Pathogen Profiling: Rapid Molecular Characterization of Staphylococcus aureus by PCR/Electrospray Ionization-Mass Spectrometry and Correlation with Phenotype

There are few diagnostic methods that readily distinguish among community-acquired methicillin (meticillin)-resistant Staphylococcus aureus strains, now frequently transmitted within hospitals. We describe a rapid and high-throughput method for bacterial profiling of staphylococcal isolates. The method couples PCR to electrospray ionization-mass spectrometry (ESI-MS) and is performed on a platform suitable for use in a diagnostic laboratory. This profiling technology produces a high-resolution genetic signature indicative of the presence of specific genetic elements that represent distinctive phenotypic features. The PCR/ESI-MS signature accurately identified genotypic determinants consistent with phenotypic traits in well-characterized reference and clinical isolates of S. aureus. Molecular identification of the antibiotic resistance genes correlated strongly with phenotypic in vitro resistance. The identification of toxin genes correlated with independent PCR analyses for the toxin genes. Finally, isolates were correctly classified into genotypic groups that correlated with genetic clonal complexes, repetitive-element-based PCR patterns, or pulsed-field gel electrophoresis types. The high-throughput PCR/ESI-MS assay should improve clinical management of staphylococcal infections.Invasive infections caused by methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) are among the most common complications of health care in the United States. Klevens et al. estimate that 94,360 invasive MRSA infections occur in the United States each year, with associated deaths in 18,650 cases (26). Infections are subcategorized as health care-associated MRSA (HA-MRSA) infections or as community-associated MRSA (CA-MRSA) infections; the latter occur in otherwise healthy people who have not experienced a hospital stay within the past 12 months (24, 35). CA-MRSA infections have largely been attributable to a few strains, designated pulsed-field types (PFTs) USA300 and USA400 (26, 29). Once introduced into a health care environment, CA-MRSA strains can be readily transmitted, blending with or replacing entrenched HA-MRSA strains (25, 35).Microbial genotyping analysis allows investigation into the clonality of an outbreak and risk factors associated with infection (5). Methods such as pulsed-field gel electrophoresis (PFGE) (39), repetitive-element-based PCR (rep-PCR) (42), and multilocus sequence typing (MLST) (10, 11) are used for microbial genotyping but are costly and labor-intensive and do not enable specific characterization of acquired genetic elements encoding virulence factors or toxins or of genes that mediate antibiotic resistance (40). A rapid technique for determining the MRSA strain genotype and its broader complement of genetic elements would enable a more comprehensive understanding of transmission dynamics and could lead to more effective actionable decisions related to bed management, prioritization of infection control resources, and treatment.Here, we describe the use of a rapid and high-throughput method to simultaneously genotype and characterize S. aureus specimens with respect to acquired genes encoding virulence factors, toxins, and antibiotic resistance determinants. The method is based on PCR coupled to electrospray ionization-mass spectrometry (ESI-MS) (8, 22, 37).The PCR/ESI-MS assay uses several novel strategies. First, broad-range primers, targeting sites that are highly conserved in all members of a microbe family, are used to amplify PCR products from groupings of microbes rather than single species. These primers are coupled with species- or strain-specific primers for the identification of specific pathogens or antibiotic targets. Second, PCR conditions are, by design, permissive and thus tolerant of mismatches, so that even sequences from novel strains can be amplified. Third, inosine and other “wild-card” nucleotides are used in primers to facilitate PCR analysis of mispaired sequences. Fourth, because MS simply measures the mass-to-charge ratio (m/z), the sequence of the amplicon need not be known in order to detect it. The technology offers advantages over routine single-target and multiplex PCR in that it is a full bioinformatics sequence analysis system.After amplification, MS is used to rapidly determine the precise mass-to-charge ratio for the amplified nucleic acid fragments present, and the A, C, T, and G contents (i.e., the base composition) of each amplicon are determined using proprietary software that creates a signature to allow organism identification and genotyping. This novel MS technology enables the rapid, sensitive, cost-effective, and simultaneous detection of a wide range of typical pathogenic organisms.We used the PCR/ESI-MS assay to analyze a well-characterized set of S. aureus strains from the CDC and geographically distinct clinical isolates from Maryland and Arizona. The PCR/ESI-MS technology effectively combines genotyping and characterization on a single high-throughput platform suitable for surveillance, infection control interventions, and patient management.     

Transition of stable pediatric patients with pulmonary arterial hypertension from intravenous epoprostenol to intravenous treprostinil

Intravenous epoprostenol was the first agent approved by the United States Food and Drug Administration for the management of pulmonary arterial hypertension (PAH). However, epoprostenol therapy carries the risks of a short half-life (<6 minutes) and side effects, including jaw pain, flushing, and headache. Recently, intravenous treprostinil has been studied, primarily in adults with PAH, and found to provide effective therapy. The effects of continuous intravenous treprostinil were retrospectively evaluated in 13 children with stable PAH who had been treated with epoprostenol for >1 year. Children were transitioned in the hospital over 24 hours using a rapid or slow strategy. The children were a mean age of 11 years (range 3 to 17) and were transitioned to treprostinil from August 2004 to August 2005. The baseline 6-minute walking distance was on average 516 +/- 115 m (n = 9) and did not change after transition. Patients were treated with treprostinil for 1.1 +/- 0.5 years. There were 2 deaths, and 2 patients transitioned to other therapy. Seven patients experienced > or =1 central-line infection. Despite a higher dose of treprostinil, the side effects were subjectively diminished. In conclusion, treprostinil provides an alternative therapy in children with PAH, with fewer side effects. However, evaluation regarding rates of infection requires further exploration.     

Outcome of Acute Graft Rejection Associated with Hemodynamic Compromise in Pediatric Heart Transplant Recipients

We sought to analyze the outcome of hemodynamically significant acute graft rejection in pediatric heart transplant recipients from a single-center experience. Acute graft rejection remains a major cause of morbidity and mortality for patients who undergo orthotopic heart transplantation and has been associated with the severity of the rejection episode. A retrospective review of all children experiencing a hemodynamically significant rejection episode after orthotopic heart transplantation was performed. Fifty-three patients with 54 grafts had 70 rejection episodes requiring intravenous inotropic support. Forty-one percent of these patients required high-dose inotropic support, with the remaining 59% of patients requiring less inotropic support. Overall graft survival to hospital discharge was 41% for patients in the high-dose group compared to 94% in the low-dose group. Six-month graft survival in patients who required high-dose inotropes remained at 41% compared to 44% in the low-dose group. Hemodynamically significant acute graft rejection in pediatric heart transplant recipients is a devastating problem with poor short- and long-term outcomes. Survival to hospital discharge is dismal in patients who require high-dose inotropic support. In contrast, survival to discharge is quite good in patients who require only low-dose inotropic support; however, six-month graft survival in this group is low secondary to a high incidence of graft failure related to worsening or aggressive transplant coronary artery disease.     

Factors influencing adolescents' decision not to smoke

ABSTRACT Objective: Although adolescents ultimately make their own decisions to smoke or not to smoke, social, economic, and environmental circumstances shape their choices. Most research on prevention of youth tobacco use focuses on predictors of smoking initiation. In this study, we explored nonsmoking attitudes, beliefs, and norms from the perspective of 16–17‐year‐old nonsmokers. Design: This qualitative study targeted nonsmoking youths because most social policy in the arena of tobacco prevention aims to support adolescents who are tobacco free. Sample: Participants were 39 nonsmokers recruited from youth organizations in an urban community and included 22 African Americans (12 females; 10 males) and 17 Caucasian Americans (10 females; 7 males). Methods: A health behavior framework guided the development of semistructured questions on attitudes, beliefs, and norms associated with nonsmoking, used in eight in‐depth group interviews. Results: Concerns for health and addiction, a positive self‐image, and perceived confidence, emerged as factors affecting participants' decisions not to smoke. The approval of parents and friends, and personal beliefs further reinforced adolescents' nonsmoking decisions. Conclusions: There were more commonalities than differences in nonsmoking attitudes across gender and race. Future studies of youth tobacco prevention should employ multifaceted approaches targeting adolescents' attitudes, families, and peer networks.     

Synthesis and pharmacological evaluation of phenylethynyl[1,2,4]methyltriazines as analogues of 3-methyl-6-(phenylethynyl)pyridine

Procedures were developed for the synthesis of 3-methyl-5-phenylethynyl[1,2,4]triazine (4), 6-methyl-3-phenylethynyl[1,2,4]triazine (5), and 5-methyl-3-phenylethynyl[1,2,4]triazine (6a) as analogues of 2-methyl-6-(phenylethynyl)pyridine (2). The compounds were evaluated for antagonism of glutamate-mediated mobilization of internal calcium in an mGluR5 in vitro efficacy assay. The most potent of the three analogues was 6a. Twenty additional analogues of 6a were synthesized and evaluated for mGluR5 antagonist efficacy. The most potent compounds were 3-(3-methylphenylethynyl)-5-methyl[1,2,4]triazine (6b), 5-(3-chlorophenylethynyl)-5-methyl[1,2,4]triazine (6c), and 3-(3-bromophenylethynyl)-5-methyl[1,2,4]triazine (6d).     

Dendritic cell paucity in mismatch repair–proficient colorectal cancer liver metastases limits immune checkpoint blockade efficacy

Liver metastasis is a major cause of mortality for patients with colorectal cancer (CRC). Mismatch repair–proficient (pMMR) CRCs make up about 95% of metastatic CRCs, and are unresponsive to immune checkpoint blockade (ICB) therapy. Here we show that mouse models of orthotopic pMMR CRC liver metastasis accurately recapitulate the inefficacy of ICB therapy in patients, whereas the same pMMR CRC tumors are sensitive to ICB therapy when grown subcutaneously. To reveal local, nonmalignant components that determine CRC sensitivity to treatment, we compared the microenvironments of pMMR CRC cells grown as liver metastases and subcutaneous tumors. We found a paucity of both activated T cells and dendritic cells in ICB-treated orthotopic liver metastases, when compared with their subcutaneous tumor counterparts. Furthermore, treatment with Feline McDonough sarcoma (FMS)-like tyrosine kinase 3 ligand (Flt3L) plus ICB therapy increased dendritic cell infiltration into pMMR CRC liver metastases and improved mouse survival. Lastly, we show that human CRC liver metastases and microsatellite stable (MSS) primary CRC have a similar paucity of T cells and dendritic cells. These studies indicate that orthotopic tumor models, but not subcutaneous models, should be used to guide human clinical trials. Our findings also posit dendritic cells as antitumor components that can increase the efficacy of immunotherapies against pMMR CRC.

Immune checkpoint blockade (ICB) therapy has revolutionized cancer treatment in recent years. Anti–PD1 (anti–programmed cell-death protein 1) and anti–CTLA-4 (anti–cytotoxic T lymphocyte-associated protein 4) are two main types of ICB therapies that can be particularly effective (1). Metastatic melanoma, which was previously an incurable disease, now has cure rates of more than 50% when patients are treated with a combination of anti–PD1 and anti–CTLA-4 (2). However, ICB therapy is only effective in less than 15% of patients who receive the therapy (3), and efforts are ongoing to uncover the underlying mechanisms of intrinsic and acquired resistance.Colorectal cancer (CRC) is the second leading cause of cancer-related death in the United States (4) and in the world (5). Metastatic spread, especially to the liver, is a major cause of mortality in patients with CRC (6). The efficacy of ICB therapy in metastatic CRCs has been limited to patients with mismatch repair–deficient (dMMR) or microsatellite instability-high (MSI-H) tumors, where a 55% objective response rate has been achieved (7). However, dMMR or MSI-H metastatic CRCs represent only about 5% of total metastatic CRC cases. The remaining 95% are mismatch repair–proficient (pMMR) or microsatellite stable (MSS) tumors (8), which are typically unresponsive to ICB therapy (8). Therefore, there is an urgent need to better understand the resistance mechanisms in pMMR and MSS metastatic CRCs, and improve the efficacy of treatments against this disease.Preclinical mouse models of cancer are effective tools for studying and improving cancer therapy. MC38 and CT26 are syngeneic mouse CRC cell lines commonly used in preclinical immunocompetent mouse models of cancer. In most preclinical studies, these cells are injected under the skin into the hind flank of mice, where they grow as subcutaneous tumors. When treated with ICB therapies such as anti–PD1 and/or anti–CTLA-4, these tumors have been shown to respond well (9, 10). However, MC38 and CT26 lack coding somatic mutations in the DNA mismatch repair genes and should be considered as pMMR CRC cell lines (11, 12). Hence, experimental preclinical models using subcutaneously implanted pMMR CRC cell lines fail to recapitulate the disease resistance to ICB therapy that is observed in patients.We hypothesized that orthotopic pMMR CRC mouse models, where pMMR CRC cells are implanted in the colon to represent primary colon tumors or in the liver to represent liver metastases, would more accurately recapitulate progression of the human disease and its response to ICB treatment in the clinic. Indeed, we report here strikingly different sensitivities to ICB treatment for pMMR CRC tumors grown orthotopically when compared with their subcutaneous counterparts. We further take advantage of these differences to define local nonmalignant components that determine the sensitivity of pMMR CRCs to treatment.