High-Throughput COVID-19 Testing of Naso-Oropharyngeal Swabs Using a Sensitive Extraction-Free Sample Preparation Method.

High-throughput diagnostic assays are required for large-scale population testing for severe acute respiratory coronavirus 2 (SARS-CoV-2). The gold standard technique for SARS-CoV-2 detection in nasopharyngeal swab specimens is nucleic acid extraction followed by real-time reverse transcription-PCR. Two high-throughput commercial extraction and detection systems are used routinely in our laboratory: the Roche cobas SARS-CoV-2 assay (cobas) and the Roche MagNA Pure 96 system combined with the SpeeDx PlexPCR SARS-CoV-2 assay (Plex). As an alternative to more costly instrumentation, or tedious sample pooling to increase throughput, we developed a high-throughput extraction-free sample preparation method for naso-oropharyngeal swabs using the PlexPCR SARS-CoV-2 assay (Direct). A collection of SARS-CoV-2-positive (n = 185) and -negative (n = 354) naso-oropharyngeal swabs in transport medium were tested in parallel to compare Plex to Direct. The overall agreement comparing the qualitative outcomes was 99.3%. The mean cycle of quantification (Cq) increase and corresponding mean reduction in viral load for Direct ORF1ab and RdRp compared to Plex was 3.11 Cq (-0.91 log10 IU/mL) and 4.78 Cq (-1.35 log10 IU/mL), respectively. We also compared Direct to a four-sample pool by combining each positive sample (n = 185) with three SARS-CoV-2-negative samples extracted with MagNA Pure 96 and tested with the PlexPCR SARS-CoV-2 assay (Pool). Although less sensitive than Plex or Pool, the Direct method is a sufficiently sensitive and viable approach to increase our throughput by 12,032 results per day. Combining cobas, Plex, and Direct, an overall throughput of 19,364 results can be achieved in a 24-h period. IMPORTANCE Laboratories have experienced extraordinary demand globally for reagents, consumables, and instrumentation, while facing unprecedented testing demand needed for the diagnosis of SARS-CoV-2 infection. A major bottleneck in testing throughput is the purification of viral RNA. Extraction-based methods provide the greatest yield and purity of RNA for downstream PCR. However, these techniques are expensive, time-consuming, and depend on commercial availability of consumables. Extraction-free methods offer an accessible and cost-effective alternative for sample preparation. However, extraction-free methods often lack sensitivity compared to extraction-based methods. We describe a sensitive extraction-free protocol based on a simple purification step using a chelating resin, combined with proteinase K and thermal treatment. We compare the sensitivity qualitatively and quantitatively to a well-known commercial extraction-based system, using a PCR assay calibrated to the 1st WHO international standard for SARS-CoV-2 RNA. This method entails high throughput and is suitable for all laboratories, particularly in jurisdictions where access to instrumentation and reagents is problematic.

Qualitative and quantitative effects of thermal treatment of naso-oropharyngeal samples before cobas SARS-CoV-2 testing.

To improve laboratory safety we thermally treated naso-oropharyngeal samples before testing with the cobas SARS-CoV-2 assay. This study aimed to determine if thermal treatment significantly affects the qualitative detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the quantitative measurement of cobas SARS-CoV-2 ORF1a and E-gene target copy number using an in-house quantitative method. A collection of positive (n = 238) and negative samples (n = 196) was tested in parallel comparing thermal treatment (75 °C for 15 minutes) to room-temperature. There were no significant differences in the final qualitative outcomes for thermal treatment versus room-temperature (99.8% agreement) despite a statistically significant reduction (P < 0.05) in target copy number following thermal treatment. The median ORF1a and E-gene reduction in target copy number was -0.07 (1.6%) and -0.22 (4.2%) log10 copies/mL respectively. The standard curves for both ORF1a and E-gene targets were highly linear (r2 = 0.99). Good correlation was observed for ORF1a (r2 = 0.96) and E-gene (r2 = 0.98) comparing thermal treatment to room-temperature control.

Bacteriology and clinical outcomes of patients with culture-positive pleural infection in Western Australia: A 6-year analysis.

BACKGROUND AND OBJECTIVE: Pleural infection is a clinical challenge; its microbiology can be complex. Epidemiological and outcome data of pleural infection in adult Australians are lacking. We describe the bacteriology and clinical outcomes of Australian adults with culture-positive pleural infection (CPPI) over a 6-year period. METHODS: Cases with CPPI were identified through Western Australian public hospitals electronic record. Culture isolates, admission dates, vital status, co-morbidities, radiology, blood and pleural fluid tests were extracted. RESULTS: In total, 601 cases (71.4% males; median age: 63 years (IQR: 50-74); median hospital stay 13 days) involving 894 bacterial isolates were identified. Hospital-acquired (HA)-CPPI was defined in 398 (66.2%) cases, community-acquired (CA)-CPPI in 164 (27.3%) cases and the remaining classified as oesophageal rupture/leak. Co-morbidities, most frequently cancer, were common (65.2%). Radiological evidence of pneumonia was present in only 43.8% of CA-CPPI and 27.3% of HA-CPPI. Of the 153 different bacterial strains cultured, Streptococcus species (32.9%) especially viridans streptococci group were most common in CA-CPPI, whereas HA-CPPI was most often associated with Staphylococcus aureus (11.6%) and Gram-negative (31.9%) infections. Mortality was high during hospitalization (CA-CPPI 13.4% vs HA-CPPI 16.6%; P = 0.417) and at 1 year (CA-CPPI 32.4% vs HA-CPPI 45.5%; P = 0.006). CONCLUSION: This is the first large multicentre epidemiological study of pleural infection in Australian adults and includes the largest cohort of HA-CPPI published to date. CPPI is caused by a diverse range of organisms which vary between CA and HA sources. CPPI is a poor prognostic indicator both in the short term and in the subsequent 12 months.

Streptococcus pneumoniae potently induces cell death in mesothelial cells.

Pleural infection/empyema is common and its incidence continues to rise. Streptococcus pneumoniae is the commonest bacterial cause of empyema in children and among the commonest in adults. The mesothelium represents the first line of defense against invading microorganisms, but mesothelial cell responses to common empyema pathogens, including S. pneumoniae, have seldom been studied. We assessed mesothelial cell viability in vitro following exposure to common empyema pathogens. Clinical isolates of S. pneumoniae from 25 patients with invasive pneumococcal disease and three reference strains were tested. All potently induced death of cultured mesothelial cells (MeT-5A) in a dose- and time-dependent manner (>90% at 107 CFU/mL after 24 hours). No significant mesothelial cell killing was observed when cells were co-cultured with Staphylococcus aureus, Streptococcus sanguinis and Streptococcus milleri group bacteria. S. pneumoniae induced mesothelial cell death via secretory product(s) as cytotoxicity could be: i) reproduced using conditioned media derived from S. pneumoniae and ii) in transwell studies when the bacteria and mesothelial cells were separated. No excess cell death was seen when heat-killed S. pneumoniae were used. Pneumolysin, a cytolytic S. pneumoniae toxin, induced cell death in a time- and dose-dependent manner. S. pneumoniae lacking the pneumolysin gene (D39 ΔPLY strain) failed to kill mesothelial cells compared to wild type (D39) controls, confirming the necessity of pneumolysin in D39-induced mesothelial cell death. However, pneumolysin gene mutation in other S. pneumoniae strains (TIGR4, ST3 and ST23F) only partly abolished their cytotoxic effects, suggesting different strains may induce cell death via different mechanisms.

Human pleural fluid is a potent growth medium for Streptococcus pneumoniae.

Empyema is defined by the presence of bacteria and/or pus in pleural effusions. However, the biology of bacteria within human pleural fluid has not been studied. Streptococcus pneumoniae is the most common cause of pediatric and frequent cause of adult empyema. We investigated whether S. pneumoniae can proliferate within human pleural fluid and if growth is affected by the cellular content of the fluid and/or characteristics of pneumococcal surface proteins. Invasive S. pneumoniae isolates (n = 24) and reference strain recovered from human blood or empyema were inoculated (1.5×106CFU/mL) into sterile human malignant pleural fluid samples (n = 11). All S. pneumoniae (n = 25) strains proliferated rapidly, increasing by a median of 3009 (IQR 1063-9846) from baseline at 24hrs in all pleural effusions tested. Proliferation was greater than in commercial pneumococcal culture media and concentrations were maintained for 48hrs without autolysis. A similar magnitude of proliferation was observed in pleural fluid before and after removal of its cellular content, p = 0.728. S. pneumoniae (D39 strain) wild-type, and derivatives (n = 12), each with mutation(s) in a different gene required for full virulence were inoculated into human pleural fluid (n = 8). S. pneumoniae with pneumococcal surface antigen A (ΔpsaA) mutation failed to grow (2207-fold lower than wild-type), p<0.001, however growth was restored with manganese supplementation. Growth of other common respiratory pathogens (n = 14) across pleural fluid samples (n = 7) was variable and inconsistent, with some strains failing to grow. We establish for the first time that pleural fluid is a potent growth medium for S. pneumoniae and proliferation is dependent on the PsaA surface protein and manganese.

Implanted brachytherapy seed movement reflecting transrectal ultrasound probe-induced prostate deformation.

PURPOSE: Compression of the prostate during transrectal ultrasound-guided permanent prostate brachytherapy is not accounted for during treatment planning. Dosimetry effects are expected to be small but have not been reported. The study aims to characterize the seed movement and prostate deformation due to probe pressure and to estimate the effects on dosimetry. METHODS AND MATERIALS: C-arm fluoroscopy imaging was performed to reconstruct the implanted seed distributions (compressed and relaxed prostate) for 10 patients immediately after implantation. The compressed prostate was delineated on ultrasound and registered to the fluoroscopy-derived seed distribution via manual seed localization. Thin-plate spline mapping, generated with implanted seeds as control points, was used to characterize the deformation field and to infer the prostate contour in the absence of probe compression. Differences in TG-43 dosimetry for the compressed prostate and that on probe removal were calculated. RESULTS: Systematic seed movement patterns were observed on probe removal. Elastic decompression was characterized by expansion in the anterior-posterior direction and contraction in the superior-inferior and lateral directions up to 4 mm. Bilateral shearing in the anterior direction was up to 6 mm, resulting in contraction of the 145 Gy prescription isodose line by 2 mm with potential consequences for the posterior-lateral margin. The average whole prostate D90 increased by 2% of prescription dose (6% max; p < 0.01). CONCLUSIONS: The current investigation presents a novel study on ultrasound probe-induced deformation. Seed movements were characterized, and the associated dosimetry effects were nonnegligible, contrary to common expectation.

Pleural empyema caused by Klebsiella oxytoca: a case series.

We report on 19 patients from Western Australia of pleural empyema with Klebsiella oxytoca, an organism never before reported in association with this condition. Median age was 65 years, 14/17 (83%) had been in hospital within 30 days prior to diagnosis, 12/18 (67%) had active cancer, 9/17 (53%) had been in intensive care and 7/17 (41%) had prior surgery. Nine patients died at the time of censure, five within 90 days of infection.

Low vancomycin MICs and fecal densities reduce the sensitivity of screening methods for vancomycin resistance in Enterococci.

Active surveillance is part of a multifaceted approach used to prevent the spread of vancomycin-resistant enterococci (VRE). The impact of fecal density, the vancomycin MIC of the isolate, and the vancomycin concentration in liquid medium on test performance are uncertain. Using fecal specimens spiked with a collection of 18 VRE (predominantly vanB) with a wide vancomycin MIC range, we compared the performances of commercial chromogenic agars (CHROMagar VRE, chromID VRE, Brilliance VRE, and VRE Select) and 1 liquid medium (Enterococcosel enrichment broth) for VRE detection. The specificity of solid media was excellent; however, the sensitivity at 48 h varied from 78 to 94%. Screening using liquid medium was less sensitive than screening with solid media, particularly as the vancomycin content increased. Sensitivity declined (i) as the fecal VRE density decreased, (ii) when the media were assessed at 24 h (versus 48 h), and (iii) for isolates with a low vancomycin MIC (sensitivity, 25 to 75% versus 100% for isolates with vancomycin MIC of <16 mg/liter versus >32 mg/liter on solid medium using 10(6) CFU/ml of feces). Depending on local epidemiology and in particular VRE vancomycin MICs, the sensitivity of culture-based methods for VRE screening of stool or rectal specimens may be suboptimal, potentially facilitating secondary transmission.

A comparison of phase, amplitude, and velocity binning for cone-beam computed tomographic projection-based motion reconstruction.

PURPOSE: We previously developed a motion estimation technique based on direct cone-beam projection analysis. It is able to reconstruct the complete motion trajectory of a radio-opaque marker, including cycle-to-cycle variability, using respiratory binning of the projection images. This paper investigates the use of phase, amplitude, and amplitude-velocity binning in the context of projection-based cone-beam motion estimation (CBME). METHODS AND MATERIALS: We simulated cone-beam computed tomographic scans of 160 tumor trajectories estimated by a CyberKnife Synchrony System (Accuray, Sunnyvale, CA), and reconstructed the complete trajectory with CBME using phase, amplitude, and amplitude-velocity binning of the projection data. Various numbers of respiratory bins, from 1 (no binning) to 100, were used for phase and amplitude binning, while 1 to 100 amplitude bins with 4 velocity bins were used for amplitude-velocity binning. From this large pool of data, we correlated the reconstruction accuracy with bin type, total number of bins, number of breathing cycles per bin, and the position of the bin within the breathing cycle. RESULTS: CBME predicted the true motion of the marker with a 3-dimensional (3D) mean root mean square (RMS) error of 0.24 mm for amplitude-velocity binning, 0.31 mm for amplitude binning, and 0.52 mm for phase binning. Reconstruction 3D RMS error increased to over 1 mm when less than 3 breathing cycles contributed to a bin. We found that reconstruction accuracy was optimized when about 20 bins were used. Accuracy also decreased in bins located around the inhale portion of the breath cycle, compared with the mid- and end-exhale positions. CONCLUSIONS: This study provides a quantitative assessment of phase, amplitude, and amplitude-velocity binning for CBME. A joint binning approach should be used to give both the accuracy of amplitude binning, as well as the robustness of phase binning, in areas of limited motion sampling.

Quantitative rt-PCR holds promise as a screening tool for patients with severe sepsis.

OBJECTIVE: The aim of the present study was to determine if the quantification of bacterial 16S rDNA could be clinically useful in predicting patients at increased risk of developing septic shock. METHODS: A retrospective study of patients with positive blood cultures taken on arrival to the ED. An EDTA sample was collected simultaneously with blood cultures and assayed by polymerase chain reaction to quantitate the bacterial 16S rDNA load. Descriptive and clinical data were collected from the medical record and this was blinded to the 16S rDNA result. Subsequently, the 16S rDNA result was compared with illness severity markers including septic shock and death to determine the relationship between the 16S rDNA load and illness severity. RESULTS: 98 patients (mean age 61 ± 20 years, range 18-92) with positive blood cultures were studied, most commonly growing Escherichia coli (n= 25) and Staphylococcus aureus (n= 23). 16 (16%) died. There were 42 (43%) 16S rDNA positive patients. A high 16S rDNA load was associated with an increased risk of developing delayed septic shock (OR 21.9, 95% CI 2.5-192.6) in comparison with either a low or negative 16S rDNA load; with a mortality OR 4.6 (95% CI 0.9-23.5). CONCLUSIONS: The quantitative assay for 16S rDNA might be a useful screening tool to detect severe sepsis in those whom it might not be clinically suspected. However, prospective studies are required to further assess the clinical usefulness of this assay.

Severity of pneumococcal pneumonia associated with genomic bacterial load.

BACKGROUND: There is a clinical need for more objective methods of identifying patients at risk for septic shock and poorer outcomes among those with community-acquired pneumonia (CAP). As viral load is useful in viral infections, we hypothesized that bacterial load may be associated with outcomes in patients with pneumococcal pneumonia. METHODS: Quantification of Streptococcus pneumoniae DNA level by real-time polymerase chain reaction (rt-PCR) was prospectively conducted on whole-blood samples from a cohort of 353 patients who were displaying CAP symptoms upon their admission to the ED. RESULTS: CAP caused by S pneumoniae was documented in 93 patients (36.5% with positive blood culture findings). A positive S pneumoniae rt-PCR assay finding was associated with a statistically significant higher mortality (odds ratio [OR], 7.08), risk for shock (OR, 6.29), and the need for mechanical ventilation (MV) [OR, 7.96]. Logistic regression, adjusted for age, sex, comorbidities, and pneumonia severity index class, revealed bacterial load as independently associated with septic shock (adjusted odds ratio [aOR], 2.42; 95% CI, 1.10 to 5.80) and the need for MV (aOR, 2.71; 95% CI, 1.17 to 6.27). An S pneumoniae bacterial load of >or= 10(3) copies per milliliter occurred in 29.0% of patients (27 of 93 patients; 95% CI, 20.8 to 38.9%) being associated with a statistically significant higher risk for septic shock (OR, 8.00), the need for MV (OR, 10.50), and hospital mortality (OR, 5.43). CONCLUSION: In patients with pneumococcal pneumonia, bacterial load is associated with the likelihood of death, the risk of septic shock, and the need for MV. High genomic bacterial load for S pneumoniae may be a useful tool for severity assessment.

Brachytherapy for carcinoma of the cervix: a Canadian survey of practice patterns in a changing era.

BACKGROUND AND PURPOSE: This survey aimed to document practices of Canadian radiation oncologists performing gynecologic brachytherapy for carcinoma of the cervix and to determine what the effect of the phasing-out of LDR after-loading systems from the commercial market is having on practice. MATERIALS AND METHODS: A 26-item questionnaire was developed to survey various aspects of brachytherapy practice to include: number of patients treated, prescription points/volume, dose and fractionation, timing, critical structure delineation, expected changes due to the phasing-out of support for low dose rate systems, and support for the development of national guidelines. A link to a web-based survey collection instrument was emailed to each radiation oncologist in Canada practicing gynecologic brachytherapy. RESULTS: A 67% response rate was achieved in this web-based survey. Radiation oncologists currently using HDR brachytherapy are most commonly delivering 5 fractions of 6 Gy in addition to an EBRT dose of 45 Gy in 25 fractions. The median total dose equivalents to Point A was 82.9 Gy for both early and advanced disease. In response to the announcement by a major vendor that they would be phasing-out service for a popular LDR after-loader, 49% of Canadian radiation oncologists who practice brachytherapy for cervix cancer are changing to an HDR technique with a further 9% changing to a PDR technique. Eighty-six percent of respondents would support the development of national guidelines for cervix brachytherapy in Canada. CONCLUSIONS: Variation in practice exists in Canada in brachytherapy for cervix cancer. Many centers are in the process of phasing-out LDR techniques in response to the withdrawal of commercial support for these systems. Support for the development of Canadian national guidelines is high.

Evaluation of linear accelerator performance standards using an outcome oriented approach.

Radiation therapy, along with other branches of medicine, is moving towards a firmer basis in evidence to optimally utilize resources. As new treatment technology and strategies place greater demands on quality assurance resources, the need to objectively evaluate equipment and process performance standards from the perspective of predicted clinical impact becomes more urgent. This study evaluates the appropriateness of recommended quality control tolerance and action levels for linear accelerators based on the calculated dosimetric impact of suboptimal equipment performance. A method is described to quantify the dosimetric changes, as reflected by the changes in the outcome surrogate, equivalent uniform dose (EUD), of machine performance deviations from the optimal, specifically in the range of tolerance and action levels promulgated by the Canadian Association of Provincial Cancer Agencies (CAPCA). Linear accelerator performance deviations were simulated for the treatment of prostate, breast, lung, and brain using 3D conformal techniques, and the impact evaluated in terms of the changes in the EUD of the target volumes and two principal organs at risk (OARs) per site. The eight key performance characteristics examined are: Output constancy, beam flatness, gantry angle, collimator angle, field size indicator, laser alignment (three directions) and, by inference, the optical distance indicator. Currently accepted CAPCA tolerance levels for these eight performance characteristics are shown to maintain average EUD deviations to within 2% for the targets and 2 Gy for the OARs. However, within the 2% or 2 Gy range, the recommended tolerance levels are found to have markedly different effects on the EUDs of the structures of interest.

Verifying monitor unit calculations for tangential whole breast fields in 3-dimensional planning.

An essential component of quality assurance in radiation therapy is verifying the accuracy of monitor unit calculations. For tangential breast fields, monitor unit differences between primary calculations and second checks are usually larger than considered acceptable at other anatomical sites. A simple model to reconcile the differences between sophisticated and simple algorithms is presented, based on estimating the the volume irradiated by the field, replacing the breast contour with a rectangular block having an equal volume but a new field width which provides almost equivalent scatter to the prescription point. This analysis can also assist the treatment planning physicist in selecting a tolerance window for verifying monitor unit calculations for tangential breast fields.

Variability in patterns of BK viral load after renal transplantation.

BK virus (BKV) may cause nephropathy in renal transplant patients, resulting in graft dysfunction and possible graft loss. We used a sensitive quantitative BKV assay to monitor plasma BK viral loads in 11 renal transplant patients for periods ranging from 37 to 189 weeks posttransplant. Five patients remained negative for BKV, and 6 developed viremia, including 1 patient with a transient viremia. Of the viremic patients, 2 were diagnosed with BKV nephropathy after increasing serial BK viral loads, prompting a renal biopsy that established the diagnosis. A 3rd patient had high initial BK viral load and biopsy-proven disease that resolved with reduced immunosuppression. Two patients did not develop nephropathy despite persistent viral loads of 10(4) copies/mL. Five of 6 patients experienced viral clearance from the plasma (BK viral load <500 copies/mL), which was associated with their renal function becoming stabilized, and the remaining patient experienced a downward trend in viral load and stable renal function. Thus, the BKV quantitative assay was useful in aiding the diagnosis of BKV nephropathy, monitoring the response to reductions in immunosuppression and identified that some patients can have persistent viremia and still develop stable renal function without specific antiviral therapy.

Feasibility of real-time polymerase chain reaction in whole blood to identify Streptococcus pneumoniae in patients with community-acquired pneumonia.

We assessed a real-time quantitative polymerase chain reaction (PCR) assay targeting the lytA and ply gene of Streptococcus pneumoniae. Both assays were applied to whole blood samples from 28 adult patients with community-acquired pneumonia. Our findings suggest the lytA PCR is more sensitive, and the quantitative aspect of the assay shows promise as an aid to clinical judgment.

Enhanced efficiency and ergonomics of an intraoperative automated prostate brachytherapy delivery technique.

INTRODUCTION: In 2003, the Tom Baker Cancer Centre started a prostate brachytherapy program using Iodine-125 seeds, intraoperative treatment planning, and an automated remote afterloader, the seedSelectron. Over a 3-month period in 2004-2005, technologic changes were implemented with the intent of reducing the time spent in the operating room and improving ergonomics for the radiation oncologist/surgeon. New commercial software including inverse planning was installed, concurrent needle insertion and seed train building was implemented, and additional hardware (a slave monitor) was connected to the system. PURPOSE: To demonstrate that, with these enhancements, dosimetry is not compromised, whereas efficiency is significantly improved. METHODS: Interactive inverse planning was used to create the treatment plans in the operating room. Seed-spacer trains were built concurrently with each needle's insertion guided by rotating the ultrasound probe to the correct sagittal plane using the Needle Navigator feature. Needles were built, inserted, and delivered one needle at a time. Needle coordinate and insert positions were verified on the live ultrasound image displayed on both the slave monitor positioned above the patient's pelvis and the operator console. Dosimetry parameters (D(90) and V(100)), numbers of seeds, and OR times were compared for 20 patients before and 11 patients after the implementation of the concurrent insertion and build protocol combined with inverse planning and the slave monitor. RESULTS: Operating room (OR) times (probe in to probe out) were reduced by 33 min and the number of seeds per unit volume by 3% on an average. The majority of the decrease in time is due to the concurrent building and insertion of needles. Before and after the new technique, average postplan D(90) and V(100) values at 4 weeks after the implant were the same to within 4 Gy and 0.1%, respectively. The range (max-min) of D(90) decreased by 20% of the mean dose and the V(100) range decreased by 6% with the new technique. Adding the slave monitor improved quality assurance of the delivery process and ergonomics for physicians. CONCLUSIONS: The concurrent insertion and build protocol, together with inverse planning and the slave monitor, have decreased OR times with greater consistency in the delivered dose distribution.