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PCR has been used successfully for the direct detection of Mycobacterium tuberculosis in uncultured patient samples. Its potential is hindered by the risk of false-positive results as a result of either amplicon carryover of cross-contamination between patient samples. In the present study, we investigated whether residual amplifiable human or M. tuberculosis DNA could remain in sterile bronchoscopes and potentially be a cause of false-positive PCR results in subsequent patient samples. Sterilized bronchoscopes were flushed with sterile saline, and the collected eluate was submitted for PCR amplification of IS6110 sequences and exon 8 of the human p53 gene. Of a total of 55 washes of sterile bronchoscopes from two institutions, 2 (3.6%) contained amplifiable M. tuberculosis DNA and 11 (20%) contained residual human DNA. These findings indicate that residual DNA can remain in sterilized bronchoscopes and can be a source of false-positive PCR results.

Molecular methods have proven extremely useful for the detection of occult tumor cells and can yield valuable clinical information as well as a better understanding of the mechanisms of metastasis and relapse of cancer (1). In the case of many hematopoietic malignancies, the presence of a unique molecular marker such as a chromosomal translocation has made this task relatively straightforward. Carcinomas generally lack such markers, however. Certain oncogene mutations have been targeted, but the lack of consistent and specific markers has remained problematic.

Molecular methods permit the detection of cells too few in number to be detected by light microscopy, immunohistochemistry, or flow cytometry (1-5). Numerous investigators are therefore developing sensitive and specific reverse transcriptase polymerase chain reaction (RT-PCR) assays for tumor cell detection. The detection of small numbers of tumor cells in blood, lymph node, and stem cell harvests may have a significant impact on our understanding of the spread of breast cancer, and eventually may impact the management of breast cancer patients as well.

A real-time PCR assay was developed targeting the bla KPC responsible for Klebsiella pneumoniae carbapenemase (KPC)-mediated carbapenem resistance and was validated for testing colonies or enrichment broth cultures. The assay accurately detects KPC-containing strains with high analytical specificity and sensitivity.

A dozen years ago, not long after the founding of the Association for Molecular Pathology (AMP), leaders of the organization recognized the need for a dedicated journal for the society. They sought a place in print that we molecular diagnosticians could call our own and where we could publish advances in a field that bridged basic science and clinical diagnostics. The initial goals were simple: to give equal value to new technology and new discoveries, to provide careful validation and comparison of clinical assays, and to promote our growing expertise, as well as that of this fledgling organization, AMP. These leaders also saw the value in partnering with a larger, established professional group that could aid us in quickly reaching beyond the borders of molecular pathology to a broader range of pathology and medical professionals. Thus, in 1999, The Journal of Molecular Diagnostics was launched (originally as The American Journal of Pathology, Part B), in collaboration with the American Society for Investigative Pathology. It has been a tremendous honor to spend 11 years serving in a leadership role for the JMD, and this issue will sadly be my last as Editor-in-Chief. It is my belief that the JMD has fulfilled those early goals, and my greatest desire is that it will continue to thrive well into the future. It has been exciting and rewarding to watch the Journal grow, along with AMP itself. Early on, AMP meetings served as a focal point for bringing together those few of us practicing molecular diagnostics (Who else could relate to the highs and lows of defining quality control and assay reproducibility when dealing with radiolabeled probes?). Over the years, the field has become much more sophisticated in its technology, clinical applications, and assay standardization, and the articles published within the JMD reflect this evolution. AMP has also become active nationally and internationally, addressing a variety of regulatory, educational, and other issues, and in parallel, the JMD has seen an increasing number of position papers from the society and other professional groups addressing many of the same issues of importance to our colleagues and trainees (see the Special Report on competency-based education in this issue). We are also at the point where an increasing number of submitted manuscripts no longer fit squarely into the traditional subdivisions of our society (Solid Tumors, Hematopathology, Genetics, and Infectious Disease) but instead fall into new areas, which will require ongoing evolution on the part of the JMD. Molecular diagnostics has grown in amazing and unanticipated directions and will continue to do so in the years to come, as will the JMD. I am very proud to see the progress that has been made by the JMD. Submissions have risen again this past year, and the quality of papers continues to go up. We have successfully moved from a quarterly to bimonthly publication, without a drop in quality or number of articles per issue. We continue to attract an increasing number of timely Reviews, as well as Commentaries on key articles, published within each issue. And perhaps the most quantitative, and scrutinized, measure of our success, the impact factor, has risen to an outstanding 3.643 (2008 JCR). The JMD has clearly established itself as the premier journal in the field of molecular diagnostics. Again, changes lie ahead, as we anticipate the revolution in scientific publishing that is coming: electronic and publish-ahead-of-print article delivery are just the beginning—I expect to have JMD content available instantaneously in my pocket in the not-so-distant future. Despite the many differences the future will bring to scientific publishing, the central purpose will remain unchanged: journals have a responsibility to provide efficient, unbiased peer review of scientific work, to relay top-quality, novel results to readers as quickly as possible, and to share useful information to advance the field as a whole. Thinking back, I am amazed by the responsibility and trust bestowed on me—by the Joint Journal Oversight Committee, the two societies, and their members, and by the innumerable authors who have entrusted our editorial team of Associate Editors and Editorial Board members with the task of evaluating their hard work. I hope we have handled each manuscript as we would our own, with fairness and a great deal of personal attention. We have strived to be open and unbiased, while maintaining and even raising the quality of published manuscripts. Occasionally issues did arise, and I hope that we handled these fairly and kindly—it is not easy to deliver negative news to one's respected colleagues. But this difficult job was made easier by the professionalism and grace shown by authors with whom we worked. The past years have been extremely gratifying, and I have learned and grown far more in knowledge and experience than I had ever imagined. While the JMD was envisioned to be a benefit to AMP, its members, and practitioners of molecular diagnostics worldwide, I truly feel as though the primary beneficiary has been me. As I say goodbye to my editorial responsibilities, my deepest thanks go out to all who have worked with me during my tenure. In particular, Nelson Fausto, who served as the first JMD Editor-in-Chief, guided me as I learned the ropes of editorial leadership as Senior Associate Editor. I thank the many Associate Editors who toiled tirelessly with me to oversee the review process and assist in manuscript decision making. I am also grateful to the three Managing Editors who have directed the JMD through its evolution: Priscilla Markwood, Maria Giorla, and Audra Cox. Finally, I am very appreciative of the leadership of the two societies for allowing me the editorial freedom to take the JMD in exciting directions. It is now time for a new leader to steer the direction of the JMD. So I extend my most enthusiastic wishes to Timothy O'Leary in his endeavors with the JMD; his leadership skills and strong molecular background make him an excellent choice to pilot the JMD into the future—a future that is very bright indeed. And although I will no longer be behind the helm, I will still read it cover to cover for years to come!

Ten weeks after the virus first appeared, the initial H1N1 influenza pandemic episode appears to be waning in the United States. Everyone involved is breathing a bit more easily knowing that this novel virus is not as virulent as we had originally feared. This episode tested our preparedness for a more virulent outbreak, which has been the subject of numerous planning meetings since the arrival of SARS and Avian Flu several years ago. It is now time to ask the hard questions: How prepared were we? Was our public health response adequate? Was our clinical response appropriate? This H1N1 pandemic episode has particular significance for those of us who work in molecular diagnostics laboratories, as it is the first pandemic in the age of molecular diagnostics. To be sure, the traditional methods of the virology laboratory are still important, but now the primary focus is on the viral sequence, including the evolution of the virus, its identification, screening tests, diagnosis, and even drug resistance when it emerges. This episode is ours. So how have molecular diagnostics laboratories performed during this pandemic? Very well, we believe. In the Chicago area, during the first 4 weeks of the pandemic, 62% of nearly 8000 patients were first screened for H1N1 by community molecular diagnostics laboratories (both academic and non-academic labs, not affiliated with public health laboratories). Indeed, more than half of the total cases of H1N1 (or probable H1N1) in Illinois were first diagnosed in community molecular diagnostics laboratories. It is reasonable to ask how this came about. What elements were in place that made this rapid, effective laboratory response possible? We can point to several key factors without which this kind of response could not have occurred. First, we need to acknowledge that there exists in this country a large network of well-developed molecular diagnostics laboratories. Some of these are located in academic centers, but others are in the community hospital setting. Characteristically, these laboratories are staffed by directors and personnel trained in molecular diagnostics with expertise in the development and validation of clinical tests. This rich resource, the foundation of modern molecular medicine, is generally undervalued, unappreciated, and, as evidenced in this most recent public health emergency, underutilized. The 2009 College of American Pathologists Survey for molecular testing for influenza boasts an enrollment of more than 100 laboratories, a 50% increase over the 2008 enrollment. This is a reasonable benchmark for the number of laboratories capable of routine molecular testing for influenza. An informal survey of Association for Molecular Pathology member laboratories during the first week of the H1N1 episode showed that 93% of the 43 respondents had a molecular assay that could detect and distinguish influenza type A from type B. Those laboratories had an aggregate test capacity of 3000 to 4000 specimens per day, and could expand their capacity to as much as 12,000 specimens per day within 30 days if needed. Thirty-six percent of the laboratories reported having the capability of distinguishing the novel H1N1 strain from seasonal H1 strains. During the first week of the outbreak, those laboratories had an aggregate test capacity approaching 2500 specimens per day, with a potential for expanding within 30 days, handling nearly 8000 specimens daily. Even with a disease incidence as low as 5%, this test capacity could have easily accommodated all of the testing that identified the nearly 28,000 infected patients in the United States that the Centers for Disease Control and Prevention has documented at this time. This is only a fraction of our true test capacity. It is important to note that reports from these laboratories are largely available within 24 hours of specimen collection, a degree of timeliness not likely to be matched by any public health laboratory. There can be no argument that accurate, timely data are crucial for making public health decisions in the first hours and days of an emerging pandemic. By the end of the first week of the H1N1 episode, our own laboratory had identified 39 cases of “probable H1N1 infection” (our laboratory initially reported cases of influenza A, not seasonal H1 or H3, as “probable H1N1, pending confirmation by the public health laboratory”), only a fraction of which had been corroborated by our state public health laboratory, and confirmed only sometime after day 8. The Centers for Disease Control and Prevention tally for all of Illinois at the end of that week was still only three cases confirmed, a number widely reported in the media, misinforming the public and the medical community of the true nature of the pandemic. The ability of clinical laboratories to respond as they did was very much tied to their ability to develop and validate their own assays, ie, laboratory-developed tests. This ability to develop new analytical procedures in response to clinical need is deeply rooted in the tradition of the pathologist as the clinical scientist. This role, this responsibility, is now shouldered primarily by us, molecular pathologists and laboratory directors who bring molecular diagnostics to patient care. The Association for Molecular Pathology survey respondents used more than five different commercial assays, both analyte-specific reagent and in vitro diagnostic tests, in their laboratories, and 18% had noncommercial laboratory-developed tests in use. Whether or not they were cleared by the US Food and Drug Administration, none of these existing assays had been approved for the detection of the novel virus. Adaptation for this use could only be accomplished by validation as a laboratory-developed test in the performing laboratory. This rapid adaptation only occurred due to the availability of the H1N1 sequence data, which is as important to the manufacturer of in vitro diagnostics as it is to the developer of a laboratory-developed test. These data need to be accessible, quickly and without limitation. Analyte-specific reagent manufacturers, prone to invoking proprietary restrictions when it comes to releasing sequence information, need to provide sequence, concentration, and purity information for their products as a matter of routine. In vitro diagnostics manufacturers, however, may encounter a dilemma in providing information that could promote unauthorized use of their products. This problem could be readily addressed through an Emergency Use Authorization as was issued by the US Food and Drug Administration for the H1N1 confirmatory assay developed by the Centers for Disease Control and Prevention; however, policies and mechanisms to follow any such course of action need to be in place beforehand. None of these arguments should be construed as calling for diminished standards with regard to laboratory testing. Test validation, as always, needs to be rigorous and thorough, but it need not be slow. Indeed, the vast majority of molecular diagnostic tests for infectious agents originate as clinical laboratory-developed tests in response to clinical need, and it is exactly that nimbleness afforded the clinical laboratory that is called for during a public health emergency. Leveraging that capability for the benefit of the public's health, however, does call for forethought and planning. Communication and collaboration with local public health laboratories are essential. The primary functions of the community molecular diagnostics laboratory and the public health laboratory are not the same. The former is focused on rapid diagnosis for patient care, and in a pandemic, for effective infection control and possible allocation of limited resources. The public health laboratory necessarily takes a broader view to understand the epidemiology of the pandemic, with an eye to formulating public policy. Both activities are necessary, but they are not independent. Understanding and coordinating their different roles not only enhances their respective values, but results in an efficiency that cannot be achieved otherwise. Preparations are currently underway within the public health laboratory network for the coming influenza season, with projections that H1N1 will be a significant player. This is an opportune time for the laboratory response network to engage the community molecular diagnostics laboratories in a dialogue to develop cohesive testing strategies. Our experience in the Chicago area suggests that the impetus to do this will not likely come from Washington or Atlanta but needs to start locally. In the next pandemic, as in the next H1N1 wave to visit this country, it is reasonable to expect that most infected individuals will be diagnosed with a test performed in a community molecular diagnostics laboratory. Appropriate treatment decisions, effective infection control measures, and prudent use of antiviral agents, all demand accurate and timely diagnoses and will drive the further implementation of molecular assays. We believe that community molecular diagnostics laboratories offer an unprecedented resource to our public health pandemic planning efforts and suggest that they be an integral part of future strategic planning.

These are exciting times for anyone in the field of molecular diagnostics. Little more than a decade ago, fledgling laboratories were offering only a handful of tests and beginning the transition from Southern blots to nucleic acid amplification technologies. Since that time molecular pathology has experienced explosive growth to become an essential element in biomedical research and medical practice. The identification of new genes and the development of new methodology for gene expression analysis, together with conceptual advances, continue to expand at a very rapid pace. The new and exciting frontier is the application of this knowledge to the prevention, diagnosis, and treatment of disease. The field of molecular diagnostics is still in its infancy but is here to stay and is gradually changing the face of medicine. In parallel with these developments, practitioners of molecular diagnostics have connected and become organized. The Association for Molecular Pathology (AMP) was founded to bring together researchers, laboratory directors, technologists, trainees, and others devoted to molecular diagnostics. AMP is now entering its fifth year and continues to grow and expand through the efforts of an enthusiastic membership. We are about to attend what we anticipate will be our biggest and best meeting yet. The first two official AMP documents have been pub

Objective: To evaluate a new, rapid, and sensitive method for the detection of T-cell clonality in patients with lesions suggestive of cutaneous T-cell lymphoma (CTCL). Design: Skin specimens were obtained from 48 patients with possible CTCL. Polymerase chain reaction amplification of the T-cell receptor g (TCRG) gene was performed using consensus primers to the V and J regions. Clonal populations having identical N-region sequences are detected by single-strand conformation polymorphism analysis using a semiautomated electrophoresis system with a silver-staining method for gel visualization. The results of clinicopathological assessment were compared with those of immunohistochemistry and polymerase chain reaction analysis. Setting: Cutaneous lymphoma clinic at a university medical center. Patients: Forty-eight patients with skin lesions suggestive of CTCL. Results: Based on the clinicopathological assessment, 26 patients were diagnosed as having CTCL. Of them, clonality was detected in 19 patients (73%) and an abnormal phenotype in 17 (68%) of 25 patients. Combining both tests, abnormal results were noted in 24 (92%) of 26 patients with CTCL. Clonality was also identified in 2 (12%) of 17 patients with presumably benign lesions on clinicopathological assessment. Conclusions: Polymerase chain reaction and single-strand conformation polymorphism analysis of the TCRG gene is a rapid and sensitive method that can contribute to the diagnosis of CTCL. The new method is especially useful when used in conjunction with immunophenotyping. Arch Dermatol. 1999;135:158-162

Molecular diagnostics is an ever-evolving field, and similarly The Journal of Molecular Diagnostics is continuing to grow and change. You may have noticed that we have increased the frequency of publication, and each issue is more interesting and full than ever before. Now in our seventh year, we look forward to bringing you even better content and expanded services. Although JMD is evolving, our emphasis remains the publication of high-quality, clinically relevant research focusing on the translation of molecular science into the clinical diagnostic setting. Because our field has grown, we are now including studies applicable to the diagnosis, monitoring, prognostic determination, and characterization of solid and hematological tumors, genetic diseases, infectious diseases, and other areas, and using a range of approaches including the analysis of nucleic acids and proteins. Pharmacogenetics is beginning to make its way into our clinical practice, and proteomic approaches are on our horizon. We welcome submissions in these burgeoning fields of study as they apply to molecular diagnostics. Additionally, the JMD is including more articles describing studies and techniques that, while not yet ready for clinical application, will keep our readership appraised of new approaches, targets, and trends before they become routine. With this issue, you will notice the addition of a Technical Advance article category. This new section of the JMD is intended to report groundbreaking new approaches and technologies that will be important in our field. This month, we feature three such studies, each of which describes a novel approach that will enable labs to more accurately and rapidly analyze nucleic acids from samples. The first article reports the use of a novel aluminum oxide tube in which nucleic acids can be rapidly isolated and amplified. The second reports and validates the application of a whole genome amplification method to facilitate analysis of DNA in plasma samples, an exciting new approach for the diagnosis of many different diseases. The third Technical Advance examines the performance and reliability of gene expression signatures measured in tissues collected in RNA preserving solution, an important issue as we try to apply expression-based analysis to human tissues collected in pathology. Additionally, the JMD will soon be featuring commentaries to highlight some of the key articles in that current issue of the Journal. Commentaries will be designed to provide background and context, highlight controversies, and emphasize the importance of the articles they accompany. We will also be bringing you more reviews and minireviews in future issues. The quality and breadth you have come to expect of original research articles in the JMD will remain unchanged. In addition to regular articles, each issue will continue to feature the now-familiar Consultations in Molecular Diagnostics. Consultations are short, case-oriented reports meant to illustrate a classic or novel application of a molecular diagnostic approach, a unique observation, interpretive challenge, or technical issue. These will continue to hold great teaching value for those of us practicing molecular diagnostics in the clinical setting. As quickly as our field evolves, it is ever more important to disseminate published research quickly, broadly, and responsibly. To that end, we are proud to announce the inauguration of JMDXpress, our online prepublication program that will allow readers to view preprint versions of articles on the Journal’s website shortly after the acceptance date, rather than weeks later in the printed Journal. While not the final version of these articles, papers featured on JMDXpress will have already gone through the rigorous peer-review and first-pass copyediting stage of publication preparation, and will enable readers to get a first look at research on the horizon. Additionally, we are pleased to announce our new affiliation with PubMed Central (PMC), which will allow research published in the JMD to reach an even greater audience. Once published articles are made freely available at the JMD website (12 months after publication), article content will also be accessible through the PMC repository. Additionally, our affiliation with PMC should allow us to better assist our authors in cooperating with the National Institutes of Health’s Public Access to Research Initiative, announced in May 2005. Through this affiliation, the JMD Editorial Office will submit articles to PMC (again, to be made publicly available at 12 months after print publication) on behalf of authors whose articles have been funded by the National Institutes of Health. In collaboration with the Association for Molecular Pathology and the American Society for Investigative Pathology, the JMD is implementing a continuing medical education (CME) program. The JMD CME Program in Molecular Diagnostics, which is accredited for 50 CME credits annually, shares the same basic educational goals as the Journal itself: to help readers grasp the latest advances in understanding the molecular pathogenesis of disease, to learn about new technological advances in molecular diagnosis, and to become familiar with the complexities of diagnostic and prognostic approaches to disease. Beginning this month, each issue will contain articles selected for their particular educational value to the molecular diagnostics community. (A triangular flag in the Table of Contents indicates these articles.) Questions pertaining to these articles are provided in both the print and online versions of the JMD. See the back of this issue for the first set of questions. We hope that you will find these exercises useful, not only for earning CME credit, but also as a basic educational resource for you, your students, residents and fellows, and your laboratory staff. We continue to look for ways in which we can add value to both your experience of the JMD and to the molecular diagnostics community as a whole. Through the continued efforts and commitment of our authors, Editorial Board, and readership, the JMD is growing and evolving. We hope you will find these new programs and features useful, and look forward with enthusiasm to the future of The Journal of Molecular Diagnostics.

Extracapsular extension of prostate cancer occurs in a significant number of men believed to have clinically localized disease. We report the ability of the reverse transcriptase-polymerase chain reaction (RT-PCR) assay to predict preoperatively the pathological stage of cases of clinically localized prostate cancer.Since October 1994, 82 consecutive men with clinically localized prostate cancer had a venous blood RT-PCR assessment before radical retropubic prostatectomy. The extracted ribonucleic acid was reverse transcribed, amplified and the amplicon identity confirmed by prostate specific antigen (PSA) directed probe hybridization. An additional 31 patients were enrolled to provide appropriate positive (T + Nx/1M2) and negative (human female and benign prostatic hyperplasia) controls. Histological examination of the entire prostatectomy specimen was performed.Positive RT-PCR assay results correlated significantly with skeletal metastases and elevated levels of serum PSA but they did not significantly improve our ability to identify prospectively patients with extracapsular extension over traditional predictors (serum PSA, Gleason score).The role of molecular techniques in prostate cancer evaluation and prognosis continues to emerge. However, in our study we demonstrate no significant advantage in preoperative staging of prostate cancer using RT-PCR assay with PSA primers.

Background Methicillin-resistant Staphylococcus aureus (MRSA) nasal colonization among inpatients is a well-established risk factor for MRSA infection during the same hospitalization, but the long-term risk of MRSA infection is uncertain. We performed a retrospective cohort study to determine the one-year risk of MRSA infection among inpatients with MRSA-positive nasal polymerase chain reaction (PCR) tests confirmed by positive nasal culture (Group 1), patients with positive nasal PCR but negative nasal culture (Group 2), and patients with negative nasal PCR (Group 3). Methodology/Principal Findings Subjects were adults admitted to a four-hospital system between November 1, 2006 and March 31, 2011, comprising 195,255 admissions. Patients underwent nasal swab for MRSA PCR upon admission; if positive, nasal culture for MRSA was performed; if recovered, MRSA was tested for Panton-Valentine Leukocidin (PVL). Outcomes included MRSA-positive clinical culture and skin and soft tissue infection (SSTI). Group 1 patients had a one-year risk of MRSA-positive clinical culture of 8.0% compared with 3.0% for Group 2 patients, and 0.6% for Group 3 patients (p

Nathan Ledeboer and colleagues assess the performance of a diagnostic platform for detecting Gram-positive bacteria in blood cultures, an important step in the diagnosis and treatment of patients with sepsis. Please see later in the article for the Editors' Summary, Background A multicenter study was conducted to evaluate the diagnostic accuracy (sensitivity and specificity) of the Verigene Gram-Positive Blood Culture Test (BC-GP) test to identify 12 Gram-positive bacterial gene targets and three genetic resistance determinants directly from positive blood culture broths containing Gram-positive bacteria. Methods and Findings 1,252 blood cultures containing Gram-positive bacteria were prospectively collected and tested at five clinical centers between April, 2011 and January, 2012. An additional 387 contrived blood cultures containing uncommon targets (e.g., Listeria spp., S. lugdunensis, vanB-positive Enterococci) were included to fully evaluate the performance of the BC-GP test. Sensitivity and specificity for the 12 specific genus or species targets identified by the BC-GP test ranged from 92.6%–100% and 95.4%–100%, respectively. Identification of the mecA gene in 599 cultures containing S. aureus or S. epidermidis was 98.6% sensitive and 94.3% specific compared to cefoxitin disk method. Identification of the vanA gene in 81 cultures containing Enterococcus faecium or E. faecalis was 100% sensitive and specific. Approximately 7.5% (87/1,157) of single-organism cultures contained Gram-positive bacteria not present on the BC-GP test panel. In 95 cultures containing multiple organisms the BC-GP test was in 71.6% (68/95) agreement with culture results. Retrospective analysis of 107 separate blood cultures demonstrated that identification of methicillin resistant S. aureus and vancomycin resistant Enterococcus spp. was completed an average of 41.8 to 42.4 h earlier using the BC-GP test compared to routine culture methods. The BC-GP test was unable to assign mecA to a specific organism in cultures containing more than one Staphylococcus isolate and does not identify common blood culture contaminants such as Micrococcus, Corynebacterium, and Bacillus. Conclusions The BC-GP test is a multiplex test capable of detecting most leading causes of Gram-positive bacterial blood stream infections as well as genetic markers of methicillin and vancomycin resistance directly from positive blood cultures. Please see later in the article for the Editors' Summary, Editors' Summary Background Severe sepsis—a life-threatening condition that is usually is triggered by a bacterial infection of the bloodstream—is a major global cause of illness and death. In the US alone, sepsis causes up to 500,000 hospital admissions and more than 250,000 deaths a year. Normally, when microbes enter the human body, the immune system efficiently kills the invaders. In sepsis, the immune system goes into overdrive and the chemicals it releases into the blood to combat the infection trigger widespread inflammation, which leads to the formation of small blood clots and leaky blood vessels that impair the flow of blood to vital organs. In the most severe cases, multiple organs fail and the patient dies. Anyone can get sepsis but people with weakened immune systems, the elderly, and the very young are most vulnerable. Symptoms of sepsis include fever, chills, rapid breathing, and a fast heart rate. In its early stages, sepsis can be treated with antibiotics alone, but people with severe sepsis need to be admitted to an intensive care unit where their vital organs can be supported while the infection is treated. Why Was This Study Done? The outcome of sepsis is affected by many factors, but fast, accurate identification of the bacterial infection and determination of its antibiotic susceptibility is essential to ensure that patients receive appropriate antibiotics. Laboratory diagnosis of bloodstream infections currently requires incubation of blood samples in a liquid medium (broth) followed by growth on solid media to identify the bacteria and to test for antibiotic sensitivity. It takes about 3 days after a positive broth culture is obtained to complete this process during which time patients are treated with broad spectrum antimicrobials, which may be ineffective. In this study, the researchers evaluate the sensitivity (a test's ability to identify patients who are positive for a specific condition) and specificity (a test's ability to identify patients who do not have a specific condition) of the Verigene BC-GP test. This multiplex, automated microarray-based nucleic acid test has been developed to directly and simultaneously identify 12 species of Gram-positive bacteria (the commonest cause of bacterial bloodstream infections) and three antibiotic resistance determinants in blood culture broths that contain Gram-positive bacteria. What Did the Researchers Do and Find? The researchers used the Verigene BC-GP test and reference culture methods to identify the bacterial species and antibiotic resistance determinants present in 1,252 blood cultures containing Gram-positive bacteria collected at five US clinical centers and in 387 contrived blood cultures that contained bacterial species rarely found in the bloodstream. Compared to the reference culture method, the sensitivity of the Verigene BC-GP test for the bacterial species included in the test ranged from 92.6% to 100%; its specificity was 94.5%–100%. Identification of the mecA gene (a genetic marker for methicillin resistance) in cultures that contained Staphylococcus aureus or S. epidermis (Gram-positive bacteria that are often methicillin resistant) was 98.6% sensitive and 94.3% specific. Identification of the vanA gene (a genetic marker for vancomycin resistance) was 100% sensitive and specific in cultures containing Enterococcus species. Only 7.5% of single-organism cultures contained Gram-positive bacteria not present in the Verigene BC-GP test panel. Importantly, results obtained with the new test agreed those obtained with reference culture methods in three-quarters of cultures that contained more than one bacterial species. Finally, in a retrospective analysis of turn-around-time, identification of methicillin-resistant S. aureus and vancomycin-resistant Enterococcus species with the Verigene BC-GP test was about 42 hours faster than with reference methods. What Do These Findings Mean? These findings show that the Verigene BC-GP test is capable of accurately identifying most leading causes of bloodstream infection with Gram-positive bacteria. Moreover, they show that the test can detect genetic markers of methicillin and vancomycin resistance directly from positive blood cultures, although they also reveal that the test cannot assign mecA positivity to a specific organism in a mixed culture, a finding that may mean that some patients are treated with unnecessary antibiotics. Overall, the researchers conclude that the Verigene BC-GP test has the potential to markedly reduce the turn-around-time for reporting bacterial identification from positive blood cultures. Its use should, therefore, improve the care of patients with sepsis by allowing physicians to prescribe appropriate antibiotics much earlier than is currently possible. Additional Information Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001478. The US National Institute of General Medical Sciences has a fact sheet on sepsis The UK National Health Service Choices website has information about sepsis The Sepsis Alliance, a US not-for-profit organization, provides information about sepsis for patients and their families, including personal stories about sepsis The not-for profit UK Sepsis Trust is another useful source of information about sepsis that includes patient stories Wikipedia has a page on Gram-positive bacteria (note: Wikipedia is a free online encyclopedia that anyone can edit; available in several languages) Medlineplus provides links to additional resources about sepsis and about bacterial infections (in English and Spanish)

Background: Triple-negative breast cancer (TNBC, estrogen receptor [ER]-, progesterone receptor [PR]-, and human epidermal growth factor receptor 2 [HER2]-negative) is often aggressive and lack molecular targets for therapy. As gene regulators, microRNAs (miRNA) are important in cancer etiology but their utility as tumor biomarkers for cancer diagnosis and prognosis is yet unclear. Methods: To characterize the differential expression pattern of miRNA in TNBC from non-TNBC, we performed a whole miRNome profiling using TaqMan based quantitative PCR array of 754 miRNAs constructed based on miRBase 19 (Life Technologies, CA). Total RNA was extracted from frozen breast tumors and only RNA samples with good quality were included in the array experiment. Rigorous post-experiment quality controls were further conducted to remove unreliable plates. Moderate t-test was used to identify TNBC-specific miRNAs and Cox proportional hazard model was used to explore miRNAs that predict progression-free survival. Benjamini and Hochberg's false discovery rate method was used to correct for multiple testing. Results: Samples from 85 patients were included in the final analysis, including 71 Caucasians, 7 African Americans, and 7 others. The average age (±SD) was 54.9 ± 9.1 years. 17 patients had stage 1 tumor, 30 stage 2A, 26 stage 2B, 7 stage 3A, and 5 stage 3B. There were 42 samples with ER+, 37 PR+, 25 HER2+, and 25 categorized as TNBC. 363 out of 754 miRNAs showed expression in at least half of the samples and were included for further analysis. Of the 363 miRNAs, we found 98 to be significantly different between TNBC and non-TNBC after multiple testing correction. The most significant miRNA (miR-190b) was down-regulated in TNBC by about 20-fold (p=1.6x10-9). In fact, most of these miRNAs are down-regulated in TNBC compared to non-TNBC. During a median follow-up of 10.9 years, 29 patients died and 7 patients survived but had progressive disease of breast cancer. After multiple testing correction, we found 6 miRNAs showing significant association with progression-free survival. Interestingly, none of these 6 miRNAs were related to TNBC, suggesting that their prognostic value is independent of breast cancer subtypes. Conclusion: Our study demonstrates the existence of a set of miRNAs specific to TNBC, which has a clear “fingerprint” to differentiate TNBC from non-TNBC. We also identified different set of miRNAs that could serve as prognostic markers. Despite the small sample set, our results promisingly indicate the potential of miRNA as diagnostic and prognostic tool for breast cancer. A replication study with larger sample size would be in order. Citation Format: Toshio F. Yoshimatsu, Katharine Yao, Karen Kaul, Mary A. Turk, Olufunmilayo I. Olopade, Dezheng Huo. MicroRNA profile of triple-negative breast cancer and prognosis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5332. doi:10.1158/1538-7445.AM2013-5332

The CA 19-9 assay detects a carbohydrate antigen on multiple protein carriers, some of which may be preferential carriers of the antigen in cancer. We tested the hypothesis that the measurement of the CA 19-9 antigen on individual proteins could improve performance over the standard CA 19-9 assay. We used antibody arrays to measure the levels of the CA 19-9 antigen on multiple proteins in serum or plasma samples from patients with pancreatic adenocarcinoma or pancreatitis. Sample sets from three different institutions were examined, comprising 531 individual samples. The measurement of the CA 19-9 antigen on any individual protein did not improve upon the performance of the standard CA 19-9 assay (82% sensitivity at 75% specificity for early-stage cancer), owing to diversity among patients in their CA 19-9 protein carriers. However, a subset of cancer patients with no elevation in the standard CA 19-9 assay showed elevations of the CA 19-9 antigen specifically on the proteins MUC5AC or MUC16 in all sample sets. By combining measurements of the standard CA 19-9 assay with detection of CA 19-9 on MUC5AC and MUC16, the sensitivity of cancer detection was improved relative to CA 19-9 alone in each sample set, achieving 67-80% sensitivity at 98% specificity. This finding demonstrates the value of measuring glycans on specific proteins for improving biomarker performance. Diagnostic tests with improved sensitivity for detecting pancreatic cancer could have important applications for improving the treatment and management of patients suffering from this disease.

The purpose of this study was to investigate the biologic activity of DAB486IL-2 when administered three times daily, in terms of toxicity, pharmacokinetics, and anti-tumor effects in patients with IL-2R expressing hematologic malignancies, especially mycosis fungoides. 20 patients were enrolled in this dose escalation phase I trial. Patient cohorts were treated at levels of 0.03 mg/kg, 0.05 mg/kg, 0.07 mg/kg and 0.09 mg/kg every 8 hours for a total of 12 doses, every 21 days as toxicity and response warranted. Eight patients experienced transient fevers associated with DAB486IL-2 administration. One patient experienced grade 3 hypotension, and a second developed fluid retention manifested as weight gain/pedal edema. Dose limiting toxicity consisted of one episode of transient grade 4 hepatic transaminase elevation, and 8 episodes of transient asymptomatic grade 3 hepatic transaminase elevation. At the maximum tolerated dose DAB486IL-2 exhibited biphasic clearance kinetics; transient receptor saturation may be one mechanism for this observation. Initial serum concentration and apparent steady state level (plateau) directly correlated with the dose administered, but no difference in area under the concentration curve with greater dose was observed. Biologic activity was noted in patients with mycosis fungoides with skin lesion clearing and relief of pruritus. One patient with mycosis fungoides, and one patient with a relapsed intermediate grade non-Hodgkin's lymphoma achieved partial responses. The novel mechanism of action, toxicity profile, and evidence of biologic activity in refractory cancer patients, support development of more active constructs of this agent; such trials are underway.

✓ Secondary non-Hodgkin's lymphoma of the central nervous system is typically a late manifestation of systemic T-cell lymphoma, with a 2-month median survival time after the development of neurological disease. Of the reported patients with this late complication, only 1% manifest spread of the disease to the brain parenchyma. The authors report a patient with an unusual initial neurological presentation of systemic T-cell lymphoproliferative disorder and associated space-occupying lesions of the brain parenchyma. The diagnosis was supported by extensive molecular, immunological, and histopathological analysis. Neurological symptoms appeared early in the course of systemic disease and were characterized by spontaneous exacerbations and remissions. The patient has survived for more than 5 years since the onset of his neurological symptoms. Histopathological characterization including immunoperoxidase staining for T-cell markers, DNA content, and cell-cycle analysis of brain tissue obtained at stereotactic biopsy were compared to those of atypical lymphoid cells of peripheral blood, bone marrow, and liver. The neurological manifestations and possible etiologies of T-cell lymphoma are discussed.

Background: Presently there is an urgent need for better biomarkers for early pancreatic cancer (PaCa) diagnosis. Hypermethylation of several genes is known to correlate with presence of neoplasia in pancreatic tissue, and tumor DNA is known to be present in bodily fluids. With this background, we wanted to develop and validate a panel of methylated gene markers that would provide clinically relevant information when applied to plasma from those undergoing diagnostic procedures for pancreatic cancer. Methods: Cell-free DNA was extracted from plasma obtained from 11 PaCa patients, 15 pancreatitis patients and 15 unaffected age-matched controls using either the automated EZ-1 (Qiagen, Valencia, CA) or the manual Charge Switch (Invitrogen, Carlsbad, CA) methods. The DNA was bisulfite treated using EZ DNA Methylation-Gold (Zymo Research, Orange, CA) then analyzed using a panel of quantitative real-time PCR assays to discriminate the methylated C or T that exists after bisulfite conversion, and normalizing the signal to the amplification level of a housekeeping gene (Col2A) to assess methylation of status of 13 genes known to be differentially methylated in pancreatic tissues. Several different combinations of 6 methylated gene markers (3-OST-2, ARF/p14, FOXE1, CDKN2/p16, ppENK, and RASSF1A) showed good discrimination between the 3 groups of patients to warrant further study with an additional 53 plasma specimens (from 21 PaCa patients, 17 pancreatitis patients, and 15 unaffected controls). Receiver operator curves were generated to examine the predictive probability between marker methylation and disease, and reliability of the regression model was evaluated by using the Hosmer-Leme show goodness-of-fit test. Results: Statistical analysis of the 6 methylated gene markers for all 94 plasma specimens yielded an AUC of 0.638 (95% CI: 0.50-0.78) to differentiate PaCa from pancreatitis, and the goodness-of-fit for the model was good (p=0.697). Conclusions: Cell-free DNA extracted from plasma is an appropriate sample for the study of gene methylation in PaCa. We utilized real-time assays to detect methylation in 6 target genes and found a good correlation between the methylation status of genes and the presence of PaCa. Further studies are needed to define the clinical utility of this approach. Funding was provided by a grant from the American Cancer Society - Illinois Division (#07-06) and by funds from the Department of Pathology and Laboratory Medicine at NorthShore. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4907.

Previous studies linked infection by Chlamydia psittaci to ocular adnexal lymphomas in Italian patients (Ferreri et al., JNCI 2004). Patients treated with doxycycline to eradicate the organism have demonstrated disease regression (Ferreri et al., J. Clin Oncol, 2005). To assess whether Chlamydia psittaci is associated with ocular adnexal lymphomas in a North American population, we studied 24 ocular adnexal lymphoma biopsy specimens from adults, 16 control lymphoma specimens from patients with systemic lymphomas not involving the ocular adnexa and five benign control ocular tissue samples. There were fifteen MALT, five diffuse, large B-cell, three follicular, and one mantle cell lymphoma specimens. The presence of Chlamydia psittaci DNA was investigated by PCR in each group. DNA was isolated from sections of formalin-fixed, paraffin-embedded tissue. Two different assays were utilized: 1) a previously published conventional PCR/gel based assay targeting a 111 bp fragment of the 16S gene [Madico, J. Clin Micro, 2000] and 2) a realtime PCR assay amplifying a 148 bp portion of the 16S gene with detection via a specific fluorescent probe. Amplification was carried out to 60 cycles. Positive controls consisted of isolated DNA from Chlamydia psittaci strain VS1, CP3, and FP. A human DNA internal control was utilized to assess DNA quality and amplification success. Both assays detected Chlamydia psittaci DNA from isolated strains. All patient samples in all categories yielded negative results using both assays. Thus, we could not confirm that Chlamydia psittaci is associated with ocular adnexal lymphomas in our patient population. Differences in the prevalence of Chlamydia psittaci infection in North America and Europe or technical differences in application of the assays may underlie the differences in our findings when compared to the Italian series. Confirmation with larger numbers of specimens is required.

A quantitative multiplex RT-PCR assay is described to measure the levels of messenger RNAs for eight human genes encoding the heat shock proteins (HSP) and molecular chaperones hsp90alpha, hsp90beta, hsp70, hsc70, mtHsp75, Grp78 (BiP), hsp60 and hsp27. The basis of this assay is reverse transcription of total RNA isolated from human cells followed by amplification with PCR. By the careful selection of pairs of oligonucleotide primers corresponding to unique regions of each heat shock gene, selectivity can be attained such that messenger RNAs of multiple heat shock genes can be analyzed simultaneously in a single reaction. This method provides both the absolute and relative levels of each heat shock message by including in the reaction, reference control RNAs corresponding to in vitro transcripts of heat shock gene plasmids carrying small internal deletions.

Pretreatment of rabbits with imidazole resulted in a twofold increase in hepatic microsomal cytochrome P -450 content, with the apparent induction of two or more distinct forms of the cytochrome [ K. K. Hajek and R. F. Novak (1982) Biochem. Biophys. Res. Commun. 108 , 664–672 ]. The metabolic properties of imidazole-induced microsomes have been compared to those of uninduced, phenobarbital- and β-naphthoflavone-induced preparations. Metabolic activity was enhanced as a consequence of increased P -450 content and as a result of the presence of different forms of the cytochrome. When rates were expressed per nanomole P -450 the following were observed: (a) p -nitroanisole O -demethylation was comparable in all preparations; (b) N,N -dimethylaniline N -demethylation was comparable in imidazole- and β-naphtho-flavone-induced, and uninduced microsomes; (c) polycyclic aromatic hydrocarbon hydroxylase activity was approximately twofold greater in imidazole-induced relative to phenobarbital-induced microsomes, but was only one-half that of β-naphthoflavone-induced microsomes; and (d) metabolism of N,N -dimethylnitrosamine was enhanced fivefold, alcohol oxidation increased three- to fivefold, and aniline hydroxylation was threefold greater in imidazole-induced microsomes compared to phenobarbital- or β-naphthoflavone-induced preparations. Eadie-Scatchard analysis yielded a single K m value for dimethylnitrosamine N -demethylase activity in imidazole-induced microsomes; in contrast, both high- and low- K m values were obtained for phenobarbital- or β-naphthoflavone-induced microsomal preparations. Dimethylnitrosamine N -demethylase activity was P -450 dependent; neither flavin monooxygenase nor monoamine oxidase appeared to contribute significantly to dimethylnitrosamine metabolism. Dimethyl sulfoxide was a competitive inhibitor of dimethylnitrosamine N -demethylase activity in imidazole-, phenobarbital-, and β-naphthoflavone-induced microsomes. Dimethyl sulfoxide competitively inhibited ethanol oxidation in imidazole-induced microsomes; it was a noncompetitive inhibitor of ethanol oxidation in phenobarbitalor β-naphthoflavone-induced microsomes.

1H FT NMR longitudinal relaxation time measurements were used to characterize the solvation sphere about the heme iron of cytochromes P-450LM2, P-450LM4, and b5. P-450LM2 and LM4 produced a 15% decrease in the T1 of the solvent HDO, while b5 gave no change. Imidazole increased the HDO T1 by 15% when added to solutions of P-450LM2 or LM4 and had no effect in the presence of b5. Acetanilide had no effect on the HDO T1 in the presence of these three hemeproteins. The T1 values of the H(2) and H(4,5) imidazole protons decreased equivalently with both P-450LM2 and LM4, but virtually no change was observed with b5. These results suggest that imidazole, in binding to the heme iron of P-450LM2 or LM4, may reduce the size of the solvation sphere and/or limit solvent accessibility.