Are Rapid Influenza Antigen Tests Still Clinically Useful in Today's Molecular Diagnostics World?

Influenza virus infection and disease historically contribute to widespread cases of seasonal morbidity and in some cases mortality. Prompt and accurate diagnosis is crucial for optimal patient management. Rapid influenza direct antigen testing (RIDT) offers a faster turn-around-time for results but test performance (ie, sensitivity and specificity) varies widely. Nucleic acid amplification testing (NAAT) can offer a viable alternative. The objective of this retrospective study was to compare the test performance of RIDT with NAAT. RIDT testing included the Directigen EZ Flu A+B or the Veritor System for Rapid Detection of Flu A+B. NAAT employed the SimplexaTM Flu A/B™ RSV assay. A total of 5,795 specimens collected from October to March for the 2012/2013 (n=953), 2013/2014 (n=2060) and 2014/2015 (n=2783) seasons were co-tested by RIDT and NAAT. Using NAAT as the gold standard, RIDT tests had a sensitivity range of 0 to 15.7% and a specificity of 98.2 to 100% for influenza type A. For influenza type B, RIDT tests had a sensitivity of 0 to 33.3% and a specificity of 98.9 to 100%. These findings suggest that RIDT has unacceptably low sensitivity for both influenza A and influenza B, despite high specificity. The key advantage of RIDT in previous years (faster turnaround time) has been challenged by newer NAAT technology that provides results in a turn-around-time comparable to RIDT, but with superior test performance.

Increasing Prevalence of Nontuberculous Mycobacteria in Respiratory Specimens from US-Affiliated Pacific Island Jurisdictions1.

Nontuberculous mycobacteria (NTM) respiratory infections represent a growing public health problem in many countries. However, there are limited published epidemiologic studies for the Western Pacific region. We reviewed respiratory specimens submitted to Diagnostic Laboratory Services in Hawaii, USA, for culture of Mycobacterium tuberculosis during August 2007-December 2011 to determine the NTM isolation rate. We observed a statistically significant increase in the rate of specimens with NTM isolated in respiratory culture (adjusted rate ratio per year 1.65, 95% CI 1.54-1.77; p<0.01). In contrast, the number of patients with respiratory cultures positive for M. tuberculosis showed no increase (adjusted rate ratio per year 0.98, 95% CI 0.94-1.01; p = 0.19). A 6-month subset of NTM isolates was identified by using a nucleic acid probe or 16S rRNA sequencing. M. avium complex and M. fortuitum were the most common NTM identified.

Correction: Environmental Nontuberculous Mycobacteria in the Hawaiian Islands.

[This corrects the article DOI: 10.1371/journal.pntd.0005068.].

Complete Genome Sequence of Mycobacterium chimaera Strain AH16.

Mycobacterium chimaera is a nontuberculous mycobacterial species that causes cardiovascular, pulmonary, and postsurgical infections. Here, we report the first complete genome sequence of M. chimaera This genome is 6.33 Mbp, with a G+C content of 67.56%, and encodes 4,926 protein-coding genes, as well as 74 tRNAs, one ncRNA, and three rRNA genes.

Environmental Nontuberculous Mycobacteria in the Hawaiian Islands.

Lung disease caused by nontuberculous mycobacteria (NTM) is an emerging infectious disease of global significance. Epidemiologic studies have shown the Hawaiian Islands have the highest prevalence of NTM lung infections in the United States. However, potential environmental reservoirs and species diversity have not been characterized. In this cross-sectional study, we describe molecular and phylogenetic comparisons of NTM isolated from 172 household plumbing biofilms and soil samples from 62 non-patient households and 15 respiratory specimens. Although non-uniform geographic sampling and availability of patient information were limitations, Mycobacterium chimaera was found to be the dominant species in both environmental and respiratory specimens. In contrast to previous studies from the continental U.S., no Mycobacterium avium was identified. Mycobacterium intracellulare was found only in respiratory specimens and a soil sample. We conclude that Hawai'i's household water sources contain a unique composition of Mycobacterium avium complex (MAC), increasing our appreciation of NTM organisms of pulmonary importance in tropical environments.

Rapid Antimicrobial Susceptibility Testing Using Forward Laser Light Scatter Technology.

The delayed reporting of antimicrobial susceptibility testing remains a limiting factor in clinical decision-making in the treatment of bacterial infection. This study evaluates the use of forward laser light scatter (FLLS) to measure bacterial growth for the early determination of antimicrobial susceptibility. Three isolates each (two clinical isolates and one reference strain) of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa were tested in triplicate using two commercial antimicrobial testing systems, the Vitek2 and the MicroScan MIC panel, to challenge the BacterioScan FLLS. The BacterioScan FLLS showed a high degree of categorical concordance with the commercial methods. Pairwise comparison with each commercial system serving as a reference standard showed 88.9% agreement with MicroScan (two minor errors) and 72.2% agreement with Vitek (five minor errors). FLLS using the BacterioScan system shows promise as a novel method for the rapid and accurate determination of antimicrobial susceptibility.

Bordetella trematum sepsis with shock in a diabetic patient with rapidly developing soft tissue infection.

Bordetella is a gram-negative, glucose non-fermenting bacillus, consisting of many host-associated species. B. trematum has previously been identified in wound infections, but rarely known to be a source of bacteremia. Currently, 16S rRNA sequencing represents the reference standard method by which identification is made. Herein, we present a case of fatal B. trematum bacteremia with septic shock. The presumed primary site of the infection was a rapidly developing left leg deep soft tissue infection without necrotizing fasciitis. B. trematum should now be considered as a significant pathogen in sepsis.

Multicenter evaluation of the BioFire FilmArray gastrointestinal panel for etiologic diagnosis of infectious gastroenteritis.

The appropriate treatment and control of infectious gastroenteritis depend on the ability to rapidly detect the wide range of etiologic agents associated with the disease. Clinical laboratories currently utilize an array of different methodologies to test for bacterial, parasitic, and viral causes of gastroenteritis, a strategy that suffers from poor sensitivity, potentially long turnaround times, and complicated ordering practices and workflows. Additionally, there are limited or no testing methods routinely available for most diarrheagenic Escherichia coli strains, astroviruses, and sapoviruses. This study assessed the performance of the FilmArray Gastrointestinal (GI) Panel for the simultaneous detection of 22 different enteric pathogens directly from stool specimens: Campylobacter spp., Clostridium difficile (toxin A/B), Plesiomonas shigelloides, Salmonella spp., Vibrio spp., Vibrio cholerae, Yersinia enterocolitica, enteroaggregative E. coli, enteropathogenic E. coli, enterotoxigenic E. coli, Shiga-like toxin-producing E. coli (stx1 and stx2) (including specific detection of E. coli O157), Shigella spp./enteroinvasive E. coli, Cryptosporidium spp., Cyclospora cayetanensis, Entamoeba histolytica, Giardia lamblia, adenovirus F 40/41, astrovirus, norovirus GI/GII, rotavirus A, and sapovirus. Prospectively collected stool specimens (n = 1,556) were evaluated using the BioFire FilmArray GI Panel and tested with conventional stool culture and molecular methods for comparison. The FilmArray GI Panel sensitivity was 100% for 12/22 targets and ≥94.5% for an additional 7/22 targets. For the remaining three targets, sensitivity could not be calculated due to the low prevalences in this study. The FilmArray GI Panel specificity was ≥97.1% for all panel targets. The FilmArray GI Panel provides a comprehensive, rapid, and streamlined alternative to conventional methods for the etiologic diagnosis of infectious gastroenteritis in the laboratory setting. The potential advantages include improved performance parameters, a more extensive menu of pathogens, and a turnaround time of as short as 1 h.

Septic arthritis and osteomyelitis due to Bordetella petrii.

A case of Bordetella petrii septic arthritis and osteomyelitis in an elbow resulted from a dirt bike accident in Hawaii. Two months of intravenous antibiotics and repeated surgeries were required to cure this infection. Our case, and literature review, suggests that extended-spectrum penicillins, tetracycline, and trimethoprim-sulfamethoxazole are good treatment options.

Culture-negative prosthetic valve endocarditis with concomitant septicemia due to a nontoxigenic Corynebacterium diphtheriae biotype gravis isolate in a patient with multiple risk factors.

A 54-year-old female with a prosthetic mitral valve presented with a 3-day history of dizziness, subjective fever, and chills. Blood cultures were positive for a pleomorphic Gram-positive rod. Initial phenotypic testing could only support the identification of a Corynebacterium species. Nucleic acid sequencing (16S rRNA) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) were conclusive for Corynebacterium diphtheriae. Definitive phenotypic testing classified the strain as nontoxigenic C. diphtheriae biotype Gravis.

Comparison of two multiplexed PCR assays for the detection of HSV-1, HSV-2, and VZV with extracted and unextracted cutaneous and mucosal specimens.

BACKGROUND: Several analyte specific reagents (ASRs) are available for the detection and differentiation of HSV-1, HSV-2, and VZV in clinical specimens. However, there is limited data on the test performance of these reagents used in multiplexed PCR assays. OBJECTIVE: This study compared the performance of two multiplexed ASR sets for detection of HSV-1, HSV-2, and VZV in dermal specimens. STUDY DESIGN: Two commercially available ASRs were combined to produce multiplexed PCR assays for simultaneous detection of HSV-1, HSV-2, and VZV. Seeded samples were used to determine the limit of detection (LOD) for each assay. Patient samples (n=156) were tested in duplicate and results for each method compared to the reference standard of culture. Both extracted and unextracted specimens were used in the study. RESULTS: Both multiplexed PCR assays showed similar test performance, with minimal LOD differences observed. The LOD was 10(3) copies/mL for HSV-1 and HSV-2 using the Focus assay compared to 5×10(3) copies/mL and 2×10(4) copies/mL, respectively for the EraGen assay. Both assays showed equal performance for VZV with a LOD of 5×10(3) copies/mL. Analytical specificity testing showed no cross reactivity with other selected DNA viruses. Both assays showed similar performance when clinical samples were tested using both extracted and unextracted specimens. CONCLUSION: Commercially available ASRs can be successfully multiplexed for the PCR detection of HSV-1, HSV-2, and VZV using dermal specimens. Either direct testing or nucleic acid extracted specimens can be used with similar performance in these assays.

A Lethal Case of Pseudomonas putida Bacteremia Due to Soft Tissue Infection.

Pseudomonas putida is an uncommon cause of skin and soft tissue infections. It is often associated with trauma or immunocompromised state. We present the first lethal case of bacteremia due to skin and soft tissue infections, which had malnutrition, immobility, and peripheral vascular disease as risk factors.

Comparison of two broadly multiplexed PCR systems for viral detection in clinical respiratory tract specimens from immunocompromised children.

BACKGROUND: The detection of viral respiratory tract infections has evolved greatly with the development of PCR based commercial systems capable of simultaneously detecting a wide variety of pathogens. OBJECTIVES: Evaluate the relative performance of two commercial broad range systems for the detection of viral agents in clinical respiratory tract specimens from immunocompromised children. STUDY DESIGN: A total of 176 patient samples were included in the analysis, representing only the first sample collected for each patient, and excluding failed reactions. Samples were de-identified and assayed in parallel using two different, broadly multiplexed PCR systems: ResPlex™ II Panel v2.0 (ResPlex), Qiagen, Hilden, Germany and FilmArray(®) Respiratory Panel (FilmArray), Idaho Technology Inc., Salt Lake City, UT. Method comparison was based upon pair-wise concordance of results according to patient age, viral target and number of targets detected. RESULTS: The two systems showed an overall concordance, by patient, of 83.8% (p=0.0001). FilmArray detected at least one target in 68.8% of samples, while ResPlex detected at least one target in 56.8%. ResPlex failed to detect 20.7% of FilmArray positives, and FilmArray failed to detect 4% of ResPlex positives. The relative performance of each system (including which system detected a higher number of positive samples) varied when stratified by target viral pathogen. CONCLUSIONS: Broadly multiplexed PCR is an effective means of detecting large numbers of clinically relevant respiratory viral pathogens.

Native valve Bacillus cereus endocarditis in a non-intravenous-drug-abusing patient.

Bacillus cereus is a rare cause of endocarditis, typically associated with intravenous drug abuse, rheumatic heart disease, prosthetic heart valves, pacemakers, or immunodeficiency. We present the first case of native valve Bacillus cereus endocarditis with no apparent risk factors. The patient had a fulminant course requiring emergent valve replacement.

A report on the first case of vancomycin-intermediate Staphylococcus aureus (VISA) in Hawai'i.

The state of Hawai'i has the highest prevalence of methicillin-resistant Staphylococcus aureus (MRSA) infection in the United States. Since vancomycin is the most frequently-prescribed antibiotic for healthcare-associated MRSA infection, there is concern for development of vancomycin resistance. We report on a 61 year-old woman with history of previous successful treatments of MRSA bacteremia with vancomycin. She was later hospitalized for catheter-related MRSA bacteremia that persisted despite vancomycin treatment. The vancomycin minimal inhibitory concentration (MIC) was initially 1-2 µg/ml, suggesting susceptibility, but changed to 4 µg/ml. At this level, the organism was classified as a vancomycin-intermediate Staphylococcus aureus (VISA). Therapy was changed from vancomycin to daptomycin, and the patient's blood cultures were sterilized. High suspicion of VISA should be raised in MRSA-infected patients who fail or have a history of vancomycin therapy so that additional susceptibility testing and appropriate antibiotic therapy can be promptly commenced to reduce the morbidity associated with VISA infection.

A probe-based method for confirmation of methicillin-resistant Staphylococcus aureus and detection of Panton-Valentine leukocidin and tst virulence genes.

Probe-based detection of mecA, lukS/F-PV (Panton-Valentine leukocidin), and tst virulence genes in 435 isolates of Staphylococcus aureus had comparable sensitivity and specificity to end-point polymerase chain reaction as a reference standard.

Increasing prevalence of nasal and rectal colonization with methicillin-resistant Staphylococcus aureus in children with cancer.

BACKGROUND: Infections with methicillin-resistant Staphylococcus aureus (MRSA), in community-settings, especially with strains carrying the Panton-Valentine Leukocidin (PVL) genes, have increased markedly in recent years. Colonization with S. aureus is a risk factor for infection. However, there are few studies that examine colonization and infection with PVL-positive strains of MRSA in cancer patients. PROCEDURE: The epidemiology of colonization and infection with MRSA was studied in children with cancer during two time periods: 2000/2001 and 2006/2007. PVL genes were screened and spa typing performed on the isolates. RESULTS: The prevalence of colonization with MRSA increased from 0.6% in 2000/2001 to 2.9% in 2006/2007 (P = 0.0003). MRSA colonization at admission was associated with infection (P < 0.0001; RR 38.32; 95% CI: 23.36-62.84). The prevalence of infection increased from 0.99% in 2000/2001 to 3.78% in 2006-2007 (P = 0.0002). Of the 32 colonized patients, 18 (56%) had infection. None of the 14 colonized but non-infected patients had dual colonization of nares and rectum, while 8 of the 18 infected patients had colonization of both of these sites (P = 0.004). Ten patients (31%) were colonized with PVL-positive strains. Patients colonized with PVL-positive strains were more likely to be colonized both in the nares and rectum (P = 0.005), and more likely to have infection (P = 0.001). Recurrent MRSA infections were seen in 22% of patients. CONCLUSION: An increasing prevalence of colonization with MRSA was observed in children with cancer at our institution. Colonization with MRSA especially with PVL-positive strains was associated with infection.

Pandemic preparedness in Hawaii: a multicenter verification of real-time RT-PCR for the direct detection of influenza virus types A and B.

OBJECTIVE: We integrated multicenter, real-time (RTi) reverse transcription polymerase chain reaction (RT-PCR) screening into a statewide laboratory algorithm for influenza surveillance and response. METHODS: Each of three sites developed its own testing strategy and was challenged with one randomized and blinded panel of 50 specimens previously tested for respiratory viruses. Following testing, each participating laboratory reported its results to the Hawaii State Department of Health, State Laboratories Division for evaluation and possible discrepant analysis. RESULTS: Two of three laboratories reported a 100% sensitivity and specificity, resulting in a 100% positive predictive value and a 100% negative predictive value (NPV) for influenza type A. The third laboratory showed a 71% sensitivity for influenza type A (83% NPV) with 100% specificity. All three laboratories were 100% sensitive and specific for the detection of influenza type B. Discrepant analysis indicated that the lack of sensitivity experienced by the third laboratory may have been due to the analyte-specific reagent probe used by that laboratory. Use of a newer version of the product with a secondary panel of 20 specimens resulted in a sensitivity and specificity of 100%. CONCLUSIONS: All three laboratories successfully verified their ability to conduct clinical testing for influenza using diverse nucleic acid extraction and RTi RT-PCR platforms. Successful completion of the verification by all collaborating laboratories paved the way for the integration of those facilities into a statewide laboratory algorithm for influenza surveillance and response.