginosa and MRSA after 48 h. Lavasorb proved less effective than Octenisept in this setting. In more mature biofilms (24 h), both antiseptics showed a delayed, partially decreased efficacy. Summarized, the hpBIOM provides essential factors for a translational research approach to be used for detailed human biofilm analyses and evaluation of antimicrobial/-biofilm properties of established and novel therapeutic strategies and products. Octenisept and Lavasorb showed an attenuated efficacy in the hpBIOM compared to planktonic conditions and previously published biofilm-studies, prompting the question for the necessity of introducing new international standards and pre-admission requirements on a translational base.

Rapid Escherichia coli Trapping and Retrieval from Bodily Fluids via a Three-Dimensional Bead-Stacked Nanodevice.

A novel micro- and nanofluidic device stacked with magnetic beads has been developed to efficiently trap, concentrate, and retrieve Escherichia coli (E. coli) from the bacterial suspension and pig plasma. The small voids between the magnetic beads are used to physically isolate the bacteria in the device. We used computational fluid dynamics, three-dimensional (3D) tomography technology, and machine learning to probe and explain the bead stacking in a small 3D space with various flow rates. A combination of beads with different sizes is utilized to achieve a high capture efficiency (∼86%) with a flow rate of 50 µL/min. Leveraging the high deformability of this device, an E. coli sample can be retrieved from the designated bacterial suspension by applying a higher flow rate followed by rapid magnetic separation. This unique function is also utilized to concentrate E. coli cells from the original bacterial suspension. An on-chip concentration factor of ∼11× is achieved by inputting 1300 µL of the E. coli sample and then concentrating it in 100 µL of buffer. Importantly, this multiplexed, miniaturized, inexpensive, and transparent device is easy to fabricate and operate, making it ideal for pathogen separation in both laboratory and point-of-care settings.

Bacterial survival in whole blood depends on plasma sensitivity and resistance to neutrophil killing.

BACKGROUND: Whole blood (WB) is held at room temperature for not more than 24 hours before blood component manufacturing. The ability of several culture collection, skin-derived, and transfusion-related bacteria to survive in WB stored at 22 ± 2°C for 24 hours was investigated in this study. STUDY DESIGN AND METHODS: Twenty-one bacteria of the species Staphylococcus epidermidis, Staphylococcus aureus, Staphylococcus capitis, Streptococcus agalactiae, Serratia liquefaciens, Serratia marcescens, Klebsiella pneumoniae, Escherichia coli, and Yersinia enterocolitica were inoculated into 7-mL aliquots of WB at a concentration of 500 colony-forming units (CFU)/mL. Spiked WB was stored aerobically at 22 ± 2°C, and bacterial viability and growth were monitored at 3, 8, and 24 hours during WB storage. Bacteria that showed decreased viability during WB incubation were further characterized for their sensitivity to plasma factors and neutrophil killing. RESULTS: There were three different scenarios for bacterial behavior during the hold of WB at 22 ± 2°C. Five bacteria proliferated (p < 0.03), 11 remained viable or showed low proliferation, and a third group of five bacteria had decreased or lost viability (p < 0.01). Three of the latter five bacteria were plasma-sensitive while the other two were plasma-resistant but susceptible to neutrophil killing (p = 0.01). CONCLUSIONS: The bactericidal activity of WB can be the result of plasma sensitivity or neutrophil killing. Bacteria with a starting inoculum of 500 CFU/mL, and able to resist WB immune factors, can proliferate to clinically significant levels posing a potential safety risk to transfusion patients. Results of this pilot study should be validated under standard WB collection and storage conditions.