Plasma Microbial Cell-Free DNA Sequencing in Immunocompromised Patients With Pneumonia: A Prospective Observational Study.

BACKGROUND: Pneumonia is a common cause of morbidity and mortality, yet a causative pathogen is identified in a minority of cases. Plasma microbial cell-free DNA sequencing may improve diagnostic yield in immunocompromised patients with pneumonia. METHODS: In this prospective, multicenter, observational study of immunocompromised adults undergoing bronchoscopy to establish a pneumonia etiology, plasma microbial cell-free DNA sequencing was compared to standardized usual care testing. Pneumonia etiology was adjudicated by a blinded independent committee. The primary outcome, additive diagnostic value, was assessed in the Per Protocol population (patients with complete testing results and no major protocol deviations) and defined as the percent of patients with an etiology of pneumonia exclusively identified by plasma microbial cell-free DNA sequencing. Clinical additive diagnostic value was assessed in the Per Protocol subgroup with negative usual care testing. RESULTS: Of 257 patients, 173 met Per Protocol criteria. A pneumonia etiology was identified by usual care in 52/173 (30.1%), plasma microbial cell-free DNA sequencing in 49/173 (28.3%) and the combination of both in 73/173 (42.2%) patients. Plasma microbial cell-free DNA sequencing exclusively identified an etiology of pneumonia in 21/173 patients (additive diagnostic value 12.1%, 95% confidence interval [CI], 7.7% to 18.0%, P < .001). In the Per Protocol subgroup with negative usual care testing, plasma microbial cell-free DNA sequencing identified a pneumonia etiology in 21/121 patients (clinical additive diagnostic value 17.4%, 95% CI, 11.1% to 25.3%). CONCLUSIONS: Non-invasive plasma microbial cell-free DNA sequencing significantly increased diagnostic yield in immunocompromised patients with pneumonia undergoing bronchoscopy and extensive microbiologic and molecular testing. CLINICAL TRIALS REGISTRATION: NCT04047719.

Transplantid.net: A Pilot Crowdsourced, Living, Online Library of Resources for the Teaching and Practice of Transplant Infectious Diseases.

The field of transplant infectious diseases is rapidly evolving, presenting a challenge for clinical practice and trainee education. Here we describe the construction of transplantid.net, a free online library, crowdsourced and continuously updated for the dual purpose of point-of-care evidence-based management and teaching.

Successful Clearance of 300 Day SARS-CoV-2 Infection in a Subject with B-Cell Depletion Associated Prolonged (B-DEAP) COVID by REGEN-COV Anti-Spike Monoclonal Antibody Cocktail.

A 59-year-old male with follicular lymphoma treated by anti-CD20-mediated B-cell depletion and ablative chemotherapy was hospitalized with a COVID-19 infection. Although the patient did not develop specific humoral immunity, he had a mild clinical course overall. The failure of all therapeutic options allowed infection to persist nearly 300 days with active accumulation of SARS-CoV-2 virus mutations. As a rescue therapy, an infusion of REGEN-COV (10933 and 10987) anti-spike monoclonal antibodies was performed 270 days from initial diagnosis. Due to partial clearance after the first dose (2.4 g), a consolidation dose (8 g) was infused six weeks later. Complete virus clearance could then be observed over the following month, after he was vaccinated with the Pfizer-BioNTech anti-COVID-19 vaccination. The successful management of this patient required prolonged enhanced quarantine, monitoring of virus mutations, pioneering clinical decisions based upon close consultation, and the coordination of multidisciplinary experts in virology, immunology, pharmacology, input from REGN, the FDA, the IRB, the health care team, the patient, and the patient's family. Current decisions to take revolve around patient's follicular lymphoma management, and monitoring for virus clearance persistence beyond disappearance of REGEN-COV monoclonal antibodies after anti-SARS-CoV-2 vaccination. Overall, specific guidelines for similar cases should be established.

Isavuconazonium for the treatment of Purpureocillium lilacinum infection in a patient with pyoderma gangrenosum.

Purpureocillium lilacinum is a rare but emerging pathogen in immunocompromised patients that primarily infects the skin and subcutaneous tissue. We present a novel case of Purpureocillium lilacinum infection in a patient with pyoderma gangrenosum who was successfully treated with isavuconazonium, followed by a literature review of 13 total cases of infection with Purpureocillium lilacinum gathered from a review of the PubMed database. Previous reports have utilized voriconazole, an antifungal with significant toxic side effects. Our case highlights the importance of culture and biopsy in patients with pyoderma gangrenosum who are unresponsive to standard treatment irrespective of pathergy risk.

MicroRNA-26b promotes transition from Kit- to Kit+ mouse spermatogonia.

During spermatogenesis, a group of undifferentiated spermatogonia undergoes an essential transition to a differentiating stage, which involves gain of Kit receptor. In the current study, we showed that a small non-coding RNA, miRNA-26b could induce transition from Kit- to Kit+ and inhibit proliferation of spermatogonia. A key transcriptional factor for undifferentiated spermatogonia, Plzf, was proven as a direct target of miR-26b. When undifferentiated spermatogonia were treated with Retinoic acid (RA), miR-26b was increased, further promoting RA-induced differentiation of spermatogonia. In addition, miR-26b could repress 5-hydroxymethylcytosine (5hmC) via repression of Tet3 in spermatogonia. These findings demonstrate that miR-26b might play a role in promoting the transition from Kit- to Kit+ SSCs.

MicroRNA-29b/Tet1 regulatory axis epigenetically modulates mesendoderm differentiation in mouse embryonic stem cells.

Ten eleven translocation (Tet) family-mediated DNA oxidation on 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) represents a novel epigenetic modification that regulates dynamic gene expression during embryonic stem cells (ESCs) differentiation. Through the role of Tet on 5hmC regulation in stem cell development is relatively defined, how the Tet family is regulated and impacts on ESCs lineage development remains elusive. In this study, we show non-coding RNA regulation on Tet family may contribute to epigenetic regulation during ESCs differentiation, which is suggested by microRNA-29b (miR-29b) binding sites on the Tet1 3' untranslated region (3' UTR). We demonstrate miR-29b increases sharply after embyoid body (EB) formation, which causes Tet1 repression and reduction of cellular 5hmC level during ESCs differentiation. Importantly, we show this miR-29b/Tet1 regulatory axis promotes the mesendoderm lineage formation both in vitro and in vivo by inducing the Nodal signaling pathway and repressing the key target of the active demethylation pathway, Tdg. Taken together, our findings underscore the contribution of small non-coding RNA mediated regulation on DNA demethylation dynamics and the differential expressions of key mesendoderm regulators during ESCs lineage specification. MiR-29b could potentially be applied to enrich production of mesoderm and endoderm derivatives and be further differentiated into desired organ-specific cells.

MicroRNAs mediated targeting on the Yin-yang dynamics of DNA methylation in disease and development.

For decades, DNA methylation at the 5 position of cytosine (5mC) catalyzed by DNA methyltransferases (DNMTs) is a well-known epigenetic modification in mammalian genome, where it modulates chromatin remodeling and transcriptional silencing. The discovery of Ten-eleven translocation (TET) enzymes that oxidize 5mC to 5-hydroxymethylcytosine (5hmC) prompts a new era of DNA demethylation research. It is now established that in DNA demethylation pathway 5mC is first converted to 5-hydroxymethylcytosine (5hmC), then 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) through TETs. Conversion to unmethylated cytosine (5C) is further facilitated by excision mechanism through thymine-DNA glycosylase (TDG) or base excision repair (BER) pathway. Our understanding of DNMTs and TETs on epigenetic dynamics of cytosine methylation has led to a completion of the methylation (Yin) - demethylation (Yang) cycle on epigenetic modifications on cytosine. However, the regulations on DNA demethylation pathway remain largely unknown. In this review, we provide the recent advances on epigenetic dynamics of DNA demethylation and its potential control from the prespective of small non-coding RNA-mediated regulation. Specifically, we will illustrate how microRNAs contribute to active DNA demethylation control in normal and disease development based on recent findings in stem cells and cancer. This article is part of a Directed Issue entitled: Epigenetics dynamics in development and disease.

Identification of human papillomavirus type 58 lineages and the distribution worldwide.

BACKGROUND: Human papillomavirus type 58 (HPV-58) accounts for a much higher proportion of cervical cancers in East Asia than other types. A classification system of HPV-58, which is essential for molecular epidemiological study, is lacking. METHODS AND RESULTS: This study analyzed the sequences of 401 isolates collected from 15 countries and cities. The 268 unique concatenated E6-E7-E2-E5-L1-LCR sequences that comprised 57% of the whole HPV-58 genome showed 4 distinct clusters. L1 and LCR produced tree topologies that best resembled the concatenated sequences and thus are the most appropriate surrogate regions for lineage classification. Moreover, short fragments from L1 (nucleotides 6014-6539) and LCR (nucleotides 7257-7429 and 7540-52) were found to contain sequence signatures informative for lineage identification. Lineage A was the most prevalent lineage across all regions. Lineage C was more frequent in Africa than elsewhere, whereas lineage D was more prevalent in Africa than in Asia. Among lineage A variants, sublineage A2 dominated in Africa, the Americas, and Europe, but not in Asia. Sublineage A1, which represents the prototype that originated from a patient with cancer, was rare worldwide except in Asia. CONCLUSIONS: HPV-58 can be classified into 4 lineages that show some degree of ethnogeographic predilection in distribution. The evolutionary, epidemiological, and pathological characteristics of these lineages warrant further study.

Distribution of human papillomavirus type 58 variants in progression of cervical dysplasia in Korean women.

This cross-sectional study examined the distribution of HPV 58 sequence variation in Korean women for the first time. Among 1,750 Korean women, 53 women were positive for HPV 58 single infection, of whom 26 were without disease, 20 were with cervical intraepithelial neoplasia (CIN) 1, and 7 with CIN 2 or 3. Altogether, 36 different nucleotide sequence variations were identified with the L1, 20 within E2, 5 within E6, and 10 within E7. Further studies on variants of oncogenic HPVs are necessary, particularly for the purpose of developing more predictive HPV detection methods.

Radiofrequency ablation induces antigen-presenting cell infiltration and amplification of weak tumor-induced immunity.

PURPOSE: To evaluate the influence of subtotal radiofrequency (RF) ablation on a tumor-specific immune response in a murine tumor model and to explore the role of intratumoral dendritic cells (ITDCs) in mediating this effect. MATERIALS AND METHODS: Animal work was performed according to an approved protocol and in compliance with the National Cancer Institute Animal Care and Use Committee guidelines and regulations. A murine urothelial carcinoma (MB49) model expressing the male minor histocompatibility (HY) antigen was inoculated subcutaneously in female mice. Fourteen days later, splenic T cells were analyzed with enzyme-linked immunosorbent spot for HY immune response (n = 57). In subsequent experiments, mice were randomized into control (n = 7), RF ablation, ITDC (n = 9), and RF ablation + ITDC (n = 9) groups and monitored for tumor growth. Eleven days after treatment, tumors were harvested for histologic and immunohistochemical analysis. Animals demonstrating complete tumor regression were rechallenged in the contralateral flank. RESULTS: Animals treated with subtotal RF ablation showed significant increases in tumor-specific class I and II responses to HY antigens and tumor regression. RF ablation, ITDC, and combined groups demonstrated similar levels of antigen-presenting cell infiltration; all groups demonstrated greater levels of infiltration compared with untreated controls. ITDC injection also resulted in tumor regression. However, combination therapy did not enhance tumor regression when compared with either treatment alone. Rechallenged mice in RF ablation, ITDC, and combination groups demonstrated significant tumor growth inhibition compared with controls. CONCLUSION: Subtotal RF ablation treatment results in enhanced systemic antitumor T-cell immune responses and tumor regression that is associated with increased dendritic cell infiltration. ITDC injection mimics the RF ablation effect but does not increase immune responses when injected immediately after RF ablation.

Distribution of human papillomavirus types in anogenital warts of men.

BACKGROUND: A wide spectrum of human papillomavirus (HPV) types can infect the male genitalia. An HPV vaccine covering HPV6 and 11 is now available. Detailed data on the distribution of these two types in anogenital warts is needed to assess the potential benefits of the vaccine. STUDY DESIGN: Anogenital wart specimens collected from 130 Chinese men were examined for HPV-type distribution by a method that covers a broad spectrum of high- and low-risk HPVs, and able to reveal multiple types from a single specimen. RESULTS: Forty-four (33.8%) of the 130 specimens had a coinfection with multiple HPV types. In 63.1% of cases, only HPV6 and/or HPV11 were/was found. In 26.2% of cases, HPV6 and/or HPV11 were/was found together with one or more other HPV types. In 10.8% of specimens, only non-6/11 HPV types were found. HPV16 and/or 18 were/was found in 12 (9.2%) specimens, with majority (8/12, 66.7%) of which existed as coinfections with HPV6/11. Other HPV types found included HPV39, 51, 52, 55, 59, 61, 62, 68, 58, 72, 81, 83, 84 and CP6108. CONCLUSIONS: A substantial proportion of HPV6/11-positive male anogenital warts are coinfected with other HPV types. The efficacy of HPV6/11 vaccine for preventing these lesions needs to be defined before the benefits of vaccinating men can be precisely assessed.

In vitro and in vivo evaluations of increased effective beam width for heat deposition using a split focus high intensity ultrasound (HIFU) transducer.

PURPOSE: To develop a novel and efficient, in vitro method for characterizing temporal and spatial heat generation of focused ultrasound exposures, and evaluate this method to compare a split focus and conventional single focus high intensity focused ultrasound transducer. MATERIALS AND METHODS: A HIFU tissue-mimicking phantom was validated by comparing respective temperature elevations generated in the phantoms and in murine tumors in vivo. The phantom was then used in combination with IR thermography to spatially and temporally characterize differences in low-level temperature elevation (e.g. 3-5 degrees C) produced by a single focus and split focus HIFU transducer, where the latter produces four simultaneous foci. In vivo experiments with heat sensitive liposomes containing doxorubicin were then carried out to determine if the larger beam width of the split focus transducer, compared to the single focus, could increase overall deployment of the drug from the liposome. RESULTS: Temperature elevations generated in the HIFU phantom were not found to be different from those measured in vivo when compensating for disparities in attenuation coefficient and specific heat, and between the two transducers by increasing the energy deposition. Exposures with the split focus transducer provided significant increases in the area treated compared to the single focus, which then translated to significant increases in drug deposition in vivo. CONCLUSIONS: Preliminary evidence was provided indicating the potential for using this novel technique for characterizing hyperthermia produced by focused ultrasound devices. Further development will be required for its suitability for correlating in vitro and in vivo outcomes.

Respiratory biofeedback during CT-guided procedures.

PURPOSE: Respiratory motion can be a complicating factor during image-guided interventions. The ability to reproduce breath-holds may facilitate safer needle-based procedures. The purpose of this study was to evaluate if respiratory biofeedback decreased variability among breath-holds and if the signals from the respiratory bellows belt can be used to measure target motion. MATERIALS AND METHODS: In phase 1 of the study, a respiratory bellows belt was applied to patients before image-guided interventional procedures. Belt stretch from respiratory motion was converted into voltage readings and displayed on a monitor as biofeedback. Patients were asked to perform inspiratory, expiratory, and midcycle breath-holds with and without the biofeedback. The variability in voltage readings between breath-holds with and without biofeedback was compared. In phase 2, the respiratory bellows belt was used during computed tomography (CT)-guided procedures with the patients blinded to the biofeedback. Voltage readings and CT series numbers were recorded as patients were asked to hold their breath during scans. The variability of CT z-axis targets was compared with the variability of voltage readings. RESULTS: A significant decrease in variability was found during expiratory breath-holds (P = .0083) with trends toward significance with midcycle and inspiratory breath-holds. A positive correlation (Kendall tau = 0.5; P = .024) was shown between CT z-axis and belt stretch variability in subjects who received smaller doses of moderate sedation compared with those who received larger doses or general anesthesia. CONCLUSIONS: Biofeedback may help the patient to have a more consistent breath-hold. The belt could decrease the error and unpredictability from craniocaudal motion of targets during image-guided interventions.

Pulsed-high intensity focused ultrasound and low temperature-sensitive liposomes for enhanced targeted drug delivery and antitumor effect.

PURPOSE: To determine if pulsed-high intensity focused ultrasound (HIFU) could effectively serve as a source of hyperthermia with thermosensitive liposomes to enhance delivery and efficacy of doxorubicin in tumors. EXPERIMENTAL DESIGN: Comparisons in vitro and in vivo were carried out between non-thermosensitive liposomes (NTSL) and low temperature-sensitive liposomes (LTSL). Liposomes were incubated in vitro over a range of temperatures and durations, and the amount of doxorubicin released was measured. For in vivo experiments, liposomes and free doxorubicin were injected i.v. in mice followed by pulsed-HIFU exposures in s.c. murine adenocarcinoma tumors at 0 and 24 h after administration. Combinations of the exposures and drug formulations were evaluated for doxorubicin concentration and growth inhibition in the tumors. RESULTS: In vitro incubations simulating the pulsed-HIFU thermal dose (42 degrees C for 2 min) triggered release of 50% of doxorubicin from the LTSLs; however, no detectable release from the NTSLs was observed. Similarly, in vivo experiments showed that pulsed-HIFU exposures combined with the LTSLs resulted in more rapid delivery of doxorubicin as well as significantly higher i.t. concentration when compared with LTSLs alone or NTSLs, with or without exposures. Combining the exposures with the LTSLs also significantly reduced tumor growth compared with all other groups. CONCLUSIONS: Combining low-temperature heat-sensitive liposomes with noninvasive and nondestructive pulsed-HIFU exposures enhanced the delivery of doxorubicin and, consequently, its antitumor effects. This combination therapy could potentially produce viable clinical strategies for improved targeting and delivery of drugs for treatment of cancer and other diseases.