Richard E. Rothman

PCR-based diagnostics for infectious diseases: uses, limitations, and future applications in acute-care settings.

Summary Molecular diagnostics are revolutionising the clinical practice of infectious disease. Their effects will be significant in acute-care settings where timely and accurate diagnostic tools are critical for patient treatment decisions and outcomes. PCR is the most well-developed molecular technique up to now, and has a wide range of already fulfilled, and potential, clinical applications, including specific or broad-spectrum pathogen detection, evaluation of emerging novel infections, surveillance, early detection of biothreat agents, and antimicrobial resistance profiling. PCR-based methods may also be cost effective relative to traditional testing procedures. Further advancement of technology is needed to improve automation, optimise detection sensitivity and specificity, and expand the capacity to detect multiple targets simultaneously (multiplexing). This review provides an up-to-date look at the general principles, diagnostic value, and limitations of the most current PCR-based platforms as they evolve from bench to bedside.

Quantitative Multiprobe PCR Assay for Simultaneous Detection and Identification to Species Level of Bacterial Pathogens

ABSTRACT We describe a novel adaptation of the TaqMan PCR assay which potentially allows for highly sensitive detection of any eubacterial species with simultaneous species identification. Our system relies on a unique multiprobe design in which a single set of highly conserved sequences encoded by the 16S rRNA gene serves as the primer pair and is used in combination with both an internal highly conserved sequence, the universal probe, and an internal variable region, the species-specific probe. A pre-PCR ultrafiltration step effectively decontaminates or removes background DNA. The TaqMan system described reliabAly detected 14 common bacterial species with a detection limit of 50 fg. Further, highly sensitive and specific pathogen detection was demonstrated with a prototype species-specific probe designed to detect Staphylococcus aureus. This assay has broad potential in the clinical arena for rapid and specific diagnosis of infectious diseases.

Global Surveillance of Emerging Influenza Virus Genotypes by Mass Spectrometry

Background Effective influenza surveillance requires new methods capable of rapid and inexpensive genomic analysis of evolving viral species for pandemic preparedness, to understand the evolution of circulating viral species, and for vaccine strain selection. We have developed one such approach based on previously described broad-range reverse transcription PCR/electrospray ionization mass spectrometry (RT-PCR/ESI-MS) technology. Methods and Principal Findings Analysis of base compositions of RT-PCR amplicons from influenza core gene segments (PB1, PB2, PA, M, NS, NP) are used to provide sub-species identification and infer influenza virus H and N subtypes. Using this approach, we detected and correctly identified 92 mammalian and avian influenza isolates, representing 30 different H and N types, including 29 avian H5N1 isolates. Further, direct analysis of 656 human clinical respiratory specimens collected over a seven-year period (1999–2006) showed correct identification of the viral species and subtypes with >97% sensitivity and specificity. Base composition derived clusters inferred from this analysis showed 100% concordance to previously established clades. Ongoing surveillance of samples from the recent influenza virus seasons (2005–2006) showed evidence for emergence and establishment of new genotypes of circulating H3N2 strains worldwide. Mixed viral quasispecies were found in approximately 1% of these recent samples providing a view into viral evolution. Conclusion/Significance Thus, rapid RT-PCR/ESI-MS analysis can be used to simultaneously identify all species of influenza viruses with clade-level resolution, identify mixed viral populations and monitor global spread and emergence of novel viral genotypes. This high-throughput method promises to become an integral component of influenza surveillance.

Influenza Forecasting with Google Flu Trends

Objective We sought to develop a practical influenza forecast model, based on real-time, geographically focused, and easy to access data, to provide individual medical centers with advanced warning of the number of influenza cases, thus allowing sufficient time to implement an intervention. Secondly, we evaluated how the addition of a real-time influenza surveillance system, Google Flu Trends, would impact the forecasting capabilities of this model. Introduction Each year, influenza results in increased Emergency Department crowding which can be mitigated through early detection linked to an appropriate response. Although current surveillance systems, such as Google Flu Trends, yield near real-time influenza surveillance, few demonstrate ability to forecast impending influenza cases. Methods Forecasting models designed to predict one week in advance were developed from weekly counts of confirmed influenza cases over seven seasons (2004 – 2011) divided into training and out-of-sample verification sets. Forecasting procedures using classical Box-Jenkins, generalized linear, and autoregressive methods were employed to develop the final model and assess the relative contribution of external variables such as, Google Flu Trends, meteorological data, and temporal information. Models were developed and evaluated through statistical measures of global deviance and log-likelihood ratio tests. An additional measure of forecast confidence, defined as the percentage of forecast values, during an influenza peak, that are within 7 influenza cases of the actual data, was examined to demonstrate practical utility of the model. Results A generalized autoregressive Poisson (GARMA) forecast model integrating previous influenza cases with Google Flu Trends information provided the most accurate influenza case predictions. Google Flu Trend data was the only source of external information providing significant forecast improvements (p = 0.00002). The final model, a GARMA intercept model with the addition of Google Flu Trends, predicted weekly influenza cases during 4 out-of-sample outbreaks within 7 cases for 80% of estimates (Figure 1). Conclusions Integer-valued autoregression of influenza cases provides a strong base forecast model, which is enhanced by the addition of Google Flu Trends confirming the predictive capabilities of search query based syndromic surveillance. This accessible and flexible forecast model can be used by individual medical centers to provide advanced warning of future influenza cases.

Quantitative PCR Assay Using Sputum Samples for Rapid Diagnosis of Pneumococcal Pneumonia in Adult Emergency Department Patients

ABSTRACT Accurate diagnosis of pneumococcal pneumonia in the acute-care setting remains a challenge due to the inadequate sensitivity of conventional diagnostic tests. Sputum cultures, which are likely to have the highest diagnostic yields of all specimen types, have been considered unreliable, due to their inability to differentiate colonization from infection. Our objective was to evaluate the potential clinical utility of a rapid quantitative real-time PCR assay using sputum samples for Streptococcus pneumoniae in adult patients with community-acquired pneumonia (CAP). A prospective clinical observational study of consecutively enrolled emergency department patients with CAP was performed; only those patients with excess good-quality sputum samples were included for evaluation. Sputum samples were tested for the presence of S. pneumoniae by using a quantitative PCR that targets the pneumolysin gene. PCR findings were compared with those of a composite reference standard comprising Gram staining of sputum samples and sputum/blood cultures. The area under the curve (AUC) and a log-transformed threshold, which provides the maximal sensitivity and specificity, were calculated. Of 487 subjects enrolled, 129 were evaluable. Receiver operating characteristic curve analysis demonstrated an AUC of 0.87. Sensitivity and specificity were 90.0 percent and 80.0 percent, respectively; positive and negative predictive values were 58.7 percent and 96.2 percent, respectively. We have demonstrated that a quantitative rapid pneumolysin PCR assay has favorable accuracy for diagnosis of pneumococcal pneumonia in adult patients with CAP; this assay may be a useful diagnostic adjunct for clinicians, particularly those practicing in the acute-care setting, where rapid pathogen identification may assist in selection of the most appropriate antibiotics.

Unsuspected gonorrhea and chlamydia in patients of an urban adult emergency department: A critical population for STD control intervention

Background Urban emergency departments (EDs) providing services to patients at high risk for sexually transmitted infection may be logical sites for intervention. Goal To determine the prevalence of gonorrhea (GC) and chlamydia (CT) in an adult ED patient population, and to assess risk factors for infection. Study Design Cross-sectional study of patients aged 18 to 44 in an urban ED, seeking care of any medical nature. Main outcome was positive for GC or CT by urine ligase chain reaction assay. Results Test results for GC and/or CT were positive in 13.6% of 434 18 to 31 year-olds and in 1.8% of 221 32 to 44 year-olds. Of 63 infected individuals identified by the study, 15 (23.8%) were treated at the ED visit. Age ≤31 detected 88% of infections. Among 18- to 31-year-old patients, predictive risk factors by multivariate analysis included age <25, >1 sex partner in the past 90 days, and a history of sexually transmitted disease. Conclusion This study identified a high prevalence of GC and CT in patients seeking ED services. Many of these infections were clinically unsuspected. These data demonstrate that the ED is a high-risk setting and may be an appropriate site for routine GC and CT screening in 18- to 31-year-old patients.

Quantitative video-oculography to help diagnose stroke in acute vertigo and dizziness: Toward an ECG for the eyes

Background and Purpose— Strokes can be distinguished from benign peripheral causes of acute vestibular syndrome using bedside oculomotor tests (head impulse test, nystagmus, test-of-skew). Using head impulse test, nystagmus, test-of-skew is more sensitive and less costly than early magnetic resonance imaging for stroke diagnosis in acute vestibular syndrome but requires expertise not routinely available in emergency departments. We sought to begin standardizing the head impulse test, nystagmus, test-of-skew diagnostic approach for eventual emergency department use through the novel application of a portable video-oculography device measuring vestibular physiology in real time. This approach is conceptually similar to ECG to diagnose acute cardiac ischemia. Methods— Proof-of-concept study (August 2011 to June 2012). We recruited adult emergency department patients with acute vestibular syndrome defined as new, persistent vertigo/dizziness, nystagmus, and (1) nausea/vomiting, (2) head motion intolerance, or (3) new gait unsteadiness. We recorded eye movements, including quantitative horizontal head impulse testing of vestibulo-ocular-reflex function. Two masked vestibular experts rated vestibular findings, which were compared with final radiographic gold-standard diagnoses. Masked neuroimaging raters determined stroke or no stroke using magnetic resonance imaging of the brain with diffusion-weighted imaging obtained 48 hours to 7 days after symptom onset. Results— We enrolled 12 consecutive patients who underwent confirmatory magnetic resonance imaging. Mean age was 61 years (range 30–73), and 10 were men. Expert-rated video-oculography–based head impulse test, nystagmus, test-of-skew examination was 100% accurate (6 strokes, 6 peripheral vestibular). Conclusions— Device-based physiological diagnosis of vertebrobasilar stroke in acute vestibular syndrome should soon be possible. If confirmed in a larger sample, this bedside eye ECG approach could eventually help fulfill a critical need for timely, accurate, efficient diagnosis in emergency department patients with vertigo or dizziness who are at high risk for stroke.

Improved Sensitivity for Molecular Detection of Bacterial and Candida Infections in Blood

ABSTRACT The rapid identification of bacteria and fungi directly from the blood of patients with suspected bloodstream infections aids in diagnosis and guides treatment decisions. The development of an automated, rapid, and sensitive molecular technology capable of detecting the diverse agents of such infections at low titers has been challenging, due in part to the high background of genomic DNA in blood. PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) allows for the rapid and accurate identification of microorganisms but with a sensitivity of about 50% compared to that of culture when using 1-ml whole-blood specimens. Here, we describe a new integrated specimen preparation technology that substantially improves the sensitivity of PCR/ESI-MS analysis. An efficient lysis method and automated DNA purification system were designed for processing 5 ml of whole blood. In addition, PCR amplification formulations were optimized to tolerate high levels of human DNA. An analysis of 331 specimens collected from patients with suspected bloodstream infections resulted in 35 PCR/ESI-MS-positive specimens (10.6%) compared to 18 positive by culture (5.4%). PCR/ESI-MS was 83% sensitive and 94% specific compared to culture. Replicate PCR/ESI-MS testing from a second aliquot of the PCR/ESI-MS-positive/culture-negative specimens corroborated the initial findings in most cases, resulting in increased sensitivity (91%) and specificity (99%) when confirmed detections were considered true positives. The integrated solution described here has the potential to provide rapid detection and identification of organisms responsible for bloodstream infections.

Rapid PCR-based diagnosis of septic arthritis by early Gram-type classification and pathogen identification.

ABSTRACT Septic arthritis (SA) is a rheumatologic emergency associated with significant morbidity and mortality. Delayed or inadequate treatment of SA can lead to irreversible joint destruction and disability. Current methods of diagnosing SA rely on synovial fluid analysis and culture which are known to be imprecise and time-consuming. We report a novel adaptation of a probe-based real-time PCR assay targeting the 16S rRNA gene for early and accurate diagnosis of bacterial SA. The assay algorithm consists of initial broad-range eubacterial detection, followed by Gram typing and species characterization of the pathogen. The platform demonstrated a high analytical sensitivity with a limit of detection of 101 CFU/ml with a panel of SA-related organisms. Gram typing and pathogen-specific probes correctly identified their respective targets in a mock test panel of 36 common clinically relevant pathogens. One hundred twenty-one clinical synovial fluid samples from patients presenting with suspected acute SA were tested. The sensitivity and specificity of the assay were 95% and 97%, respectively, versus synovial fluid culture results. Gram-typing probes correctly identified 100% of eubacterial positive samples as to gram-positive or gram-negative status, and pathogen-specific probes correctly identified the etiologic agent in 16/20 eubacterial positive samples. The total assay time from sample collection to result is 3 h. We have demonstrated that a real-time broad-based PCR assay has high analytical and clinical performance with an improved time to detection versus culture for SA. This assay may be a useful diagnostic adjunct for clinicians, particularly those practicing in the acute care setting where rapid pathogen detection and identification would assist in disposition and treatment decisions.

Trimethoprim–Sulfamethoxazole versus Placebo for Uncomplicated Skin Abscess

BACKGROUND U.S. emergency department visits for cutaneous abscess have increased with the emergence of methicillin-resistant Staphylococcus aureus (MRSA). The role of antibiotics for patients with a drained abscess is unclear. METHODS We conducted a randomized trial at five U.S. emergency departments to determine whether trimethoprim-sulfamethoxazole (at doses of 320 mg and 1600 mg, respectively, twice daily, for 7 days) would be superior to placebo in outpatients older than 12 years of age who had an uncomplicated abscess that was being treated with drainage. The primary outcome was clinical cure of the abscess, assessed 7 to 14 days after the end of the treatment period. RESULTS The median age of the participants was 35 years (range, 14 to 73); 45.3% of the participants had wound cultures that were positive for MRSA. In the modified intention-to-treat population, clinical cure of the abscess occurred in 507 of 630 participants (80.5%) in the trimethoprim-sulfamethoxazole group versus 454 of 617 participants (73.6%) in the placebo group (difference, 6.9 percentage points; 95% confidence interval [CI], 2.1 to 11.7; P=0.005). In the per-protocol population, clinical cure occurred in 487 of 524 participants (92.9%) in the trimethoprim-sulfamethoxazole group versus 457 of 533 participants (85.7%) in the placebo group (difference, 7.2 percentage points; 95% CI, 3.2 to 11.2; P<0.001). Trimethoprim-sulfamethoxazole was superior to placebo with respect to most secondary outcomes in the per-protocol population, resulting in lower rates of subsequent surgical drainage procedures (3.4% vs. 8.6%; difference, -5.2 percentage points; 95% CI, -8.2 to -2.2), skin infections at new sites (3.1% vs. 10.3%; difference, -7.2 percentage points; 95% CI, -10.4 to -4.1), and infections in household members (1.7% vs. 4.1%; difference, -2.4 percentage points; 95% CI, -4.6 to -0.2) 7 to 14 days after the treatment period. Trimethoprim-sulfamethoxazole was associated with slightly more gastrointestinal side effects (mostly mild) than placebo. At 7 to 14 days after the treatment period, invasive infections had developed in 2 of 524 participants (0.4%) in the trimethoprim-sulfamethoxazole group and in 2 of 533 participants (0.4%) in the placebo group; at 42 to 56 days after the treatment period, an invasive infection had developed in 1 participant (0.2%) in the trimethoprim-sulfamethoxazole group. CONCLUSIONS In settings in which MRSA was prevalent, trimethoprim-sulfamethoxazole treatment resulted in a higher cure rate among patients with a drained cutaneous abscess than placebo. (Funded by the National Institute of Allergy and Infectious Diseases; ClinicalTrials.gov number, NCT00729937.).