Group A Streptococcus Testing in Pediatrics: the Move to Point-of-Care Molecular Testing.

Each year, there are an estimated 11 million visits to ambulatory care centers for pharyngitis in children between the ages of 3 and 18 years. While there are many causes of pediatric pharyngitis, group A streptococcal pharyngitis represents 15 to 30% of infections and is the only cause for which treatment is recommended. Unfortunately, clinical suspicion is insufficient for the accurate diagnosis of group A streptococcal pharyngitis, and laboratory testing for confirmation of Streptococcus pyogenes infection is required to prevent complications of infection. Traditionally, throat swabs are inoculated onto agar plates for isolation of the large-zone beta-hemolytic streptococcus. However, traditional culture methods present a potential delay in treatment due to turnaround times of 18 to 48 h. In order to improve turnaround times and enhance antimicrobial stewardship, multiple point-of-care assays have been developed. This review describes current point-of-care testing for group A streptococcal pharyngitis, including rapid antigen detection tests and more recent molecular methods. Additional attention is given to the diagnostic considerations when choosing a method for group A streptococcal point-of-care testing, implementation of molecular group A streptococcal testing, and the institutional cost of immunoassays compared to those of newer molecular methods.

Multicenter evaluation of the RAPIDEC® CARBA NP assay for the detection of carbapenemase production in clinical isolates of Enterobacterales and Pseudomonas aeruginosa.

Carbapenem-resistant Gram-negative bacilli are a major public health problem. Accurate and rapid detection of carbapenemase-producing organisms can facilitate appropriate infection prevention measures. The objective was to evaluate the performance of the RAPIDEC® CARBA NP assay (RAPIDEC), a screening assay that utilizes a pH indicator to detect carbapenem hydrolysis within 2 h. A multicenter study evaluated 306 clinical bacterial strains of Enterobacterales (n = 257) and Pseudomonas aeruginosa (n = 49). The RAPIDEC was compared to a composite reference standard-the Clinical Laboratory Standards Institute (CLSI) Carba NP assay, PCR for specific carbapenemase genes (blaKPC, blaNDM, blaOXA-48-like, blaVIM and blaIMP), and phenotypic carbapenem susceptibility testing. The assay was evaluated using two culture incubation times for the bacterial isolates: "routine"(cultures incubated 18-24 h) and "short" (cultures incubated 4-5 h). For the routine incubation, the overall percent agreement was 98.7% with a positive percent agreement (PPA) of 99.6% and a negative percent agreement (NPA) of 97.4%; there were five false positives and one false negative. For the short incubation, the overall percent agreement was 98.0% with a PPA of 98.5% and a NPA of 97.3%; there were five false positives and four false negatives. RAPIDEC results for the P. aeruginosa isolates were 100% concordant with the reference standard for both incubation times. The RAPIDEC assay is an accurate and rapid (≤ 2 h) assay for the detection of the most common carbapenemases in clinical isolates. Growth from a short incubation culture may be used to reliably detect carbapenemase production in clinical strains.

Evaluation of telavancin susceptibility in isolates of Staphylococcus aureus with reduced susceptibility to vancomycin.

Historically, vancomycin has been considered a primary therapeutic option for treating infections with Staphylococcus aureus, but isolates with reduced vancomycin susceptibility (SA-RVS) (MIC ≥ 4 µg/mL) have emerged. Telavancin, a semisynthetic lipoglycopeptide, is an alternative treatment option for S. aureus, but data examining telavancin activity against SA-RVS are limited. In the present study, we characterize 300 isolates of S. aureus isolates (50 vancomycin-susceptible (VSSA) isolates and 250 SA-RVS isolates) from a large tertiary care, academic medical center, 51.8% of which were methicillin resistant (MRSA). Sixteen (6.4%) SA-RVS isolates were non-susceptible to telavancin, whereas all VSSA isolates were susceptible. Additionally, 3.6% of SA-RVS isolates were non-susceptible to daptomycin, with three (1.2%) isolates testing non-susceptible to both telavancin and daptomycin. When tested against other classes of antimicrobials, there were no statistical differences in susceptibility of VSSA and SA-RVS isolates, except for the fluoroquinolones (ciprofloxacin and moxifloxacin). Molecular characterization of the isolates showed that SCCmec types II and IV together represented over half of the SA-RVS isolates; 12.0% of the VSSA isolates were SCCmec type II. Using RepPCR, we detected 16 distinct strain types in this isolate collection, and tst-1 (gene encoding the Staphylococcus toxic shock syndrome super-antigen) carriage was low (5.4%). Overall, we show that in addition to reduced vancomycin susceptibility, a small, but clinically significant, proportion of SA-RVS isolates also demonstrate reduced susceptibility to both telavancin and daptomycin.

In vitro analysis of growth patterns of invasive fungal species on commonly used endonasal hemostatic agents.

OBJECTIVE: Previous studies have not examined the potential role of endonasal hemostatic agents in facilitating growth of fungal species. We aim to determine the possibility of these to serve as a nutrient source for fungal growth. METHODS: Cultures of Aspergillus, Fusarium, and Mucor were harvested and placed in solution in sterile saline at standardized high and low concentrations. Thrombin gelatin matrix, carboxyl methylcelluose, and potato starch derivative agents were prepared following manufacturer instructions and applied to two separate Petri dishes per agent. Each substrate was then inoculated with either high or low concentrations of fungal species. Negative and positive control plates with each organism were included. Dishes were sealed, incubated, and examined daily for fourteen days for microscopic and macroscopic growth. RESULTS: Thrombin gelatin matrix was relatively resilient to growth, although Fusarium growth was noted on all packing material by day three. Carboxyl methylcellulose also supported growth of high-concentration Mucor appreciated on day five. The potato starch derivative supported fulminant growth of all fungal species. CONCLUSIONS: Endonasal hemostatic agents may be nutrient sources that facilitate growth of fungal species. This may be a consideration in a surgeon's decision to use a hemostatic agent. Prompt initial post-operative debridement may be warranted in select patients. Our findings serve as a model for further testing of fungal growth on other hemostatic materials. Future studies are needed to confirm the clinical significance of these findings in vivo.

Impact of total laboratory automation on workflow and specimen processing time for culture of urine specimens.

Total laboratory automation (TLA) has the potential to reduce specimen processing time, improve standardization of cultures, and decrease turnaround time (TAT). The objective of this study was to perform a detailed interrogation of the impact of TLA implementation in all aspects of the workflow for routine culture of urine specimens. Using a detailed motion capture study, the time required for major steps of processing and result reporting were prospectively assessed for urine samples prior to (n = 215) and after (n = 203) implementation of the BD Kiestra TLA system. Specimens were plated on all shifts, but cultures were read only during the day shift for both time periods. Significant increases were noted in the time from receipt to inoculation (23.0 min versus 32.0 min, p < 0.001) and total processing time (28.0 min versus 66.0 min, p < 0.0001) for urine specimens post-TLA. Rates of positive (18.6% versus 16.3%) and negative (71.2% versus 79.3%) urine cultures remained stable through the pre- and post-TLA time periods (p = 0.58). There were no changes in TAT for organism identification or susceptibility results. The time to final report was decreased from 43.8 h pre-TLA to 42.0 h post-TLA, which was attributed to significant decreases in TAT for negative cultures (42.0 h versus 37.5 h, p = 0.01). These findings demonstrate that changes in laboratory workflow are necessary to maximize efficiency of TLA and optimize TAT.

When Good Bugs Go Bad: Epidemiology and Antimicrobial Resistance Profiles of Corynebacterium striatum, an Emerging Multidrug-Resistant, Opportunistic Pathogen.

Infections with Corynebacterium striatum have been described in the literature over the last 2 decades, with the majority being bacteremia, central line infections, and occasionally, endocarditis. In recent years, the frequency of C. striatum infections appears to be increasing; a factor likely contributing to this is the increased ease and accuracy of the identification of Corynebacterium spp., including C. striatum, from clinical cultures. The objective of this study was to retrospectively characterize C. striatum isolates recovered from specimens submitted as part of routine patient care at a 1,250-bed, tertiary-care academic medical center. Multiple strain types were recovered, as demonstrated by repetitive-sequence-based PCR. Most of the strains of C. striatum characterized were resistant to antimicrobials commonly used to treat Gram-positive organisms, such as penicillin, ceftriaxone, meropenem, clindamycin, and tetracycline. The MIC50 for ceftaroline was >32 µg/ml. Although there are no interpretive criteria for susceptibility with telavancin, it appeared to have potent in vitro efficacy against this species, with MIC50 and MIC90 values of 0.064 and 0.125 µg/ml, respectively. Finally, as previously reported in case studies, we demonstrated rapid in vitro development of daptomycin resistance in 100% of the isolates tested (n = 50), indicating that caution should be exhibited when using daptomycin for the treatment of C. striatum infections. C. striatum is an emerging, multidrug-resistant pathogen that can be associated with a variety of infection types.

Evaluation of Genotypic and Phenotypic Methods to Detect Carbapenemase Production in Gram-Negative Bacilli.

BACKGROUND: Carbapenemase-producing gram-negative bacteria (CP-GNB) are an urgent and expanding public health threat. Rapid and accurate identification of these organisms facilitates infection prevention efforts in healthcare facilities. The objective of our study was to evaluate methods to detect and identify CP-GNB. METHODS: We examined 189 carbapenem-resistant GNB(CR-GNB), including Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii complex, using 3 different methods: 2 methods to screen isolates of GNB for carbapenemase production [the carbapenem inactivation method (CIM) and 2 chromogenic agars] and a molecular method (Cepheid GeneXpert Carba-R) to identify the mechanism of carbapenem resistance and the associated resistance genes (blaKPC, blaNDM, blaIMP, blaOXA-48-like, and blaVIM). RESULTS: The CIM was a simple and inexpensive phenotypic screen to differentiate between CR-GNB and CP-GNB, with improved analytical performance characteristics and inter-reader correlation compared to the modified Hodge test. Both chromogenic agars evaluated (HardyCHROM CRE and chromID CARBA) were able to support growth of most of the organisms tested, including isolates possessing the blaOXA-48-like gene. However, these media had a low analytical specificity for carbapenemase production, with breakthrough of CR-GNB that did not produce a carbapenemase. The Xpert Carba-R assay was rapid and easy to perform, and demonstrated 100% positive and negative agreement for characterization of genetic determinants of carbapenem resistance. CONCLUSIONS: Screening by CIM followed by the Xpert Carba-R PCR is an accurate method for detecting and characterizing CP-GNB, including Enterobacteriaceae, P. aeruginosa, and A. baumannii complex.

Evaluation of the Vitek MS Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry System for Identification of Clinically Relevant Filamentous Fungi.

Invasive fungal infections have a high rate of morbidity and mortality, and accurate identification is necessary to guide appropriate antifungal therapy. With the increasing incidence of invasive disease attributed to filamentous fungi, rapid and accurate species-level identification of these pathogens is necessary. Traditional methods for identification of filamentous fungi can be slow and may lack resolution. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has emerged as a rapid and accurate method for identification of bacteria and yeasts, but a paucity of data exists on the performance characteristics of this method for identification of filamentous fungi. The objective of our study was to evaluate the accuracy of the Vitek MS for mold identification. A total of 319 mold isolates representing 43 genera recovered from clinical specimens were evaluated. Of these isolates, 213 (66.8%) were correctly identified using the Vitek MS Knowledge Base, version 3.0 database. When a modified SARAMIS (Spectral Archive and Microbial Identification System) database was used to augment the version 3.0 Knowledge Base, 245 (76.8%) isolates were correctly identified. Unidentified isolates were subcultured for repeat testing; 71/319 (22.3%) remained unidentified. Of the unidentified isolates, 69 were not in the database. Only 3 (0.9%) isolates were misidentified by MALDI-TOF MS (including Aspergillus amoenus [n = 2] and Aspergillus calidoustus [n = 1]) although 10 (3.1%) of the original phenotypic identifications were not correct. In addition, this methodology was able to accurately identify 133/144 (93.6%) Aspergillus sp. isolates to the species level. MALDI-TOF MS has the potential to expedite mold identification, and misidentifications are rare.

Comparison of Sample Preparation Methods, Instrumentation Platforms, and Contemporary Commercial Databases for Identification of Clinically Relevant Mycobacteria by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry.

When mycobacteria are recovered in clinical specimens, timely species-level identification is required to establish the clinical significance of the isolate and facilitate optimization of antimicrobial therapy. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has recently been reported to be a reliable and expedited method for identification of mycobacteria, although various specimen preparation techniques and databases for analysis are reported across studies. Here we compared two MALDI-TOF MS instrumentation platforms and three databases: Bruker Biotyper Real Time Classification 3.1 (Biotyper), Vitek MS Plus Saramis Premium (Saramis), and Vitek MS v3.0. We evaluated two sample preparation techniques and demonstrate that extraction methods are not interchangeable across different platforms or databases. Once testing parameters were established, a panel of 157 mycobacterial isolates (including 16 Mycobacterium tuberculosis isolates) was evaluated, demonstrating that with the appropriate specimen preparation, all three methods provide reliable identification for most species. Using a score cutoff value of ≥1.8, the Biotyper correctly identified 133 (84.7%) isolates with no misidentifications. Using a confidence value of ≥90%, Saramis correctly identified 134 (85.4%) isolates with one misidentification and Vitek MS v3.0 correctly identified 140 (89.2%) isolates with one misidentification. The levels of accuracy were not significantly different across the three platforms (P = 0.14). In addition, we show that Vitek MS v3.0 requires modestly fewer repeat analyses than the Biotyper and Saramis methods (P = 0.04), which may have implications for laboratory workflow.

Brown-Pigmented Mycobacterium mageritense as a Cause of Prosthetic Valve Endocarditis and Bloodstream Infection.

Mycobacterium spp. are a rare cause of endocarditis. Herein, we describe a case of Mycobacterium mageritense prosthetic valve endocarditis. This organism produced an unusual brown pigment on solid media. Cultures of valve tissue for acid-fast bacilli might be considered in some cases of apparently culture-negative prosthetic valve endocarditis.

Continued evolution of West Nile virus, Houston, Texas, USA, 2002-2012.

We investigated the genetics and evolution of West Nile virus (WNV) since initial detection in the United States in 1999 on the basis of continual surveillance studies in the Houston, Texas, USA, metropolitan area (Harris County) as a surrogate model for WNV evolution on a national scale. Full-length genomic sequencing of 14 novel 2010-2012 WNV isolates collected from resident birds in Harris County demonstrates emergence of 4 independent genetic groups distinct from historical strains circulating in the greater Houston region since 2002. Phylogenetic and geospatial analyses of the 2012 WNV isolates indicate closer genetic relationship with 2003-2006 Harris County isolates than more recent 2007-2011 isolates. Inferred monophyletic relationships of these groups with several 2006-2009 northeastern US isolates supports potential introduction of a novel WNV strain in Texas since 2010. These results emphasize the need to maintain WNV surveillance activities to better understand WNV transmission dynamics in the United States.

Molecular evolution of lineage 2 West Nile virus.

Since the 1990s West Nile virus (WNV) has become an increasingly important public health problem and the cause of outbreaks of neurological disease. Genetic analyses have identified multiple lineages with many studies focusing on lineage 1 due to its emergence in New York in 1999 and its neuroinvasive phenotype. Until recently, viruses in lineage 2 were not thought to be of public health importance due to few outbreaks of disease being associated with viruses in this lineage. However, recent epidemics of lineage 2 in Europe (Greece and Italy) and Russia have shown the increasing importance of this lineage. There are very few genetic studies examining isolates belonging to lineage 2. We have sequenced the full-length genomes of four older lineage 2 WNV isolates, compared them to 12 previously published genomic sequences and examined the evolution of this lineage. Our studies show that this lineage has evolved over the past 300-400 years and appears to correlate with a change from mouse attenuated to virulent phenotype based on previous studies by our group. This evolution mirrors that which is seen in lineage 1 isolates, which have also evolved to a virulent phenotype over the same period of time.

Evolution of new genotype of West Nile virus in North America.

Previous studies of North American isolates of West Nile virus (WNV) during 1999-2005 suggested that the virus had reached genetic homeostasis in North America. However, genomic sequencing of WNV isolates from Harris County, Texas, during 2002-2009 suggests that this is not the case. Three new genetic groups have been identified in Texas since 2005. Spread of the southwestern US genotype (SW/WN03) from the Arizona/Colorado/northern Mexico region to California, Illinois, New Mexico, New York, North Dakota, and the Texas Gulf Coast demonstrates continued evolution of WNV. Thus, WNV continues to evolve in North America, as demonstrated by selection of this new genotype. Continued surveillance of the virus is essential as it continues to evolve in the New World.

Revisiting the "leading edge" of hospital privacy curtains in the medical intensive care unit.

BACKGROUND: Privacy curtains within medical intensive care unit (MICU) rooms are a potential contributor to health care associated infections. The "leading edge" of a hospital curtain, estimated to be the edge most frequently touched, likely plays a role in health care associated infections at hospitals. The aims of this study were to (1) compare the bacterial load of the edge vs the middle of curtains in the MICU, and (2) determine the identity and distribution of relevant pathogens colonizing them. METHODS: The edge and middle sections of 8 curtains in MICU rooms (4 contact precaution and 4 noncontact precaution) were sampled for culture on patient and staff sides. Bacterial loads of edges and middles were compared. Select isolates were further analyzed for species identification. RESULTS: There was a statistically significant difference for the contact (t = 2.10, P = .047) and noncontact (t = 2.62, P = .016) rooms, with the edges having a significantly higher median than the middles. Pathogens such as methicillin-resistant Staphylococcus aureus, Enterococcus, Klebsiella, and Acinetobacter were found on the curtains, though at lower rates than in previous studies. Opportunistic fungi were also found on all curtains. CONCLUSIONS: Results of this study confirm that hospital curtains, most notably the edge but also the middle, are contaminated with pathogens, and that these areas are frequently touched by health care workers in between hand hygiene.

Repeat Molecular Testing for Respiratory Pathogens: Diagnostic Gain or Diminishing Returns?

BACKGROUND: Upper respiratory tract infections are common, and the ability to accurately and rapidly diagnose the causative pathogen has important implications for patient management. METHODS: We evaluated the test-ordering practices for 2 commonly utilized nucleic acid amplification tests (NAATs) for the detection of respiratory pathogens: the Xpert Flu Assay for influenza A/B (Flu assay) and the Biofire FilmArray respiratory panel assay (RP assay), which detects 20 different targets. Our study examined repeat testing; that is, testing within 7 days from an initial test. RESULTS: Our study found that repeat testing is common for each of the individual assays: 3.0% of all Flu assays and 10.0% of all RP assays were repeat testing. Of repeat testing, 8/293 (2.7%) of repeat Flu assays and 75/1257 (6.0%) of RP assays resulted diagnostic gains, i.e., new detections. However, for the RP assay, these new detections were not always clinically actionable. The most frequently discrepant organisms were rhinovirus/enterovirus (28/102, 27.5%), followed by respiratory syncytial virus (12/102, 11.8%) and coronavirus OC43 (11/102, 10.8%). Furthermore, there were 3,336 instances in which a patient was tested using both a Flu assay and RP assay, of which only 44 (1.3%) had discrepant influenza results. CONCLUSIONS: Our findings suggest opportunities exist to better guide ordering practices for respiratory pathogen testing, including limiting repeat testing, with the goal of optimization of clinical yield, and diagnostic stewardship.

Use of electrocautery does not diminish the transmission rate of Cutibacterium acnes compared to a scalpel blade.

Many methods are used during shoulder surgery to prevent wound contamination with Cutibacterium acnes, but there are no accepted standards for prevention. Some surgeons use an electrosurgical instrument instead of a scalpel blade during open shoulder surgery in an effort to prevent deep tissue contamination with C. acnes. We sought to compare the transference rate of C. acnes between a scalpel blade at room temperature and an electrosurgical blade heated to 41°C (temperature of electrosurgical blade after standard deltopectoral approach). In our model, using a scalpel blade versus a heated electrosurgical blade resulted in no difference in pathogen transference.

Complete Genome Sequences of Mycobacteriophages Clautastrophe, Kingsolomon, Krypton555, and Nicholas.

We report here the complete genome sequences of four subcluster L3 mycobacteriophages newly isolated from soil samples, using Mycobacterium smegmatis mc2155 as the host. Comparative genomic analyses with four previously described subcluster L3 phages reveal strong nucleotide similarity and gene conservation, with several large insertions/deletions near their right genome ends.

Pathology Consultation on Influenza Diagnostics.

OBJECTIVES: To describe the strengths and limitations of the available influenza diagnostics, with a focus on rapid antigen detection assays and nucleic acid detection assays. METHODS: A case-based presentation is used to illustrate the potential limitations of rapid antigen detection assays for influenza. RESULTS: Influenza is a seasonal illness; estimates attribute influenza to approximately 200,000 hospitalizations and 41,000 deaths in the United States annually. Antigen detection assays for influenza are rapid and convenient, and thus are widely used in a variety of health care settings, even though the sensitivity of these assays may be suboptimal. The United States Food and Drug Administration has recently created new guidelines intended to improve the oversight and performance characteristics of influenza antigen detection assays. Molecular assays, although more costly and complex, are more sensitive and may be designed to simultaneously detect multiple respiratory pathogens within a single assay. CONCLUSIONS: Diagnostic assays for influenza can vary greatly with regards to analytical performance characteristics, complexity, turnaround time and cost. This can have important patient care and infection prevention implications.