Bradly P. Nicholson

Multiplex PCR to diagnose bloodstream infections in patients admitted from the emergency department with sepsis.

ABSTRACT Sepsis is caused by a heterogeneous group of infectious etiologies. Early diagnosis and the provision of appropriate antimicrobial therapy correlate with positive clinical outcomes. Current microbiological techniques are limited in their diagnostic capacities and timeliness. Multiplex PCR has the potential to rapidly identify bloodstream infections and fill this diagnostic gap. We identified patients from two large academic hospital emergency departments with suspected sepsis. The results of a multiplex PCR that could detect 25 bacterial and fungal pathogens were compared to those of blood culture. The results were analyzed with respect to the likelihood of infection, sepsis severity, the site of infection, and the effect of prior antibiotic therapy. We enrolled 306 subjects with suspected sepsis. Of these, 43 were later determined not to have infectious etiologies. Of the remaining 263 subjects, 70% had sepsis, 16% had severe sepsis, and 14% had septic shock. The majority had a definite infection (41.5%) or a probable infection (30.7%). Blood culture and PCR performed similarly with samples from patients with clinically defined infections (areas under the receiver operating characteristic curves, 0.64 and 0.60, respectively). However, blood culture identified more cases of septicemia than PCR among patients with an identified infectious etiology (66 and 46, respectively; P = 0.0004). The two tests performed similarly when the results were stratified by sepsis severity or infection site. Blood culture tended to detect infections more frequently among patients who had previously received antibiotics (P = 0.06). Conversely, PCR identified an additional 24 organisms that blood culture failed to detect. Real-time multiplex PCR has the potential to serve as an adjunct to conventional blood culture, adding diagnostic yield and shortening the time to pathogen identification.

Complex Genetic Interactions in a Quantitative Trait Locus

Whether in natural populations or between two unrelated members of a species, most phenotypic variation is quantitative. To analyze such quantitative traits, one must first map the underlying quantitative trait loci. Next, and far more difficult, one must identify the quantitative trait genes (QTGs), characterize QTG interactions, and identify the phenotypically relevant polymorphisms to determine how QTGs contribute to phenotype. In this work, we analyzed three Saccharomyces cerevisiae high-temperature growth (Htg) QTGs (MKT1, END3, and RHO2). We observed a high level of genetic interactions among QTGs and strain background. Interestingly, while the MKT1 and END3 coding polymorphisms contribute to phenotype, it is the RHO2 3′UTR polymorphisms that are phenotypically relevant. Reciprocal hemizygosity analysis of the Htg QTGs in hybrids between S288c and ten unrelated S. cerevisiae strains reveals that the contributions of the Htg QTGs are not conserved in nine other hybrids, which has implications for QTG identification by marker-trait association. Our findings demonstrate the variety and complexity of QTG contributions to phenotype, the impact of genetic background, and the value of quantitative genetic studies in S. cerevisiae.

A host transcriptional signature for presymptomatic detection of infection in humans exposed to influenza H1N1 or H3N2.

There is great potential for host-based gene expression analysis to impact the early diagnosis of infectious diseases. In particular, the influenza pandemic of 2009 highlighted the challenges and limitations of traditional pathogen-based testing for suspected upper respiratory viral infection. We inoculated human volunteers with either influenza A (A/Brisbane/59/2007 (H1N1) or A/Wisconsin/67/2005 (H3N2)), and assayed the peripheral blood transcriptome every 8 hours for 7 days. Of 41 inoculated volunteers, 18 (44%) developed symptomatic infection. Using unbiased sparse latent factor regression analysis, we generated a gene signature (or factor) for symptomatic influenza capable of detecting 94% of infected cases. This gene signature is detectable as early as 29 hours post-exposure and achieves maximal accuracy on average 43 hours (p = 0.003, H1N1) and 38 hours (p-value = 0.005, H3N2) before peak clinical symptoms. In order to test the relevance of these findings in naturally acquired disease, a composite influenza A signature built from these challenge studies was applied to Emergency Department patients where it discriminates between swine-origin influenza A/H1N1 (2009) infected and non-infected individuals with 92% accuracy. The host genomic response to Influenza infection is robust and may provide the means for detection before typical clinical symptoms are apparent.

A case-control study of community-associated Clostridium difficile infection: no role for proton pump inhibitors.

BACKGROUND The epidemiology of community-associated Clostridium difficile infection is not well known. We performed a multicenter, case-control study to further describe community-associated C. difficile infection and assess novel risk factors. METHODS We conducted this study at 5 sites from October 2006 through November 2007. Community-associated C. difficile infection included individuals with diarrhea, a positive C. difficile toxin, and no recent (12 weeks) discharge from a health care facility. We selected controls from the same clinics attended by cases. We collected clinical and exposure data at the time of illness and cultured residual stool samples and performed ribotyping. RESULTS Of 1041 adult C. difficile infections, 162 (15.5%) met criteria for community-associated: 66 case and 114 control patients were enrolled. Case patients were relatively young (median 64 years), female (56%), and frequently required hospitalization (38%). Antimicrobials, malignancy, exposure to high-risk persons, and remote health care exposure were independently associated with community-associated C. difficile infection. In 40% of cases, we could not confirm recent antibiotic exposure. Stomach-acid suppressants were not associated with community-associated infection, and 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors appeared protective. Prevalence of the hypervirulent NAP-1/027 strain was infrequent (17%). CONCLUSIONS Community-associated C. difficile infection resulted in a substantial health care burden. Antimicrobials are a significant risk factor for community-associated infection. However, other unique factors also may contribute, including person-to-person transmission, remote health care exposures, and 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors. A role for stomach-acid suppressants in community-associated C. difficile infection is not supported.

A Host-Based RT-PCR Gene Expression Signature to Identify Acute Respiratory Viral Infection

To improve the diagnosis of respiratory viral infection, a multiplex RT-PCR assay based on the host response was derived from experimentally infected subjects and validated in patients with febrile illness. Diagnosing the Cause of Coughs and Sneezes Diagnosis of viral respiratory infections remains a challenge. Early differentiation between a viral and bacterial etiology of respiratory symptoms would help to direct therapy more appropriately and prevent overuse of antibiotics. Measuring the host immune response to infection is an alternative to pathogen-based diagnostic testing and may improve diagnostic accuracy. Now, Zaas et al. have developed a reverse transcription polymerase chain reaction (RT-PCR) assay for blood RNA that can classify respiratory viral infections based on the host immune response. They developed their assay using two groups of individuals experimentally infected with either influenza A H3N2/Wisconsin or influenza A H1N1/Brisbane. They then validated their RT-PCR diagnostic in a sample of adults presenting to the emergency department with fever, who had microbiologically confirmed viral or bacterial illness. The sensitivity of the RT-PCR assay was 89% [95% confidence interval (CI), 72 to 98%], and the specificity was 94% (95% CI, 86 to 99%). These data establish an important “proof of concept” that host expression of a relatively small set of genes measured by RT-PCR can be used to classify viral respiratory illness in unselected individuals presenting at an emergency department for evaluation of fever. The development of this new assay and its validation in an independent “real-world” patient population is an important step on the translational pathway to establishing this platform for diagnostic testing in the clinic. Improved ways to diagnose acute respiratory viral infections could decrease inappropriate antibacterial use and serve as a vital triage mechanism in the event of a potential viral pandemic. Measurement of the host response to infection is an alternative to pathogen-based diagnostic testing and may improve diagnostic accuracy. We have developed a host-based assay with a reverse transcription polymerase chain reaction (RT-PCR) TaqMan low-density array (TLDA) platform for classifying respiratory viral infection. We developed the assay using two cohorts experimentally infected with influenza A H3N2/Wisconsin or influenza A H1N1/Brisbane, and validated the assay in a sample of adults presenting to the emergency department with fever (n = 102) and in healthy volunteers (n = 41). Peripheral blood RNA samples were obtained from individuals who underwent experimental viral challenge or who presented to the emergency department and had microbiologically proven viral respiratory infection or systemic bacterial infection. The selected gene set on the RT-PCR TLDA assay classified participants with experimentally induced influenza H3N2 and H1N1 infection with 100 and 87% accuracy, respectively. We validated this host gene expression signature in a cohort of 102 individuals arriving at the emergency department. The sensitivity of the RT-PCR test was 89% [95% confidence interval (CI), 72 to 98%], and the specificity was 94% (95% CI, 86 to 99%). These results show that RT-PCR–based detection of a host gene expression signature can classify individuals with respiratory viral infection and sets the stage for prospective evaluation of this diagnostic approach in a clinical setting.

Multicenter Evaluation of the LightCycler Methicillin-Resistant Staphylococcus aureus (MRSA) Advanced Test as a Rapid Method for Detection of MRSA in Nasal Surveillance Swabs

ABSTRACT The rate of methicillin-resistant Staphylococcus aureus (MRSA) infection continues to rise in many health care settings. Rapid detection of MRSA colonization followed by appropriate isolation can reduce transmission and infection. We compared the performance of the new Roche LightCycler MRSA advanced test to that of the BD GeneOhm MRSA test and culture. Double-headed swabs were used to collect anterior nasal specimens from each subject. For both tests, DNA was extracted and real-time PCR was performed according to manufacturers instructions. For culture, one swab of the pair was plated directly to CHROMagar MRSA. The swab paired with the BD GeneOhm MRSA test was also placed into an enrichment broth and then plated to CHROMagar MRSA. Colonies resembling staphylococci were confirmed as S. aureus by standard methods. Discrepant specimens had further testing with additional attempts to grow MRSA as well as sample amplicon sequencing. Agreement between results for the two swabs was 99.3% for those with valid results. A total of 1,402 specimens were tested using direct culture detection of MRSA as the gold standard; 187 were culture positive for MRSA. The LightCycler MRSA advanced test had relative sensitivity and specificity of 95.2% (95% confidence interval [CI]: 91.1% to 97.8%) and 96.4% (95% CI: 95.2% to 97.4%), respectively. The BD GeneOhm assay had relative sensitivity and specificity of 95.7% (95% CI: 91.7% to 98.1%) and 91.7% (95% CI: 90.0% to 93.2%), respectively. Following discrepancy analysis, the relative sensitivities of the LightCycler MRSA advanced test and the BD GeneOhm MRSA assay were 92.2 and 93.2%, respectively; relative specificities were 98.9 and 94.2%, respectively. Specificity was significantly better (P < 0.001) with the LightCycler MRSA advanced test. The sensitivity of direct culture was 80.4%. The LightCycler MRSA advanced test is a useful tool for sensitive and rapid detection of MRSA nasal colonization.

Cytosine deaminase MX cassettes as positive/negative selectable markers in Saccharomyces cerevisiae

We describe positive/negative selectable cytosine deaminase MX cassettes for use in Saccharomyces cerevisiae. The basis of positive selection for cytosine deaminase (Fcy1) activity is that (a) fcy1 strains are unable to grow on medium containing cytosine as a sole nitrogen source and (b) fcy1 ura3 strains are unable to grow on medium containing cytosine as the sole pyrimidine source. Conversely, as 5‐fluorocytosine (5FC) is toxic to cytosine deaminase‐producing cells, fcy1 strains are resistant to 5FC. FCY1MX and FCA1MX cassettes, containing open reading frames (ORFs) of S. cerevisiae FCY1 and Candida albicans FCA1, respectively, were constructed and used to disrupt targeted genes in S. cerevisiae fcy1 strains. In addition, new direct repeat cassettes, kanPR, FCA1PR, FCY1PR and CaURA3PR, were developed to allow efficient deletion of target genes in cells containing MX3 repeats. Finally, the FCY1‐ and FCA1MX3 or PR direct repeat cassettes can be readily recycled after 5FC counter‐selection on both synthetic and rich media. Copyright

Repeat endocarditis: analysis of risk factors based on the International Collaboration on Endocarditis - Prospective Cohort Study.

Repeat episodes of infective endocarditis (IE) can occur in patients who survive an initial episode. We analysed risk factors and 1-year mortality of patients with repeat IE. We considered 1874 patients enrolled in the International Collaboration on Endocarditis - Prospective Cohort Study between January 2000 and December 2006 (ICE-PCS) who had definite native or prosthetic valve IE and 1-year follow-up. Multivariable analysis was used to determine risk factors for repeat IE and 1-year mortality. Of 1874 patients, 1783 (95.2%) had single-episode IE and 91 (4.8%) had repeat IE: 74/91 (81%) with new infection and 17/91 (19%) with presumed relapse. On bivariate analysis, repeat IE was associated with haemodialysis (p 0.002), HIV (p 0.009), injection drug use (IDU) (p < 0.001), Staphylococcus aureus IE (p 0.003), healthcare acquisition (p 0.006) and previous IE before ICE enrolment (p 0.001). On adjusted analysis, independent risk factors were haemodialysis (OR, 2.5; 95% CI, 1.2-5.3), IDU (OR, 2.9; 95% CI, 1.6-5.4), previous IE (OR, 2.8; 95% CI, 1.5-5.1) and living in the North American region (OR, 1.9; 95% CI, 1.1-3.4). Patients with repeat IE had higher 1-year mortality than those with single-episode IE (p 0.003). Repeat IE is associated with IDU, previous IE and haemodialysis. Clinicians should be aware of these risk factors in order to recognize patients who are at risk of repeat IE.

Longitudinal analysis of leukocyte differentials in peripheral blood of patients with acute respiratory viral infections

BACKGROUND Leukocyte counts and differentials are commonly acquired in patients with suspected respiratory viral infections and may contribute diagnostic information. However, most published work is limited to a single timepoint at initial presentation to a medical provider, which may correspond to widely varying points in the course of disease. OBJECTIVES To examine the temporal development and time-dependent utility of routine leukocyte differentials in the diagnosis of respiratory viral infections. STUDY DESIGN We analyzed data from recent experimental human challenges with influenza A/H3N2, human rhinovirus (HRV), and respiratory syncytial virus (RSV). Routine clinical lab cell counts and differentials were measured daily from the time period immediately prior to inoculation through the eventual resolution of symptomatic disease. RESULTS Approximately 50% of challenged individuals developed symptoms and viral shedding consistent with clinical disease. Subpopulations of WBC showed marked differences between symptomatic and asymptomatic individuals over time, but these changes were much more profound and consistent in influenza infection. Influenza-infected subjects develop both relative lymphopenia and relative monocytosis, both of which closely mirror symptom development in time. A lymphocyte:monocyte ratio of <2 correctly classifies 100% of influenza (but not RSV or HRV) infected subjects at the time of maximal symptoms. CONCLUSIONS Leukocyte differentials may suggest a viral etiology in patients with upper respiratory infection, but are not sufficient to allow differentiation between common viruses. Timing of data acquisition relative to the disease course is a key component in determining the utility of these tests.

Etiology of Pediatric Fever in Western Kenya: A Case–Control Study of Falciparum Malaria, Respiratory Viruses, and Streptococcal Pharyngitis

In Kenya, more than 10 million episodes of acute febrile illness are treated annually among children under 5 years. Most are clinically managed as malaria without parasitological confirmation. There is an unmet need to describe pathogen-specific etiologies of fever. We enrolled 370 febrile children and 184 healthy controls. We report demographic and clinical characteristics of patients with Plasmodium falciparum, group A streptococcal (GAS) pharyngitis, and respiratory viruses (influenza A and B, respiratory syncytial virus [RSV], parainfluenza [PIV] types 1-3, adenovirus, human metapneumovirus [hMPV]), as well as those with undifferentiated fever. Of febrile children, 79.7% were treated for malaria. However, P. falciparum was detected infrequently in both cases and controls (14/268 [5.2%] versus 3/133 [2.3%], P = 0.165), whereas 41% (117/282) of febrile children had a respiratory viral infection, compared with 24.8% (29/117) of controls (P = 0.002). Only 9/515 (1.7%) children had streptococcal infection. Of febrile children, 22/269 (8.2%) were infected with > 1 pathogen, and 102/275 (37.1%) had fevers of unknown etiology. Respiratory viruses were common in both groups, but only influenza or parainfluenza was more likely to be associated with symptomatic disease (attributable fraction [AF] 67.5% and 59%, respectively). Malaria was overdiagnosed and overtreated. Few children presented to the hospital with GAS pharyngitis. An enhanced understanding of carriage of common pathogens, improved diagnostic capacity, and better-informed clinical algorithms for febrile illness are needed.