Target capture sequencing of SARS-CoV-2 genomes using the ONETest Coronaviruses Plus.

We introduce a target capture next-generation sequencing methodology, the ONETest Coronaviruses Plus, to sequence the SARS-CoV-2 genome and select loci of other respiratory viruses. We applied the ONETest on 70 respiratory samples (collected in Florida, USA between May and July, 2020), in which SARS-CoV-2 had been detected by a PCR assay. For 48 of the samples, we also applied the ARTIC protocol. Of the 70 ONETest libraries, 45 (64%) had a (near-)complete sequence (>29,000 bases and >90% covered by >9 reads). Of the 48 ARTIC libraries, 25 (52%) had a (near-)complete sequence. In 19 out of 25 (76%) samples in which both the ONETest and ARTIC yielded (near-)complete sequences, the lineages assigned were identical. As a target capture approach, the ONETest is less prone to loss of sequence coverage than amplicon approaches, and thus can provide complete genomic information more often to track and monitor SARS-CoV-2 variants.

Performance of a Semiquantitative Multiplex Bacterial and Viral PCR Panel Compared With Standard Microbiological Laboratory Results: 396 Patients Studied With the BioFire Pneumonia Panel.

BACKGROUND: Microbiologic results are critical to optimal management of patients with lower respiratory tract infection, but standard methods may take several days. The multiplex polymerase chain reaction BioFire Pneumonia (PN) panel detects 15 common bacterial species semiquantitatively as copy number/mL, 8 viral species, and 7 resistance genes in about an hour within the clinical laboratory. METHODS: We tested 396 unique endotracheal or bronchoalveolar lavage specimens with the BioFire Pneumonia panel and compared the bacterial detections to conventional gram stain and culture results. RESULTS: Of the 396 patients, 138 grew at least 1 bacterium that had a target on the PN panel, and 136/138 (98.6%) were detected by the panel. A total of 177 isolates were recovered in culture and the PN panel detected 174/177 (98.3%). A further 20% of patients had additional targets detected that were not found on standard culture (specificity 69%, positive predictive value 63%, and negative predictive value 98.9%). Copy number was strongly related to standard semiquantitative growth on plates reported by the laboratory (eg, 1+, 2+, 3+ growths) and was significantly higher in those specimens that grew a potential pathogen. Both higher copy number and bacterial detections found by the PN panel, but not found in culture, were strongly positively related to the level of white blood cells reported in the initial gram stain. CONCLUSIONS: Higher copy number and bacterial detections by the PN panel are related to the host respiratory tract inflammatory response. If laboratories can achieve a rapid turnaround time, the PN panel should have a significant impact both on patient management and on antibiotic stewardship.

Performance and Impact of a CLIA-waived, Point-of-care Respiratory PCR Panel in a Pediatric Clinic.

BACKGROUND: Laboratory-based respiratory polymerase chain reaction (PCR) panels are rarely used in outpatient pediatric practice due to prolonged turn-around times and cost of medical equipment. The BioFire FilmArray Respiratory Panel EZ (RP EZ) is a Clinical Laboratory Improvement Amendments-waived respiratory pathogen PCR panel which rapidly tests for 14 common respiratory organisms. The aim of this study was to identify the distribution of organisms seen in pediatric clinics and to determine if utilization of this point-of-care test improved disease management, while exploring impact on clinic workflow. METHODS: From January 2018 through January 2019, when clinically appropriate, patients were tested by the RP EZ and/or antigen tests (Clinic A) or antigen test only (Clinic B). Residual samples from Clinic B antigen tests were frozen and later tested on the RP EZ for definitive pathogen identification. Patient data and prescription records were extracted from the electronic health record. RESULTS: A total of 430 patients had RP EZ tests performed, and at least 1 organism was detected in 70.4% of patients. The most common organisms identified were human rhinovirus/enterovirus, influenza, and respiratory syncytial virus. Appropriate treatment occurred for 93.6% of patients when the RP EZ was performed (Clinic A) versus 87.9% of patients who had only antigen tests performed (Clinic B, P = 0.0445). Utilization of RP EZ testing also significantly reduced appointment duration time (48.0 versus 54.9 minutes, P = 0.0009). Three false-positive influenza B results were identified by antigen testing. CONCLUSIONS: A point-of-care PCR panel improved patient care by providing an accurate diagnosis and shortened appointment duration.

Semi-quantitative Influenza A population averages from a multiplex respiratory viral panel (RVP): potential for reflecting target sequence changes affecting the assay.

BACKGROUND: Yearly influenza virus mutations potentially affect the performance of molecular assays, if nucleic acid changes involve the sequences in the assay. Because individual patient viral loads depend on variables such as duration of illness, specimen type, age, and immunosuppression, we examined seasonal population averages of positive tests to smooth inherent variability. METHODS: We studied the population seasonal averages of the semi-quantitative nAMPs for the influenza matrix and hemagglutinin genes in the GenMark (Carlsbad, CA) Respiratory Viral Panel assay between 3 institutions over 3 Influenza seasons. RESULTS: Population average nAMPs were strikingly consistent between separate institutions, but differed substantially between H3N2 and H1N1 seasons. In the 2012-2013 and 2014-2015 influenza seasons, matrix gene H3N2 nAMP averages were 50-70% less than those of the same assay in the 2013-2014 H1N1 season. Influenza strains representative of these seasons were grown in tissue culture and when the supernatant virus was adjusted to the same copy number using a TaqMan assay, the same relative differences were reproduced in the RVP assay. Because the sequences for the PCR and PCR product detection in the GenMark assay are proprietary, the manufacturer provided single stranded DNA matching the capture probe for the representative H3N2 (3 mismatches) and H1N1 strains (2 different mismatches). Equimolar concentrations of these synthetic DNA sequences gave average nAMP values that closely correlated with the average nAMPS of the representative strains and their respective seasonal averages. CONCLUSIONS: Seasonal averages of semi-quantitative data may provide a means to follow assay performance as a reflection of the effects of molecular drift.

Comparison of two multiplex methods for detection of respiratory viruses: FilmArray RP and xTAG RVP.

We compared the FilmArray RP (Idaho Technology, Inc., Salt Lake City, UT) and the xTAG RVP (Luminex Corporation, Toronto, Canada) multiplex respiratory virus PCR methods for the detection of respiratory viruses in a set of 200 patient specimens frozen at -70 °C after standard viral culture and antigen detection methods were done. Both systems detected between 40 to 50% more viruses than traditional methods, primarily rhinoviruses and human metapneumovirus. The FilmArray RP detected significantly more total viruses either alone or as part of mixed infections than the xTAG RVP, as well as an additional 21.6% more respiratory syncytial viruses. The xTAG RVP requires 5 to 6 h with 2.5 to 3 h of hands-on time, while the FilmArray RP takes about an hour with 3 to 5 min of hands-on time, making it much easier to perform.

Daptomycin-reversible rifampicin resistance in vancomycin-resistant Enterococcus faecium.

OBJECTIVES: In a previous study, we observed marked synergy between daptomycin and rifampicin against 73% of rifampicin-resistant, vancomycin-resistant Enterococcus faecium (VRE), with approximately 100-fold reductions in rifampicin MICs observed at one-eighth to one-fourth daptomycin MIC. The purpose of this study was to determine whether the synergy between daptomycin and rifampicin could be explained by enhanced entry of rifampicin into the cell or was related to amino acid substitutions in the rifampicin-binding site in the beta subunit (rpo beta) of the RNA polymerase. METHODS: We developed a bioassay for rifampicin to measure cell-bound rifampicin levels as well as metabolic inactivation of rifampicin. In addition, we sequenced the rifampicin-binding site in the rpo beta of VRE strains with and without synergy between daptomycin and rifampicin. RESULTS: Cell-bound rifampicin levels were the same in rifampicin-susceptible VRE as in rifampicin-resistant VRE showing daptomycin synergy and were not affected by the presence of daptomycin. In contrast, rifampicin-resistant VRE without daptomycin synergy had undetectable cell-bound rifampicin. Sequencing the rpo beta rifampicin-binding site revealed that the synergistic strains had the same sequence as rifampicin-susceptible wild-type E. faecium. The daptomycin synergy-resistant strains all had mutations in known rifampicin-binding sites. CONCLUSIONS: Daptomycin is able to reverse rifampicin resistance in some strains of VRE, but the mechanism could not be explained by an effect of daptomycin on entry of rifampicin into or transport out of the cell, by inactivation of rifampicin or by mutation involving the rifampicin-binding site.

Real-time polymerase chain reaction detection of herpes simplex virus in cerebrospinal fluid and cost savings from earlier hospital discharge.

Neonatal herpes simplex virus (HSV) can be a devastating illness and may be difficult to diagnose in those cases without a typical skin rash. As a result, physicians often rely on HSV polymerase chain reaction of cerebrospinal fluid to rule out HSV encephalitis. We developed a real-time polymerase chain reaction assay for HSV using the SmartCycler II (Cepheid, Sunnyvale, CA). End point dilution studies showed sensitivity comparable to that of two national reference laboratories that use LightCycler. In-house turnaround time was approximately 1.5 days versus approximately 5.2 days for sending the test to a reference laboratory. We hypothesized that the rapid availability of a negative test result would allow physicians to discharge appropriate patients earlier. Six months after implementation, clinical case analysis identified 12 pediatric patients who were discharged earlier based on more rapid test results, with a projected savings of approximately 55.2 hospital days throughout the first year. Actual length of stay for patients tested in-house was significantly less than that of historical controls and was projected to save approximately 70.2 hospital days in the first year. Including projected annual laboratory cost/test savings of approximately $11,000, a total savings of $38,000 to $43,000 was estimated for the first year of implementation, more than offsetting startup instrument and development cost.

Synergy of daptomycin with oxacillin and other beta-lactams against methicillin-resistant Staphylococcus aureus.

We previously observed marked synergy between daptomycin and both rifampin and ampicillin against vancomycin-resistant enterococci (VRE). Because the synergy between daptomycin and ampicillin was observed for 100% of VRE strains with high-level ampicillin resistance (ampicillin MIC of > or =128 microg/ml), we looked for synergy between daptomycin and other beta-lactams against 18 strains of methicillin-resistant Staphylococcus aureus (MRSA) by employing a time-kill method using Mueller-Hinton broth supplemented to 50 mg of Ca2+/liter. All strains were resistant to oxacillin (16 of 18 strains were resistant at drug concentrations of > or =256 microg/ml), and all strains were susceptible to daptomycin (the MIC at which 90% of the tested isolates were inhibited was 1 microg/ml). Daptomycin was tested at concentrations of 2, 1, 0.5, 0.25, 0.125, and 0.0625 microg/ml alone or in combination with oxacillin at a fixed concentration of 32 microg/ml. Synergy was found for all 18 strains with daptomycin at one-half the MIC in combination with 32 microg of oxacillin/ml, and synergy was found for 11 of 18 strains (61%) with daptomycin at one-fourth the MIC or less in combination with oxacillin. At 24 h, the daptomycin-oxacillin combination with daptomycin at one-half the MIC showed bactericidal activity against all 18 strains, and the combination with one-fourth the daptomycin MIC showed bactericidal activity against 9 of 18 strains. We also used a novel screening method to look for synergy between daptomycin and other beta-lactams. In this approach, daptomycin was incorporated into Ca(2+)-supplemented Mueller-Hinton agar at subinhibitory concentrations, and synergy was screened by comparing test antibiotic Kirby-Bauer disks on agar with and without daptomycin. By this method, daptomycin with ampicillin-sulbactam, ticarcillin-clavulanate, or piperacillin-tazobactam showed synergy comparable to or greater than daptomycin with oxacillin. For seven of the eight strains tested, time-kill studies confirmed synergy between daptomycin and ampicillin-sulbactam with ampicillin in the range of 2 to 8 microg/ml. The combination of daptomycin and beta-lactams may be useful for the treatment of MRSA infection, but further studies are needed to elucidate the mechanisms and to determine the in vivo efficacy of the combination.

Daptomycin synergy with rifampicin and ampicillin against vancomycin-resistant enterococci.

We used a novel screening method to look for synergy between daptomycin and 18 other antibiotics against 19 strains of high-level vancomycin-resistant enterococci (VRE) (vancomycin MIC > or = 256 mg/L). In this approach, daptomycin was incorporated into Ca(2+)-supplemented Mueller-Hinton agar at subinhibitory concentrations, and synergy was screened by comparing test antibiotic Etest MICs on agar with and without daptomycin. A striking reduction in the rifampicin MIC was seen in 11/15 (73.3%) VRE that were resistant to rifampicin, from > or =12 mg/L to a mean +/- s.d. of 0.22 +/- 0.21 mg/L at daptomycin 0.25 x MIC and 0.85 +/- 0.90 mg/L at daptomycin 0.125 x MIC. Synergy was also observed for 13/19 (68%) isolates with ampicillin (MIC > or = 128 mg/L). There was no significant synergy between daptomycin and any other antibiotic by this screening method. If confirmed by further studies, daptomycin with either rifampicin or ampicillin may be useful in the management of infections caused by VRE.