Comparison of a Novel Rapid Lateral Flow Assay to Enzyme Immunoassay Results for Early Diagnosis of Coccidioidomycosis.

BACKGROUND: Coccidioidomycosis (CM) is a common cause of community-acquired pneumonia where CM is endemic. Manifestations include self-limited pulmonary infection, chronic fibrocavitary pulmonary disease, and disseminated coccidioidomycosis. Most infections are identified by serological assays including enzyme-linked immunoassay (EIA), complement fixation, and immunodiffusion. These are time-consuming and take days to result, impeding early diagnosis. A new lateral flow assay (LFA; Sona; IMMY, Norman, OK) improves time-to-result to 1 hour. METHODS: We prospectively enrolled 392 patients with suspected CM, compared the LFA with standard EIA and included procalcitonin evaluation. RESULTS: Compared with standard EIA, LFA demonstrates 31% sensitivity (95% confidence interval [CI], 20-44%) and 92% specificity (95% CI, 88-95%). Acute pulmonary disease (74%) was the most common clinical syndrome. Hospitalized patients constituted 75% of subjects, and compared with outpatients, they more frequently had ≥3 previous healthcare facility visits (P = .05), received antibacterials (P < .01), and had >3 antibacterial courses (P < .01). Procalcitonin (PCT) was <0.25 ng/mL in 52 (83%) EIA-positive patients, suggesting infection was not bacterial. CONCLUSIONS: When CM is a possible diagnosis, LFA identified nearly one-third of EIA-positive infections. Combined with PCT <0.25 ng/mL, LFA could reduce unnecessary antibacterial use by 77%.

Interlaboratory agreement of coccidioidomycosis enzyme immunoassay from two different manufacturers.

Coccidioidomycosis, a fungal infection endemic to the Southwestern United States, is challenging to diagnose. The coccidioidomycosis enzyme immunoassay (EIA) test is the least expensive and simplest to perform to detect coccidioidomycosis antibodies in the serum. Concerns regarding falsely positive immunoglobulin (Ig) M EIA test results have led to questions about the agreement of commercially available EIA test kits among laboratories. We sought to evaluate the laboratory agreement of the EIA test at three laboratories using both IMMY and Meridian EIA test kits. Sensitivity and specificity of EIA IgM and IgG were calculated as secondary outcomes. The percent agreement of the EIA IgM and IgG test results among all three laboratories was 90% and 89% for IMMY test kits, respectively, and 67% and 80.5% for Meridian test kits, respectively. Agreement between IgM and IgG combined test results was 85.5% and 70.5%, for IMMY and Meridian, respectively. Combined IgM and IgG assays demonstrated a sensitivity of 68% (62.7%-76%) and a specificity of 99.3% (98%-100%) [IMMY] and a sensitivity of 72.4% (57.3%-87.3%) and a specificity of 91.3% (74%-100%) [Meridian]. In summary, results from the IMMY EIA test kit agreed more often across laboratories than Meridian EIA results, especially for the IgM assay. Isolated positive IgM EIA results using the Meridian test kit should be interpreted with caution and consideration of clinical information and test methodology. Further study of the sensitivity and specificity of coccidioidomycosis EIA test kits is warranted.

Designing Studies Acceptable for Abstraction and Inclusion in Evidence-Based Laboratory Practice Guidelines.

Expansion of technologies, changing infrastructure, and dwindling resources have produced the need for health care reform and changes in clinical laboratories. The health care model will have to shift increasingly from a fee-for-service model to a value-based model. Laboratories will have to focus more on evidence-based outcome studies evaluating the effects of their preanalytical and postanalytical practices on real patient outcomes. Although well-designed clinical trials and multicenter studies are needed to determine the effects of laboratory processes on outcomes, there has been concern that too few well-designed studies have been published. To help improve the quality of study design and to facilitate reporting transparency, several method statements have been developed. The Standards for Reporting of Diagnostic Accuracy Studies (STARD) initiative was recently updated, listing 30 items deemed crucial for transparent reporting of studies, thereby allowing the creation of a robust database for clinical practice guidelines. Three methods describing the assessment of the quality of data on which to base recommendations for such guidelines are also available. Close attention must be given to study design so that parameters ensuring study quality are met, thereby allowing inclusion of the study data in the formulation of evidence-based laboratory best practices guidelines.

Multicenter Clinical Validation of a Cartridge-Based Real-Time PCR System for Detection of Coccidioides spp. in Lower Respiratory Specimens.

Available methods for the diagnosis of coccidioidomycosis have significant shortcomings relative to accuracy and timeliness. We retrospectively and prospectively evaluated the diagnostic performance and reproducibility of a new cartridge-based real-time PCR assay for Coccidioides spp. directly in lower respiratory secretions and compared them to today's "gold standard," fungal culture. The GeneSTAT Coccidioides assay uses a 106-bp target sequence repeated multiple times (∼60×) per genome, thus lowering the limit of detection (LOD) for extracted DNA to 10 genome equivalents/ml. A total of 332 prospective and retrospective individual patient specimens were tested. The retrospective samples consisted of 100 bronchoalveolar lavage or bronchial wash (BAL/BW) (51 positive and 49 negative by culture) specimens that had been collected previously and stored at -70°C. These samples were tested by the GeneSTAT Coccidioides assay across three clinical test sites. The sensitivity was 100%, and the specificity ranged between 93.8% and 100%. There was minimal variance in the percent agreement across the three sites, 95.6% to 100%. Additionally, a total of 232 fresh (prospective) deidentified BAL/BW specimens were tested across the three clinical sites, which included a number of specimens from Southern California to provide a diversity of isolates. Specimens were tested by fungal culture, with any isolates of Coccidioides, except for one, being confirmed by molecular means (AccuProbe). The sensitivity of the GeneSTAT Coccidioides assay across the three sites was 100% (4/4) for positive fresh specimens, and the overall specificity of the assay was 99.6% (227/228), ranging from 98.1% to 100%. In testing for cross-reactivity, the assay was 100% specific when screened against 47 different bacterial, viral, and fungal species.

Aerobic Actinomycetes of Clinical Significance.

The group of Gram-positive bacillary organisms broadly known as "aerobic actinomycetes" consists of heterogeneous and taxonomically divergent genera. They are found in a wide variety of natural and man-made environments but are rarely considered a part of the normal human flora, with infections normally originating from exogenous sources. An extensive number of genera have been described, but only a minority of these has been associated with human or veterinary health. The association with human disease is usually of an opportunistic nature, either through accidental means of inoculation or through involvement with immunocompromising conditions in the host. They cause a wide spectrum of diseases in humans, which may differ greatly between the genera and even between species, but which also may have a great amount of overlap. The occurrence of such infections is probably greater than appreciated, since many may go unrecognized. Etiologic prevalence of specific genera and species varies geographically within the United States and worldwide. Traditional phenotypic identification methods for separation of the many genera and species of aerobic actinomycetes have found great difficulties. Recent use of chemotaxonomic analyses and emerging technologies such as molecular analysis of nucleic acids, and more recently proteomics for identification to the genus/species level, has provided a far more robust technique to understand the organisms' relatedness, distribution, epidemiology, and pathogenicity in humans.

Identification of mycobacterium spp. and nocardia spp. from solid and liquid cultures by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS).

Identification of microorganisms by MALDI-TOF MS has been widely accepted in clinical microbiology. However, for Mycobacterium spp. and Nocardia spp. such identification has not yet reached the optimal level of routine testing. Here we describe the development of an identification tool for 49 and 15 species of Mycobacterium spp. and Nocardia spp., respectively. During database construction, a number of ambiguous reference identifications were revealed and corrected via molecular analyses. Eventually, more than 2000 individual mass spectra acquired from 494 strains were included in a reference database and subjected to bio-statistical analyses. This led to correct species identification and correct combination of species into several complexes or groups, such as the Mycobacterium tuberculosis complex. With the Advanced Spectrum Classifier algorithm, class-specific bin weights were determined and tested by cross-validation experiments with good results. When challenged with independent isolates, overall identification performance was 90% for identification of Mycobacterium spp. and 88% for Nocardia spp. However, for a number of Mycobacterium sp. isolates, no identification could be achieved and in most cases, this could be attributed to the production of polymers that masked the species-specific protein peak patterns. For the species where >20 isolates were tested, correct identification reached 95% or higher. With the current spectral database, the identification of Mycobacterium spp. and Nocardia spp. by MALDI-TOF MS can be performed in routine clinical diagnostics although in some complicated cases verification by sequencing remains mandatory.

KlebSeq, a Diagnostic Tool for Surveillance, Detection, and Monitoring of Klebsiella pneumoniae.

Health care-acquired infections (HAIs) kill tens of thousands of people each year and add significantly to health care costs. Multidrug-resistant and epidemic strains are a large proportion of HAI agents, and multidrug-resistant strains of Klebsiella pneumoniae, a leading HAI agent, have caused an urgent public health crisis. In the health care environment, patient colonization by K. pneumoniae precedes infection, and transmission via colonization leads to outbreaks. Periodic patient screening for K. pneumoniae colonization has the potential to curb the number of HAIs. In this report, we describe the design and validation of KlebSeq, a highly informative screening tool that detects Klebsiella species and identifies clinically important strains and characteristics by using highly multiplexed amplicon sequencing without a live-culturing step. We demonstrate the utility of this tool on several complex specimen types, including urine, wound swabs and tissue, and several types of respiratory and fecal specimens, showing K. pneumoniae species and clonal group identification and antimicrobial resistance and virulence profiling, including capsule typing. Use of this amplicon sequencing tool to screen patients for Klebsiella carriage could inform health care staff of the risk of infection and outbreak potential. KlebSeq also serves as a model for next-generation molecular tools for public health and health care, as expansion of this tool can be used for several other HAI agents or applications.

Effectiveness of Practices To Increase Timeliness of Providing Targeted Therapy for Inpatients with Bloodstream Infections: a Laboratory Medicine Best Practices Systematic Review and Meta-analysis.

BACKGROUND: Bloodstream infection (BSI) is a major cause of morbidity and mortality throughout the world. Rapid identification of bloodstream pathogens is a laboratory practice that supports strategies for rapid transition to direct targeted therapy by providing for timely and effective patient care. In fact, the more rapidly that appropriate antimicrobials are prescribed, the lower the mortality for patients with sepsis. Rapid identification methods may have multiple positive impacts on patient outcomes, including reductions in mortality, morbidity, hospital lengths of stay, and antibiotic use. In addition, the strategy can reduce the cost of care for patients with BSIs. OBJECTIVES: The purpose of this review is to evaluate the evidence for the effectiveness of three rapid diagnostic practices in decreasing the time to targeted therapy for hospitalized patients with BSIs. The review was performed by applying the Centers for Disease Control and Prevention's (CDC's) Laboratory Medicine Best Practices Initiative (LMBP) systematic review methods for quality improvement (QI) practices and translating the results into evidence-based guidance (R. H. Christenson et al., Clin Chem 57:816-825, 2011, http://dx.doi.org/10.1373/clinchem.2010.157131). SEARCH STRATEGY: A comprehensive literature search was conducted to identify studies with measurable outcomes. A search of three electronic bibliographic databases (PubMed, Embase, and CINAHL), databases containing "gray" literature (unpublished academic, government, or industry evidence not governed by commercial publishing) (CIHI, NIHR, SIGN, and other databases), and the Cochrane database for English-language articles published between 1990 and 2011 was conducted in July 2011. DATES OF SEARCH: The dates of our search were from 1990 to July 2011. SELECTION CRITERIA: Animal studies and non-English publications were excluded. The search contained the following medical subject headings: bacteremia; bloodstream infection; time factors; health care costs; length of stay; morbidity; mortality; antimicrobial therapy; rapid molecular techniques, polymerase chain reaction (PCR); in situ hybridization, fluorescence; treatment outcome; drug therapy; patient care team; pharmacy service, hospital; hospital information systems; Gram stain; pharmacy service; and spectrometry, mass, matrix-assisted laser desorption-ionization. Phenotypic as well as the following key words were searched: targeted therapy; rapid identification; rapid; Gram positive; Gram negative; reduce(ed); cost(s); pneumoslide; PBP2; tube coagulase; matrix-assisted laser desorption/ionization time of flight; MALDI TOF; blood culture; EMR; electronic reporting; call to provider; collaboration; pharmacy; laboratory; bacteria; yeast; ICU; and others. In addition to the electronic search being performed, a request for unpublished quality improvement data was made to the clinical laboratory community. MAIN RESULTS: Rapid molecular testing with direct communication significantly improves timeliness compared to standard testing. Rapid phenotypic techniques with direct communication likely improve the timeliness of targeted therapy. Studies show a significant and homogeneous reduction in mortality associated with rapid molecular testing combined with direct communication. AUTHORS' CONCLUSIONS: No recommendation is made for or against the use of the three assessed practices of this review due to insufficient evidence. The overall strength of evidence is suggestive; the data suggest that each of these three practices has the potential to improve the time required to initiate targeted therapy and possibly improve other patient outcomes, such as mortality. The meta-analysis results suggest that the implementation of any of the three practices may be more effective at increasing timeliness to targeted therapy than routine microbiology techniques for identification of the microorganisms causing BSIs. Based on the included studies, results for all three practices appear applicable across multiple microorganisms, including methicillin-resistant Staphylococcus aureus (MRSA), methicillin-sensitive S. aureus (MSSA), Candida species, and Enterococcus species.

Mycobacterium haemophilum as a novel etiology of cervical lymphadenitis in an otherwise healthy adult patient.

We describe a case and summarize six additional cases of cervical lymphadenitis in otherwise healthy adults caused by Mycobacterium haemophilum. The organism causes cervicofacial lymphadenitis in healthy children and severe disease in immunocompromised patients but has not been previously reported to cause cervical lymphadenitis in nonimmunocompromised, healthy adults.

Transmission of coccidioidomycosis to a human via a cat bite.

We report an unusual case of coccidioidomycosis in the arm of a veterinary assistant without pulmonary symptoms. The patient had been bitten on the hand by a cat which was later diagnosed with disseminated disease. The patient responded to fluconazole therapy and remained asymptomatic at 2 months after cessation of therapy.

First report of cfr-mediated resistance to linezolid in human staphylococcal clinical isolates recovered in the United States.

Linezolid resistance has dominantly been mediated by mutations in 23S rRNA or ribosomal protein L4 genes. Recently, cfr has demonstrated the ability to produce a phenotype of resistance to not only oxazolidinones, but also other antimicrobial classes (phenicols, lincosamides, pleuromutilins, and streptogramin A). We describe the first detection of cfr-mediated linezolid resistance in Staphylococcus aureus and Staphylococcus epidermidis recovered from human infection cases monitored during the 2007 LEADER Program.

Genotype prevalence and risk factors for severe clinical adenovirus infection, United States 2004-2006.

BACKGROUND: Recently, epidemiological and clinical data have revealed important changes with regard to clinical adenovirus infection, including alterations in antigenic presentation, geographical distribution, and virulence of the virus. METHODS: In an effort to better understand the epidemiology of clinical adenovirus infection in the United States, we adopted a new molecular adenovirus typing technique to study clinical adenovirus isolates collected from 22 medical facilities over a 25-month period during 2004-2006. A hexon gene sequence typing method was used to characterize 2237 clinical adenovirus-positive specimens, comparing their sequences with those of the 51 currently recognized prototype human adenovirus strains. In a blinded comparison, this method performed well and was much faster than the classic serologic typing method. RESULTS: Among civilians, the most prevalent adenovirus types were types 3 (prevalence, 34.6%), 2 (24.3%), 1 (17.7%), and 5 (5.3%). Among military trainees, the most prevalent types were types 4 (prevalence, 92.8%), 3 (2.6%), and 21 (2.4%). CONCLUSIONS: For both populations, we observed a statistically significant increasing trend of adenovirus type 21 detection over time. Among adenovirus isolates recovered from specimens from civilians, 50% were associated with hospitalization, 19.6% with a chronic disease condition, 11% with a bone marrow or solid organ transplantation, 7.4% with intensive care unit stay, and 4.2% with a cancer diagnosis. Multivariable risk factor modeling for adenovirus disease severity found that age <7 years (odds ratio [OR], 3.2; 95% confidence interval [CI], 1.4-7.4), chronic disease (OR, 3.6; 95% CI, 2.6-5.1), recent transplantation (OR, 2.7; 95% CI, 1.3-5.2), and adenovirus type 5 (OR, 2.7; 95% CI, 1.5-4.7) or type 21 infection (OR, 7.6; 95% CI, 2.6-22.3) increased the risk of severe disease.

Laboratory aspects in the diagnosis of coccidioidomycosis.

Coccidioides immitis and Coccidioides posadasii, the two recognized causes of coccidioidomycosis, may be detected by direct microscopy, culture, and serologic documentation. Two useful stains include the Grocott methenamine silver (GMS) and the calcofluor white (CFW). Other useful stains used in histopathologic studies include hematoxylin-eosin (H&E) and periodic acid Schiff (PAS). Nucleic acid amplification tests (NAATs) have been introduced for detection of Coccidioides spp. in specimens, but are not yet commercially available. Isolation of Coccidioides spp. by culture is not difficult as many fungal as well as routine bacteriologic media are available. For the safe isolation of Coccidioides spp., the laboratory should maintain a biological safety level 2 or 3. Identification of Coccidioides spp. uses the organisms' phenotypic or genotypic characteristics. Phenotypic identification to genus level may be achieved by visualization of spherules in specimens and/or by the presence of arthroconidia in culture. Isolates may be confirmed as Coccidioides spp. by molecular probes. Separation of species into C. immitis and C. posadasii is best achieved by specialized molecular techniques which are not normally available in routine clinical laboratories. Humoral antibodies can be used for the diagnosis and prognosis of coccidioidomycosis. Although positive serologic results may be helpful in the diagnosis of coccidioidomycosis, negative serologic results cannot be used to rule out the disease. Enzyme immunoassays (EIA) and immunodiffusion methods are commonly used for detection of both IgM and IgG antibody groups. Sequential complement fixation (CF) studies for IgG class of antibody are useful for the prognosis of coccidioidomycosis.

Antimicrobial resistance: current status and future direction.

BACKGROUND: Antimicrobial resistance is initiated through mutations in bacterial genes, culminating in end products that help circumvent the action of specific antimicrobial agents. Resistant mutants can proliferate under a number of circumstances but primarily through the action of selective pressure from the overuse of antimicrobial agents. METHODS: The results of surveillance studies over approximately the last ten years were evaluated. CONCLUSION: Resistance rates in the group of microorganisms associated with respiratory tract infections had been increasing rapidly over the past 10 years, but, recently, many seem to have reached a plateau. However, newer, more invasive clones of methicillin resistant Staphylococcus aureus (MRSA), differing from health care-associated MRSA (HA-MRSA), and typically associated with community-acquisition (CA-MRSA), recently have begun to proliferate. Burgeoning use of fluoroquinolones has impacted the Gram-negative bacilli (e.g., Pseudomonas aeruginosa, Escherichia coli, and Salmonella), causing their resistance rates to approach the critical point. A better understanding of the epidemiology of resistance and responsible use of antimicrobial agents are mandatory if the continuing rates of increasing resistance are to be abrogated.