Identification of a therapeutic interfering particle-A single-dose SARS-CoV-2 antiviral intervention with a high barrier to resistance.

Viral-deletion mutants that conditionally replicate and inhibit the wild-type virus (i.e., defective interfering particles, DIPs) have long been proposed as single-administration interventions with high genetic barriers to resistance. However, theories predict that robust, therapeutic DIPs (i.e., therapeutic interfering particles, TIPs) must conditionally spread between cells with R0 >1. Here, we report engineering of TIPs that conditionally replicate with SARS-CoV-2, exhibit R0 >1, and inhibit viral replication 10- to 100-fold. Inhibition occurs via competition for viral replication machinery, and a single administration of TIP RNA inhibits SARS-CoV-2 sustainably in continuous cultures. Strikingly, TIPs maintain efficacy against neutralization-resistant variants (e.g., B.1.351). In hamsters, both prophylactic and therapeutic intranasal administration of lipid-nanoparticle TIPs durably suppressed SARS-CoV-2 by 100-fold in the lungs, reduced pro-inflammatory cytokine expression, and prevented severe pulmonary edema. These data provide proof of concept for a class of single-administration antivirals that may circumvent current requirements to continually update medical countermeasures against new variants.

Targeted isolation of diverse human protective broadly neutralizing antibodies against SARS-like viruses.

The emergence of current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) and potential future spillovers of SARS-like coronaviruses into humans pose a major threat to human health and the global economy. Development of broadly effective coronavirus vaccines that can mitigate these threats is needed. Here, we utilized a targeted donor selection strategy to isolate a large panel of human broadly neutralizing antibodies (bnAbs) to sarbecoviruses. Many of these bnAbs are remarkably effective in neutralizing a diversity of sarbecoviruses and against most SARS-CoV-2 VOCs, including the Omicron variant. Neutralization breadth is achieved by bnAb binding to epitopes on a relatively conserved face of the receptor-binding domain (RBD). Consistent with targeting of conserved sites, select RBD bnAbs exhibited protective efficacy against diverse SARS-like coronaviruses in a prophylaxis challenge model in vivo. These bnAbs provide new opportunities and choices for next-generation antibody prophylactic and therapeutic applications and provide a molecular basis for effective design of pan-sarbecovirus vaccines.

Broadly neutralizing anti-S2 antibodies protect against all three human betacoronaviruses that cause deadly disease.

Pan-betacoronavirus neutralizing antibodies may hold the key to developing broadly protective vaccines against novel pandemic coronaviruses and to more effectively respond to SARS-CoV-2 variants. The emergence of Omicron and subvariants of SARS-CoV-2 illustrates the limitations of solely targeting the receptor-binding domain (RBD) of the spike (S) protein. Here, we isolated a large panel of broadly neutralizing antibodies (bnAbs) from SARS-CoV-2 recovered-vaccinated donors, which targets a conserved S2 region in the betacoronavirus spike fusion machinery. Select bnAbs showed broad in vivo protection against all three deadly betacoronaviruses, SARS-CoV-1, SARS-CoV-2, and MERS-CoV, which have spilled over into humans in the past two decades. Structural studies of these bnAbs delineated the molecular basis for their broad reactivity and revealed common antibody features targetable by broad vaccination strategies. These bnAbs provide new insights and opportunities for antibody-based interventions and for developing pan-betacoronavirus vaccines.

Compartmentalized metabolism supports midgestation mammalian development.

Mammalian embryogenesis requires rapid growth and proper metabolic regulation1. Midgestation features increasing oxygen and nutrient availability concomitant with fetal organ development2,3. Understanding how metabolism supports development requires approaches to observe metabolism directly in model organisms in utero. Here we used isotope tracing and metabolomics to identify evolving metabolic programmes in the placenta and embryo during midgestation in mice. These tissues differ metabolically throughout midgestation, but we pinpointed gestational days (GD) 10.5-11.5 as a transition period for both placenta and embryo. Isotope tracing revealed differences in carbohydrate metabolism between the tissues and rapid glucose-dependent purine synthesis, especially in the embryo. Glucose's contribution to the tricarboxylic acid (TCA) cycle rises throughout midgestation in the embryo but not in the placenta. By GD12.5, compartmentalized metabolic programmes are apparent within the embryo, including different nutrient contributions to the TCA cycle in different organs. To contextualize developmental anomalies associated with Mendelian metabolic defects, we analysed mice deficient in LIPT1, the enzyme that activates 2-ketoacid dehydrogenases related to the TCA cycle4,5. LIPT1 deficiency suppresses TCA cycle metabolism during the GD10.5-GD11.5 transition, perturbs brain, heart and erythrocyte development and leads to embryonic demise by GD11.5. These data document individualized metabolic programmes in developing organs in utero.

Animal models for COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the aetiological agent of coronavirus disease 2019 (COVID-19), an emerging respiratory infection caused by the introduction of a novel coronavirus into humans late in 2019 (first detected in Hubei province, China). As of 18 September 2020, SARS-CoV-2 has spread to 215 countries, has infected more than 30 million people and has caused more than 950,000 deaths. As humans do not have pre-existing immunity to SARS-CoV-2, there is an urgent need to develop therapeutic agents and vaccines to mitigate the current pandemic and to prevent the re-emergence of COVID-19. In February 2020, the World Health Organization (WHO) assembled an international panel to develop animal models for COVID-19 to accelerate the testing of vaccines and therapeutic agents. Here we summarize the findings to date and provides relevant information for preclinical testing of vaccine candidates and therapeutic agents for COVID-19.

Targeted protein S-nitrosylation of ACE2 inhibits SARS-CoV-2 infection.

Prevention of infection and propagation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a high priority in the Coronavirus Disease 2019 (COVID-19) pandemic. Here we describe S-nitrosylation of multiple proteins involved in SARS-CoV-2 infection, including angiotensin-converting enzyme 2 (ACE2), the receptor for viral entry. This reaction prevents binding of ACE2 to the SARS-CoV-2 spike protein, thereby inhibiting viral entry, infectivity and cytotoxicity. Aminoadamantane compounds also inhibit coronavirus ion channels formed by envelope (E) protein. Accordingly, we developed dual-mechanism aminoadamantane nitrate compounds that inhibit viral entry and, thus, the spread of infection by S-nitrosylating ACE2 via targeted delivery of the drug after E protein channel blockade. These non-toxic compounds are active in vitro and in vivo in the Syrian hamster COVID-19 model and, thus, provide a novel avenue to pursue therapy.

A single-administration therapeutic interfering particle reduces SARS-CoV-2 viral shedding and pathogenesis in hamsters.

The high transmissibility of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a primary driver of the COVID-19 pandemic. While existing interventions prevent severe disease, they exhibit mixed efficacy in preventing transmission, presumably due to their limited antiviral effects in the respiratory mucosa, whereas interventions targeting the sites of viral replication might more effectively limit respiratory virus transmission. Recently, intranasally administered RNA-based therapeutic interfering particles (TIPs) were reported to suppress SARS-CoV-2 replication, exhibit a high barrier to resistance, and prevent serious disease in hamsters. Since TIPs intrinsically target the tissues with the highest viral replication burden (i.e., respiratory tissues for SARS-CoV-2), we tested the potential of TIP intervention to reduce SARS-CoV-2 shedding. Here, we report that a single, postexposure TIP dose lowers SARS-CoV-2 nasal shedding, and at 5 days postinfection, infectious virus shed is below detection limits in 4 out of 5 infected animals. Furthermore, TIPs reduce shedding of Delta variant or WA-1 from infected to uninfected hamsters. Cohoused "contact" animals exposed to infected, TIP-treated animals exhibited significantly lower viral loads, reduced inflammatory cytokines, no severe lung pathology, and shortened shedding duration compared to animals cohoused with untreated infected animals. TIPs may represent an effective countermeasure to limit SARS-CoV-2 transmission.

Polymerase Chain Reaction on Respiratory Tract Specimens of Immunocompromised Patients to Diagnose Pneumocystis Pneumonia: A Systematic Review and Meta-analysis.

BACKGROUND: This meta-analysis examines the comparative diagnostic performance of polymerase chain reaction (PCR) for the diagnosis of Pneumocystis pneumonia (PCP) on different respiratory tract samples, in both human immunodeficiency virus (HIV) and non-HIV populations. METHODS: A total of 55 articles met inclusion criteria, including 11 434 PCR assays on respiratory specimens from 7835 patients at risk of PCP. QUADAS-2 tool indicated low risk of bias across all studies. Using a bivariate and random-effects meta-regression analysis, the diagnostic performance of PCR against the European Organisation for Research and Treatment of Cancer-Mycoses Study Group definition of proven PCP was examined. RESULTS: Quantitative PCR (qPCR) on bronchoalveolar lavage fluid provided the highest pooled sensitivity of 98.7% (95% confidence interval [CI], 96.8%-99.5%), adequate specificity of 89.3% (95% CI, 84.4%-92.7%), negative likelihood ratio (LR-) of 0.014, and positive likelihood ratio (LR+) of 9.19. qPCR on induced sputum provided similarly high sensitivity of 99.0% (95% CI, 94.4%-99.3%) but a reduced specificity of 81.5% (95% CI, 72.1%-88.3%), LR- of 0.024, and LR+ of 5.30. qPCR on upper respiratory tract samples provided lower sensitivity of 89.2% (95% CI, 71.0%-96.5%), high specificity of 90.5% (95% CI, 80.9%-95.5%), LR- of 0.120, and LR+ of 9.34. There was no significant difference in sensitivity and specificity of PCR according to HIV status of patients. CONCLUSIONS: On deeper respiratory tract specimens, PCR negativity can be used to confidently exclude PCP, but PCR positivity will likely require clinical interpretation to distinguish between colonization and active infection, partially dependent on the strength of the PCR signal (indicative of fungal burden), the specimen type, and patient population tested.

A novel CSP C-terminal epitope targeted by an antibody with protective activity against Plasmodium falciparum.

Potent and durable vaccine responses will be required for control of malaria caused by Plasmodium falciparum (Pf). RTS,S/AS01 is the first, and to date, the only vaccine that has demonstrated significant reduction of clinical and severe malaria in endemic cohorts in Phase 3 trials. Although the vaccine is protective, efficacy declines over time with kinetics paralleling the decline in antibody responses to the Pf circumsporozoite protein (PfCSP). Although most attention has focused on antibodies to repeat motifs on PfCSP, antibodies to other regions may play a role in protection. Here, we expressed and characterized seven monoclonal antibodies to the C-terminal domain of CSP (ctCSP) from volunteers immunized with RTS,S/AS01. Competition and crystal structure studies indicated that the antibodies target two different sites on opposite faces of ctCSP. One site contains a polymorphic region (denoted α-ctCSP) and has been previously characterized, whereas the second is a previously undescribed site on the conserved ß-sheet face of the ctCSP (denoted ß-ctCSP). Antibodies to the ß-ctCSP site exhibited broad reactivity with a diverse panel of ctCSP peptides whose sequences were derived from field isolates of P. falciparum whereas antibodies to the α-ctCSP site showed very limited cross reactivity. Importantly, an antibody to the ß-site demonstrated inhibition activity against malaria infection in a murine model. This study identifies a previously unidentified conserved epitope on CSP that could be targeted by prophylactic antibodies and exploited in structure-based vaccine design.

Population genomics of the pathogenic yeast Candida tropicalis identifies hybrid isolates in environmental samples.

Candida tropicalis is a human pathogen that primarily infects the immunocompromised. Whereas the genome of one isolate, C. tropicalis MYA-3404, was originally sequenced in 2009, there have been no large-scale, multi-isolate studies of the genetic and phenotypic diversity of this species. Here, we used whole genome sequencing and phenotyping to characterize 77 isolates of C. tropicalis from clinical and environmental sources from a variety of locations. We show that most C. tropicalis isolates are diploids with approximately 2-6 heterozygous variants per kilobase. The genomes are relatively stable, with few aneuploidies. However, we identified one highly homozygous isolate and six isolates of C. tropicalis with much higher heterozygosity levels ranging from 36-49 heterozygous variants per kilobase. Our analyses show that the heterozygous isolates represent two different hybrid lineages, where the hybrids share one parent (A) with most other C. tropicalis isolates, but the second parent (B or C) differs by at least 4% at the genome level. Four of the sequenced isolates descend from an AB hybridization, and two from an AC hybridization. The hybrids are MTLa/α heterozygotes. Hybridization, or mating, between different parents is therefore common in the evolutionary history of C. tropicalis. The new hybrids were predominantly found in environmental niches, including from soil. Hybridization is therefore unlikely to be associated with virulence. In addition, we used genotype-phenotype correlation and CRISPR-Cas9 editing to identify a genome variant that results in the inability of one isolate to utilize certain branched-chain amino acids as a sole nitrogen source.

The effect of cold storage and cooking on the viability of Clostridioides difficile spores in consumer foods.

The increased detection of clinical cases of Clostridioides difficile coupled with the persistence of clostridial spores at various stages along the food chain suggest that this pathogen may be foodborne. This study examined C. difficile (ribotypes 078 and 126) spore viability in chicken breast, beef steak, spinach leaves and cottage cheese during refrigerated (4 °C) and frozen (-20 °C) storage with and without a subsequent sous vide mild cooking (60 °C, 1 h). Spore inactivation at 80 °C in phosphate buffer solution, beef and chicken were also investigated to provide D80°C values and determine if PBS was a suitable model system for real food matrices. There was no decrease in spore concentration after chilled or frozen storage and/or sous vide cooking at 60 °C. Non-log-linear thermal inactivation was observed for both C. difficile ribotypes at 80 °C in phosphate buffer solution (PBS), beef and chicken. The predicted PBS D80°C values of 5.72±[2.90, 8.55] min and 7.50±[6.61, 8.39] min for RT078 and RT126, respectively, were in agreement with the food matrices D80°C values of 5.65 min (95% CI range from 4.29 to 8.89 min) for RT078 and 7.35 min (95% CI range from 6.81 to 7.01 min) for RT126. It was concluded that C. difficile spores survive chilled and frozen storage and mild cooking at 60 °C but may be inactivated at 80 °C. Moreover thermal inactivation in PBS was representative of that observed in real food matrices (beef and chicken).

Molecular mechanisms of acquired antifungal drug resistance in principal fungal pathogens and EUCAST guidance for their laboratory detection and clinical implications.

The increasing incidence and changing epidemiology of invasive fungal infections continue to present many challenges to their effective management. The repertoire of antifungal drugs available for treatment is still limited although there are new antifungals on the horizon. Successful treatment of invasive mycoses is dependent on a mix of pathogen-, host- and antifungal drug-related factors. Laboratories need to be adept at detection of fungal pathogens in clinical samples in order to effectively guide treatment by identifying isolates with acquired drug resistance. While there are international guidelines on how to conduct in vitro antifungal susceptibility testing, these are not performed as widely as for bacterial pathogens. Furthermore, fungi generally are recovered in cultures more slowly than bacteria, and often cannot be cultured in the laboratory. Therefore, non-culture-based methods, including molecular tests, to detect fungi in clinical specimens are increasingly important in patient management and are becoming more reliable as technology improves. Molecular methods can also be used for detection of target gene mutations or other mechanisms that predict antifungal drug resistance. This review addresses acquired antifungal drug resistance in the principal human fungal pathogens and describes known resistance mechanisms and what in-house and commercial tools are available for their detection. It is emphasized that this approach should be complementary to culture-based susceptibility testing, given the range of mutations, resistance mechanisms and target genes that may be present in clinical isolates, but may not be included in current molecular assays.

Electronic Cigarettes Induce Mitochondrial DNA Damage and Trigger TLR9 (Toll-Like Receptor 9)-Mediated Atherosclerosis.

OBJECTIVE: Electronic cigarette (e-cig) use has recently been implicated in promoting atherosclerosis. In this study, we aimed to investigate the mechanism of e-cig exposure accelerated atherosclerotic lesion development. Approach and Results: Eight-week-old ApoE-/- mice fed normal laboratory diet were exposed to e-cig vapor (ECV) for 2 hours/day, 5 days/week for 16 weeks. We found that ECV exposure significantly induced atherosclerotic lesions as examined by Oil Red O staining and greatly upregulated TLR9 (toll-like receptor 9) expression in classical monocytes and in the atherosclerotic plaques, which the latter was corroborated by enhanced TLR9 expression in human femoral artery atherosclerotic plaques from e-cig smokers. Intriguingly, we found a significant increase of oxidative mitochondria DNA lesion in the plasma of ECV-exposed mice. Administration of TLR9 antagonist before ECV exposure not only alleviated atherosclerosis and the upregulation of TLR9 in plaques but also attenuated the increase of plasma levels of inflammatory cytokines, reduced the plaque accumulation of lipid and macrophages, and decreased the frequency of blood CCR2+ (C-C chemokine receptor type 2) classical monocytes. Surprisingly, we found that cytoplasmic mitochondrial DNA isolated from ECV extract-treated macrophages can enhance TLR9 activation in reporter cells and the induction of inflammatory cytokine could be suppressed by TLR9 inhibitor in macrophages. CONCLUSIONS: E-cig increases level of damaged mitochondrial DNA in circulating blood and induces the expression of TLR9, which elevate the expression of proinflammatory cytokines in monocyte/macrophage and consequently lead to atherosclerosis. Our results raise the possibility that intervention of TLR9 activation is a potential pharmacological target of ECV-related inflammation and cardiovascular diseases.

Kappa Opioid Receptor Expression and Function in Cells of the Immune System.

The kappa opioid receptor (KOR) is expressed on a number of hematopoietic cell populations, based on both protein binding analysis and the detection of kappa opioid receptor gene (Oprk1) transcripts. There are prominent Oprk1 splice variants that are expressed in the mouse and human brain cells and leukocytes. The activation of KOR results in reduced antibody production, an inhibition of phagocytic cell activity, an inhibition of T cell development, alterations in the production of various pro-inflammatory cytokines, chemokines, and the receptors for these mediators. Finally, the activation of KOR also leads to the regulation of receptor functional activity of chemokine receptors through the process of heterologous desensitization. The functional activity of KOR is important for the regulation of inflammatory responses and may provide opportunities for the development of therapeutics for the treatment of inflammatory disease states.

Acute versus delayed radial head arthroplasty for the treatment of radial head fractures.

BACKGROUND: Radial head arthroplasty (RHA) is an important tool in the acute treatment of comminuted radial head and neck fractures. RHA is also performed in a delayed manner after failed open reduction and internal fixation, for fracture malunion or nonunion, and other chronic post-traumatic elbow disorders where restoration of the lateral column of the elbow is considered necessary. The relative efficacy and longevity of acute vs. delayed RHA is unknown. We sought to compare clinical, radiographic, and patient-reported outcomes between these groups. METHODS: We identified patients ≥18 years old who underwent an RHA between 2000 and 2018 and then extracted 135 total elbows with a mean follow-up of 2.3 years that sustained isolated radial head fractures (30%), terrible triad injuries (66%), or Essex-Lopresti injuries (4%). The acute cohort (RHA: <12 weeks) contained 101 elbows that underwent surgery at a mean of 0.6 weeks (range, 0 days to 7 weeks, 96% <2 weeks) from injury, whereas the delayed cohort (RHA: 12 weeks to 2 years) contained 34 elbows that underwent surgery at a mean of 36 weeks (range, 14-82 weeks) from injury. Patients in the acute group had a higher percentage of terrible triad injuries (75% vs. 40%, P < .001) and Mason 3 fractures (98% vs. 45%, P < .001). RESULTS: At the final follow-up, 13 of 101 patients in the acute cohort (13%) and 7 of 34 patients in the delayed cohort (21%) required implant revision or resection. A total of 25 patients (25%) in the acute cohort and 12 patients (35%) in the delayed cohort required a reoperation. Kaplan-Meier 2-year survival estimates free of implant resection or revision (90% acute, 86% delayed) and reoperation (76% acute, 70% delayed) were similar between groups. In patients with 5-year follow-up, there was an increased rate of revision or resection in the delayed group (30% vs. 13%). Two-year survival estimates free of radiographic loosening were 80% in the acute cohort vs. 57% in the delayed cohort (P = .04). Mayo Elbow Performance Score at 2 years demonstrated mean scores of 83 and 79 in the acute and delayed groups, respectively, with 71% of the acute cohort and 64% of the delayed cohort achieving good or excellent scores. CONCLUSIONS: Our results demonstrated that although 2-year Kaplan-Meier survival free of revision or resection estimates and reoperation rates was equivalent between the groups, the delayed group experienced worse Mayo Elbow Performance Score outcomes, a higher revision or resection rate at 5 years, and an increased rate of radiographic loosening.

Does proximal versus distal injury location of the medial ulnar collateral ligament of the elbow differentially impact elbow stability? An ultrasound-guided and robot-assisted biomechanical study.

BACKGROUND: The location (proximal vs. distal) of elbow medial ulnar collateral ligament (MUCL) tears impacts clinical outcomes of nonoperative treatment. The purposes of our study were to (1) determine whether selective releases of the MUCL could be performed under ultrasound (US) guidance without disrupting overlying soft tissues, (2) assess the difference in medial elbow stability for proximal and distal releases of the MUCL using stress US and a robotic testing device, and (3) elucidate the flexion angle that resulted in the greatest amount of medial elbow laxity after MUCL injury. METHODS: Sixteen paired, fresh-frozen elbow specimens were used. Valgus laxity was evaluated with both US and robotic-assisted measurements before and after selective MUCL releases. A percutaneous US-guided technique was used to perform proximal MUCL releases in 8 elbows and to perform distal MUCL releases in their matched pairs. The robot was used to determine the elbow flexion angle at which the maximum valgus displacement occurred for both proximally and distally released specimens. Open dissection was then performed to assess the accuracy of the percutaneous releases. RESULTS: Percutaneous US-guided releases were successfully performed in 15 of 16 specimens. The proximal release resulted in greater valgus angle displacement (11° ± 2°) than the distal release (8° ± 2°) between flexion angles of 30° and 70° (P < .0001 at 30°, P < .0001 at 40°, P = .001 at 50°, P = .005 at 60°, and P = .020 at 70°). Valgus displacement between release locations did not reach the level of statistical significance between 80° and 120° (P = .051 at 80°, P = .131 at 90°, P = .245 at 100°, P = .400 at 110°, and P = .532 at 120°). When we compared the values for the mean increase in US delta gap (stressed - supported state) from before to after MUCL release, the proximally released elbows had larger increases than the distally released elbows (5.0 mm proximal vs. 3.7 mm distal, P = .032). After MUCL release, maximum mean valgus displacement occurred at 49° of flexion. CONCLUSIONS: US-guided selective releases of the MUCL can be performed reliably without violating the overlying musculature. Valgus instability is not of greater magnitude for distal releases when compared with proximal releases. This findings suggests there must be alternative factors to explain the difference in clinical prognosis between distal and proximal tears. The observed flexion angle for maximum valgus laxity could have important implications for elbow positioning during US or fluoroscopic stress examination, as well as surgical repair or reconstruction of the MUCL.

Human and Porcine Transmission of Clostridioides difficile Ribotype 078, Europe.

Genomic analysis of a diverse collection of Clostridioides difficile ribotype 078 isolates from Ireland and 9 countries in Europe provided evidence for complex regional and international patterns of dissemination that are not restricted to humans. These isolates are associated with C. difficile colonization and clinical illness in humans and pigs.

Unconventional T cells - New players in antifungal immunity.

Life-threatening invasive fungal diseases (IFD) are increasing in incidence, especially in immunocompromised patients and successful resolution of IFD requires a variety of different immune cells. With the limited repertoire of available antifungal drugs there is a need for more effective therapeutic strategies. This review interrogates the evidence on the human immune response to the main pathogens driving IFD, with a focus on the role of unconventional lymphocytes e.g. natural killer (NK) cells, gamma/delta (γδ) T cells, mucosal associated invariant T (MAIT) cells, invariant natural killer T (iNKT) cells and innate lymphoid cells (ILC). Recent discoveries and new insights into the roles of these novel lymphocyte groups in antifungal immunity will be discussed, and we will explore how an improved understanding of antifungal action by lymphocytes can inform efforts to improve antifungal treatment options.

Overcoming the Challenges of Pyrazinamide Susceptibility Testing in Clinical Mycobacterium tuberculosis Isolates.

Pyrazinamide (PZA) is one of the first-line agents used for the treatment of tuberculosis. However, current phenotypic PZA susceptibility testing in the Bactec MGIT 960 system is unreliable, and false resistance is well documented. Rapid identification of resistance-associated mutations can confirm the phenotypic result. This study aimed to investigate the use of genotypic methods in combination with phenotypic susceptibility testing for confirmation of PZA-resistant Mycobacterium tuberculosis isolates. Sanger sequencing and/or whole-genome sequencing were performed to detect mutations in pncA, rpsA, panD, and clpC1. Isolates were screened for heteroresistance, and PZA susceptibility testing was performed using the Bactec MGIT 960 system using a reduced inoculum to investigate false resistance. Overall, 40 phenotypically PZA-resistant isolates were identified. Of these, PZA resistance was confirmed in 22/40 (55%) isolates by detecting mutations in the pncA, rpsA, and panD genes. Of the 40 isolates, 16 (40%) were found to be susceptible using the reduced inoculum method (i.e., false resistance). No mutations were detected in two PZA-resistant isolates. False resistance was observed in isolates with MICs close to the critical concentration. In particular, East African Indian strains (lineage 1) appeared to have an elevated MIC that is close to the critical concentration. While this study illustrates the complexity and challenges associated with PZA susceptibility testing of M. tuberculosis, we conclude that a combination of genotypic and phenotypic drug susceptibility testing methods is required for accurate detection of PZA resistance.

Protein kinase C-delta inhibition is organ-protective, enhances pathogen clearance, and improves survival in sepsis.

Sepsis is a leading cause of morbidity and mortality in intensive care units. Previously, we identified Protein Kinase C-delta (PKCδ) as an important regulator of the inflammatory response in sepsis. An important issue in development of anti-inflammatory therapeutics is the risk of immunosuppression and inability to effectively clear pathogens. In this study, we investigated whether PKCδ inhibition prevented organ dysfunction and improved survival without compromising pathogen clearance. Sprague Dawley rats underwent sham surgery or cecal ligation and puncture (CLP) to induce sepsis. Post-surgery, PBS or a PKCδ inhibitor (200µg/kg) was administered intra-tracheally (IT). At 24 hours post-CLP, there was evidence of lung and kidney dysfunction. PKCδ inhibition decreased leukocyte influx in these organs, decreased endothelial permeability, improved gas exchange, and reduced blood urea nitrogen/creatinine ratios indicating organ protection. PKCδ inhibition significantly decreased bacterial levels in the peritoneal cavity, spleen and blood but did not exhibit direct bactericidal properties. Peritoneal chemokine levels, neutrophil numbers, or macrophage phenotypes were not altered by PKCδ inhibition. Peritoneal macrophages isolated from PKCδ inhibitor-treated septic rats demonstrated increased bacterial phagocytosis. Importantly, PKCδ inhibition increased survival. Thus, PKCδ inhibition improved survival and improved survival was associated with increased phagocytic activity, enhanced pathogen clearance, and decreased organ injury.