Karen L. Kaul

Universal Surveillance for Methicillin-Resistant Staphylococcus aureus in 3 Affiliated Hospitals

Context Efforts to reduce the frequency of methicillin-resistant Staphylococcus aureus (MRSA) infections have failed until now. Contribution After a baseline year, the authors screened all intensive care unit admissions for MRSA colonization using polymerase chain reaction. In year 3, they screened all hospital admissions. They placed patients who tested positive for MRSA on contact precautions. The prevalence density of MRSA clinical infection was 8.9, 7.4, and 3.9 per 10000 patient-days in years 1, 2, and 3, respectively. Methicillin-sensitive S. aureus infection rates did not change. Caution There was no concomitant, unscreened control group. Implication Screening for MRSA colonization is associated with substantially reduced rates of MRSA clinical infection. The Editors Methicillin-resistant Staphylococcus aureus (MRSA) is now endemic in many U.S. hospitals (1, 2). Colonization with this organism is a risk factor for eventual MRSA clinical infection (3), which is associated with high cost (4) and poor clinical outcomes (5). The burden of health careassociated MRSA disease is high and may be increasing: In their multiregion survey of invasive MRSA disease, investigators for the Centers for Disease Control and Prevention noted substantial increases in both community- and health careassociated infections at several sites when comparing data from 2001 to 2002 with data from 2004 to 2005 (6). Driven by the emerging concern that community-associated MRSA has entered the hospital environment (7), the medical community and the public are seeking to limit the spread of this organism with increasing urgency (8). In the United Kingdom, the Department of Health has instituted mandatory reporting of MRSA infections in hospitals (9), and in the United States, state legislatures are considering (or have passed bills) requiring active surveillance for MRSA (1012). Consumer organizations (13) and the media (14) also seek action. The Healthcare Infection Control Practices Advisory Committee of the Centers for Disease Control and Prevention (15) recently published guidelines recommending expanded surveillance of asymptomatic patients in settings in which multidrug-resistant organisms are poorly controlled with other measures. However, the evidence supporting this practice is limited to surveillance on circumscribed (for example, intensive care only) populations in small, single-center studies at large academic hospitals (1618). Because rates of MRSA infection remained unacceptably high despite conventional interventions, we implemented expanded surveillance at our 3-hospital health care organization in 2 steps. For 12 months, we implemented organization-wide, intensive care unit (ICU)based MRSA surveillance. On 1 August 2005, we initiated the first program (to our knowledge) of universal surveillance of all hospital admissions in the United States. We aimed to determine whether expanded surveillance was associated with changes in the rate of MRSA clinical disease. Methods We measured the utility of expanded surveillance for MRSA by using a 3-period before-and-after design (Figure 1). Period 1 (no active surveillance) was the baseline. In periods 2 and 3, we introduced ICU-based surveillance and universal admission surveillance, respectively. We compared MRSA disease rates during and after hospitalization in the 3 periods. Figure 1. Intervention timeline. ICU = intensive care unit; MRSA = methicillin-resistant Staphylococcus aureus. Outcomes The primary outcome was aggregate hospital-associated MRSA infection rate, defined as the sum of all MRSA bloodstream, respiratory, urinary tract, and surgical site clinical infections occurring more than 48 hours after admission through day 30 after discharge. Secondary outcomes were rates of health careassociated MRSA and methicillin-susceptible Staphylococcus aureus (MSSA) bacteremia, rates of aggregate MRSA infections occurring up to 180 days after discharge, and adherence to MRSA surveillance. We defined adherence as the percentage of admissions (ICU or whole house, depending on the period) in which surveillance testing was done. Study Sites Evanston Northwestern Healthcare, Evanston, Illinois, is a 3-hospital organization with approximately 40000 annual admissions, 75 affiliated off-site offices, 450 staff physicians, and more than 1000 affiliated physicians. Hospital 1 is an academic facility with several residency programs, 476 beds, and a high proportion of surgical patients. Hospital 2 is a primary care teaching hospital with 143 beds that serves a large population of long-term care facility residents (the elderly) relative to the other 2 hospitals. Hospital 3 is a community hospital with 239 beds. The total number of ICU beds was 45 (5.2% of all beds). Surveillance, Isolation, and Decolonization During the baseline year (period 1), routine surveillance for MRSA colonization did not occur. Patients who were MRSA-colonized on the basis of clinical cultures were placed in contact isolation, and decolonization was not attempted. During all periods, contact isolation consisted of a private room or a shared room with another MRSA-colonized patient. Gowns and gloves were required for all room entries, and patient rooms were supplied with dedicated equipment (for example, stethoscopes) for staff use. During period 2, a policy of nasal surveillance for MRSA colonization was enforced for all ICU admissions. Test turnaround time was 2.5 days. Colonized patients were isolated; decolonization therapy was not standard policy. During period 3, a policy of nasal surveillance for MRSA colonization was enforced for all hospitalizations on entry into a ward room (that is, day 1 of admission). A nurse or patient care technician obtained the specimen. Average test turnaround time was 0.67 day. Nursing staff were notified of results by telephone. Adherence was promoted through in-service education for all nursing staff and educational rounds for physicians (19). During this period, we monitored adherence and provided feedback in real time to underperforming nursing units. The infection control department recommended treatment of colonized patients with a 5-day regimen comprising mupirocin calcium, 2% twice daily to the nares, and a chlorhexidine 4% wash or shower every 2 days during period 3. Patients who were discharged before therapy was complete were sent home with prescriptions to complete this regimen. Because we felt that the decision to decolonize should be at the discretion of the physician, we did not monitor adherence to decolonization, nor did we define it as an outcome of the study. However, we had access to pharmacy data for most patients who tested positive for MRSA during the first 12 months of period 3, and we used it to determine adherence to at least 1 chlorhexidine wash and 4 or more doses of mupirocin (a quantity that seems as effective as 10 doses [20]). Patients were not removed from isolation after decolonization therapy unless another test (done at least 7 days after decolonization therapy during the same hospitalization or on repeated hospitalization) was negative for MRSA. Laboratory Methods Polymerase chain reaction tests for S. aureus colonization have better sensitivity than culture-based assays, but they may yield more false-positive results (21). Real-time polymerase chain reaction was used for MRSA detection in periods 2 (22) and 3 (21). Our in-house method and the commercial assay (BD-GeneOhm real-time polymerase chain reaction test, Becton Dickinson, Franklin Lakes, New Jersey) have equal sensitivity (21, 22). For the commercial assay, we modified the package insert protocol for specimen processing to facilitate high-volume testing (21). Data Collection Demographic Characteristics Administrative data were used to determine admission, procedure, and demographic data for all patients hospitalized from 1 August 2003 to 30 April 2007. We used International Classification of Diseases, Ninth Revision, diagnostic and procedure codes to generate comorbidity data according to the method of Elixhauser and coworkers (23) with Healthcare Cost and Utilization Project comorbidity software, version 3.2 (Agency for Healthcare Research and Quality, Rockville, Maryland) (24). Infections To measure the effect of our intervention, we were interested in true clinical disease due to MRSA. Therefore, we reviewed the records of all patients with positive inpatient or outpatient clinical cultures for MRSA from 1 August 2003 to 30 April 2007. Infections were determined as follows: bacteremia = any positive blood culture; bloodstream infection = positive blood culture in the absence of a positive clinical culture from another site; respiratory tract infection = positive respiratory culture, compatible chest radiograph, and decision to treat; urinary tract infection = positive urine culture and either a decision to treat or growth of more than 100000 colony-forming units/mL plus at least 50 leukocytes per high-power field; and surgical site infection = positive culture of a surgical site. These infection types, although not encompassing all MRSA infections at our organization, represent the major body sites affected by culture-demonstrable MRSA disease. Our primary outcome measure was the rate of clinical hospital-associated MRSA infections. Infections occurring more than 2 days after the admission date and within 30 days after discharge were considered hospital-associated. Rates of hospital-associated MRSA and hospital-associated MSSA were expressed as prevalence density of infections, that is, the number of infections per 10000 inpatient-days. Patients were counted once every 30-day period. In a separate analysis of the timing of MRSA infections, disease prevalence (that is, infections per 10000 admissions) was measured during admission and in 6 postdischarge, 30-day time frames. Statistical Analysis Rates of MRSA Infection For 1 hospital-associated infection analysis, we compared infection

Detection of Toxigenic Clostridium difficile in Stool Samples by Real-Time Polymerase Chain Reaction for the Diagnosis of C. difficile-Associated Diarrhea

BACKGROUND Clostridium difficile-associated diarrhea (CDAD) is the major cause of health care-associated infectious diarrhea. Current laboratory testing lacks a single assay that is sensitive, specific, and rapid. The purpose of this work was to design and validate a sensitive and specific real-time polymerase chain reaction (PCR) diagnostic test for CDAD. METHODS This observational validation study of a new real-time PCR assay occurred from July 2004 through April 2006 and involved the testing of 1368 stool samples. As the final validation portion of the investigation, 350 inpatients were prospectively interviewed for clinical findings for 365 episodes of diarrheal illness. Test results and clinical criteria were used to assess the performance of 4 assays. RESULTS Using clinical criteria requiring at least 3 loose stools in 1 day as part of the reference standard for a positive test result supporting CDAD, the sensitivity, specificity, and positive and negative predictive values were 73.3%, 97.6%, 73.3%, and 97.6%, respectively, for enzyme immunoassay; 93.3%, 97.4%, 75.7%, and 99.4%, respectively, for real-time PCR; 76.7%, 97.1%, 69.7%, and 97.9%, respectively, for cell culture cytotoxin assay; and 100.0%, 95.9%, 68.2%, and 100.0%, respectively, for anaerobic culture (for toxigenic C. difficile strains). The real-time PCR and anaerobic culture assays were significantly more sensitive than the enzyme immunoassay (P<.01 to P<.05). CONCLUSIONS With an assay turnaround time of <4 h, real-time PCR is a more sensitive and equally rapid test, compared with enzyme immunoassay, and is a feasible laboratory option to replace enzyme immunoassay for toxigenic C. difficile detection in clinical practice, as well as for use during the development of new therapeutic agents.

Real-Time PCR for Detection and Identification of Plasmodium spp.

ABSTRACT Rapid and accurate detection of malaria parasites in blood is needed to institute proper therapy. We developed and used a real-time PCR assay to detect and distinguish four Plasmodium spp. that cause human disease by using a single amplification reaction and melting curve analysis. Consensus primers were used to amplify a species-specific region of the multicopy 18S rRNA gene, and SYBR Green was used for detection in a LightCycler instrument. Patient specimens infected at 0.01 to 0.02% parasitemia densities were detected, and analytical sensitivity was estimated to be 0.2 genome equivalent per reaction. Melting curve analysis based on nucleotide variations within the amplicons provided a basis for accurate differentiation of Plasmodium falciparum, P. vivax, P. ovale, and P. malariae. For assay validation, 358 patient blood samples from the National University Hospital in Singapore and Evanston Northwestern Healthcare in Illinois were analyzed. Of 76 blinded patient samples with a microscopic diagnosis of P. falciparum, P. vivax, or P. ovale infection, 74 (97.4%) were detected by real-time PCR, including three specimens containing mixed P. falciparum-P. vivax infections. No Plasmodium DNA was amplified in any of the 82 specimens sent for malaria testing but that were microscopically negative for Plasmodium infection. In addition, 200 blood samples from patients whose blood was collected for reasons other than malaria testing were also determined to be negative by real-time PCR. Real-time PCR with melting curve analysis could be a rapid and objective supplement to the examination of Giemsa-stained blood smears and may replace microscopy following further validation.

Performance of the BD GeneOhm Methicillin-Resistant Staphylococcus aureus Test before and during High-Volume Clinical Use

ABSTRACT We evaluated the use of the BD GeneOhm MRSA real-time PCR assay (BD Diagnostics, San Diego, CA) for the detection of nasal colonization with methicillin-resistant Staphylococcus aureus (MRSA). The initial evaluation consisted of 403 paired nasal swabs and was done using the specimen preparation provided with the kit and an in-house lysis method that was specifically developed to accommodate large-volume testing using a minimal amount of personnel time. One swab was placed in an achromopeptidase (ACP) lysis solution, and the other was first used for culture and then prepared according to the kit protocol. PCR was performed on both lysates, and results were compared to those for culture. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the PCR assay were 98%, 96%, 77%, and 99.7% with the kit lysate and 98%, 95%, 75%, and 99.7% with the ACP lysate (P, not significant), respectively. The second evaluation was done after implementation of all-admission surveillance using PCR with ACP lysis and a sampling of 1,107 PCR-negative samples and 215 PCR-positive samples that were confirmed by culture. The results of this sampling showed an NPV of 99.9% and a PPV of 73.5% (prevalence, 6%), consistent with our initial findings. The BD GeneOhm MRSA assay is an accurate and rapid way to detect MRSA nasal colonization. When one is dealing with large specimen numbers, the ACP lysis method offers easier processing without negatively affecting the sensitivity or specificity of the PCR assay.

Siah2-Dependent Concerted Activity of HIF and FoxA2 Regulates Formation of Neuroendocrine Phenotype and Neuroendocrine Prostate Tumors

Neuroendocrine (NE) phenotype, seen in >30% of prostate adenocarcinomas (PCa), and NE prostate tumors are implicated in aggressive prostate cancer. Formation of NE prostate tumors in the TRAMP mouse model was suppressed in mice lacking the ubiquitin ligase Siah2, which regulates HIF-1alpha availability. Cooperation between HIF-1alpha and FoxA2, a transcription factor expressed in NE tissue, promotes recruitment of p300 to transactivate select HIF-regulated genes, Hes6, Sox9, and Jmjd1a. These HIF-regulated genes are highly expressed in metastatic PCa and required for hypoxia-mediated NE phenotype, metastasis in PCa, and the formation of NE tumors. Tissue-specific expression of FoxA2 combined with Siah2-dependent HIF-1alpha availability enables a transcriptional program required for NE prostate tumor development and NE phenotype in PCa.

Molecular Detection of Prostate Epithelial Cells from the Surgical Field and Peripheral Circulation during Radical Prostatectomy

PURPOSE Prostate cancer progression despite organ confined pathological assessment has been reported in a variable number of men after radical retropubic prostatectomy. To study this phenomenon, we used the prostate specific antigen (PSA) reverse transcriptase-polymerase chain reaction assay. MATERIALS AND METHODS We prospectively assayed the peripheral venous blood before, during and after surgical manipulation as well as the intraoperative field blood for PSA reverse transcriptase-polymerase chain reaction-positive cells in 22 men undergoing radical retropubic prostatectomy. RESULTS PSA reverse transcriptase-polymerase chain reaction-positive cells were identified in 20 of the 22 operative field samples (91%) and 4 of 16 (25%) had evidence of intraoperative hematogenous dissemination (p = 0.046). No significant association was identified among Gleason score, pathological stage and the PSA reverse transcriptase-polymerase chain reaction result. CONCLUSIONS Our results suggest that tumor cell spillage and less frequently hematogenous dissemination may be associated with operative manipulation of the prostate during radical retropubic prostatectomy and may potentially represent mechanisms of failure after radical retropubic prostatectomy.

The Prevalence and Nature of Glycan Alterations on Specific Proteins in Pancreatic Cancer Patients Revealed Using Antibody-Lectin Sandwich Arrays

Changes to the glycan structures of proteins secreted by cancer cells are known to be functionally important and to have potential diagnostic value. However, an exploration of the population variation and prevalence of glycan alterations on specific proteins has been lacking because of limitations in conventional glycobiology methods. Here we report the use of a previously developed antibody-lectin sandwich array method to characterize both the protein and glycan levels of specific mucins and carcinoembryonic antigen-related proteins captured from the sera of pancreatic cancer patients (n = 23) and control subjects (n = 23). The MUC16 protein was frequently elevated in the cancer patients (65% of the patients) but showed no glycan alterations, whereas the MUC1 and MUC5AC proteins were less frequently elevated (30 and 35%, respectively) and showed highly prevalent (up to 65%) and distinct glycan alterations. The most frequent glycan elevations involved the Thomsen-Friedenreich antigen, fucose, and Lewis antigens. An unexpected increase in the exposure of α-linked mannose also was observed on MUC1 and MUC5ac, indicating possible N-glycan modifications. Because glycan alterations occurred independently from the protein levels, improved identification of the cancer samples was achieved using glycan measurements on specific proteins relative to using the core protein measurements. The most significant elevation was the cancer antigen 19-9 on MUC1, occurring in 19 of 23 (87%) of the cancer patients and one of 23 (4%) of the control subjects. This work gives insight into the prevalence and protein carriers of glycan alterations in pancreatic cancer and points to the potential of using glycan measurements on specific proteins for highly effective biomarkers.

Differential methylation of cell‐free circulating DNA among patients with pancreatic cancer versus chronic pancreatitis

Although patients with chronic pancreatitis (CP) have an increased risk of pancreatic cancer (PanCa), the timely detection of PanCa often is difficult, because the symptoms of CP and PanCa are very similar. Moreover, secondary inflammation may be identified in PanCa, further complicating diagnosis. To improve the survival of patients with PanCa, a reliable test to differentiate CP from PanCa is needed. In this article, the authors describe a methylation profile of cell‐free plasma DNA that distinguished CP from PanCa with >90% accuracy.

Direct Detection of Staphylococcus aureus from Adult and Neonate Nasal Swab Specimens Using Real-Time Polymerase Chain Reaction

Nasal carriage of Staphylococcus aureus is considered a source of subsequent infection in health care settings. Utilization of real-time polymerase chain reaction (PCR) for detection of S. aureus has the potential to dramatically affect infection control practice by rapidly identifying S. aureus-colonized patients. We developed and validated the use of real-time PCR for detection of S. aureus colonization in two patient populations. Paired nasal swabs were collected from 299 neonates and from 151 adult patients at Evanston Hospital. One swab was used for culture and the other placed into a bacterial lysis solution containing achromopeptidase. The DNA liberated was used as the template for real-time PCR with primers for the femA gene. SYBR Green was used for amplicon detection. In the neonatal population the sensitivity, specificity, predictive value positive and predictive value negative for culture and PCR was 92% versus 96%, 100% versus 100%, 100% versus 100%, and 98% versus 99%, respectively. In the adults the results were 90% versus 100%, 100% versus 98%, 100% versus 96%, and 95% versus 100%, respectively. Real-time PCR was able to detect S. aureus in 2 hours compared to 1 to 4 days for culture and provided sensitivity equal to or greater than culture.

Real-Time PCR Can Rapidly Detect Methicillin-Susceptible and Methicillin-Resistant Staphylococcus aureus Directly From Positive Blood Culture Bottles

We developed, validated, and implemented real-time polymerase chain reaction (PCR) detection of the femA gene for Staphylococcus aureus and the mecA gene for methicillin resistance directly from BACTEC (Becton Dickinson, Sparks, MD) blood culture bottles showing gram-positive cocci in clusters. For the 332 positive blood cultures tested, the assay had 100% sensitivity and specificity for identifying methicillin-susceptible (n=28) and methicillin-resistant (n=28) S aureus, and overall was 98% sensitive and 94% specific, with 3 uninterpretable test results when identification of coagulase-negative staphylococci was included. PCR detection yields rapid (2-3 hours) results and accurate identification of S aureus directly from signal-positive blood culture bottle samples.