Fully automatic d-lactate assay using a modified commercially available method.

Intestinal infarction is the fast-evolving endpoint of impaired blood perfusion to an intestinal segment which may have fatal outcome. Early diagnosis and treatment within 6 h reduce mortality. Currently, d-lactate is a promising biomarker, however, not available in the acute clinical setting. The aim of this study is implementation of d-lactate analysis in a routine clinical setting. We used a spectrophotometric method, based on enzymatic oxidation of d-lactate by d-lactate dehydrogenase (D-LDH) coupled to the reduction of nicotinamide-adenine dinucleotide (NAD+). The amount of NADH formed in this reaction is equivalent to d-lactate. The primary concern in this method is interfering NADH formed by oxidation of l-lactate by l-lactate dehydrogenase (L-LDH). A commercially available kit for d-lactate measurement was implemented on our existing automated routine laboratory equipment including pH-inactivation of L-LDH. Our setup fulfilled clinical quality goals. We were able to measure d-lactate with an acceptable performance of the analysis and a short turn-around time. The method can be used to distinguish between the expected cut-off for intestinal ischemia around 0.3 mM and the upper reference limit of 0.05 mM. With a turnaround time of just 9 min, the analysis has potential as a readily available detection of circulating d-lactate for early diagnosis of intestinal ischemia.

A Comparative Study of Some Procedures for Isolation of Fruit DNA of Sufficient Quality for PCR-Based Assays.

Food fraud has been and still is a problem in the food industry. It is detectable by several approaches, such as high performance liquid chromatography (HPLC), chemometric assays, or DNA-based techniques, each with its own drawbacks. This work addresses one major drawback of DNA-based methods, in particular their sensitivity to inhibitors contained in particular matrices from which DNA is isolated. We tested five commercial kits and one in-house method characterized by different ways of sample homogenization and DNA capture and purification. Using these methods, DNA was isolated from 10 different fruit species commonly used in plant-based foodstuffs. The quality of the DNA was evaluated by UV-VIS spectrophotometry. Two types of qPCR assays were used for DNA quality testing: (i) Method specific for plant ITS2 region, (ii) methods specific for individual fruit species. Based mainly on the results of real-time PCR assays, we were able to find two column-based kits and one magnetic carrier-based kit, which consistently provided fruit DNA isolates of sufficient quality for PCR-based assays useful for routine analysis and identification of individual fruit species in food products.

Identification and antimicrobial susceptibility testing of Gram-positive and Gram-negative bacteria from positive blood cultures using the Accelerate Pheno™ system.

Rapid identification and antimicrobial susceptibility testing remain a crucial step for early efficient therapy of bloodstream infections. Traditional methods require turnaround times of at least 2 days, while rapid procedures are often associated with extended hands-on time. The Accelerate Pheno™ System provides microbial identification results within 90 min and susceptibility data in approximately 7 h directly from positive blood cultures with only few minutes of hands-on time. The aim of this study was, therefore, to evaluate the performance of the Accelerate Pheno™ System in identification and antimicrobial susceptibility testing of both Gram-positive and Gram-negative bacteria directly from clinical blood culture samples. We analyzed 108 and 67 blood culture bottles using the Accelerate PhenoTest™ BC kit with software version v1.0 and the FDA-cleared version v1.2, respectively. Reliable identification was achieved for Enterobacteriaceae, staphylococci, and enterococci, with 76/80 (95%), 42/46 (91%), and 10/11 (91%) correct identifications. Limitations were observed in the identification of streptococci, including Streptococcus pneumoniae and Streptococcus pyogenes, and coagulase-negative staphylococci. Antimicrobial susceptibility results for Enterobacteriaceae, for amikacin, ertapenem, ciprofloxacin, gentamicin, meropenem, and piperacillin-tazobactam ranged between 86 and 100% categorical agreement. Using v1.2, results for ceftazidime showed 100% concordance with the reference method. For staphylococci, the overall performance reached 92% using v1.2. Qualitative tests for detection of methicillin or macrolide-lincosamide-streptogramin B (MLSB) resistance caused major and very major errors for isolates. Overall, the present data show that the Accelerate Pheno™ system can, in combination with Gram stain, be used as a rapid complementation to standard microbial diagnosis of bloodstream infections.

The applicability of the Migratest® kit for evaluating the chemotactic activity of peripheral blood neutrophils in goats on the example of animals supplemented with ß-hydroxy-ß-methylbutyrate (HMB).

The objective of this study was to determine the applicability of the Migratest® kit for evaluating the chemotactic activity of peripheral blood neutrophils in goats. The experiment was performed on 14 goat kids aged 30±2 days, divided into two groups of 7 animals each: C - control group, and E - experimental group, supplemented with ß-hydroxy-ß-methylbutyrate (HMB), a typical immunostimulant which influences the phagocytic activity of peripheral neutrophils. The feed administered to experimental goat kids was supplemented with HMB at 40 mg/kg BW, whereas control goat kids were administered standard farm-made feed without supplementation. Blood was sampled from the jugular vein immediately before the experiment (day 0) and on experimental days 15, 30 and 60 to determine the chemotactic activity of peripheral blood neutrophils in goats. The results of the study indicate that the Migratest® kit can be used to evaluate the influence of immunomodulators on the chemotactic activity of peripheral blood neutrophils in goats. The results of the assay are most effectively presented by calculating the chemotactic index which accounts for the chemotaxis or migration of neutrophils in the presence or absence of a chemotactic factor, respectively, and the percentage of granulocytes that migrate towards fMLP. The results of both presentation methods appear to be identical.

Effect of Mailed Human Papillomavirus Test Kits vs Usual Care Reminders on Cervical Cancer Screening Uptake, Precancer Detection, and Treatment: A Randomized Clinical Trial.

Importance: In the United States, more than 50% of cervical cancers are diagnosed in underscreened women. Cervical cancer screening guidelines now include primary human papillomavirus (HPV) testing as a recommended strategy. Home-based HPV self-sampling is a viable option for increasing screening compliance and effectiveness; however, US data are needed to inform health care system implementation. Objective: To evaluate effectiveness of mailed HPV self-sampling kits vs usual care reminders for in-clinic screening to increase detection and treatment of cervical intraepithelial neoplasia grade 2 or worse (CIN2+) and uptake of cervical cancer screening. Design, Setting, and Participants: Randomized clinical trial conducted in Kaiser Permanente Washington, a US integrated health care delivery system. Women aged 30 to 64 years with health plan enrollment for 3 years and 5 months or more, a primary care clinician, no Papanicolaou test within 3 years and 5 months, and no hysterectomy were identified through electronic medical records and enrolled from February 25, 2014, to August 29, 2016, with follow-up through February 26, 2018. Interventions: The control group received usual care (annual patient reminders and ad hoc outreach from primary care clinics). The intervention group received usual care plus a mailed HPV self-sampling kit. Main Outcomes and Measures: Two primary outcomes were (1) CIN2+ detection within 6 months of screening and (2) treatment within 6 months of CIN2+ detection. Screening uptake within 6 months of randomization was a secondary outcome. Results: A total of 19 851 women (mean [SD] age, 50.1 [9.5] years) were included, with 9960 randomized to the intervention group and 9891 randomized to the control group. All women randomized were included in analysis. In the intervention group, 12 participants with CIN2+ were detected compared with 8 in the control group (relative risk, 1.49; 95% CI, 0.61-3.64) and 12 cases were treated vs 7 in the control group (relative risk, 1.70; 95% CI, 0.67-4.32). Screening uptake was higher in the intervention group (2618 participants [26.3%] vs 1719 participants [17.4%]; relative risk, 1.51; 95% CI, 1.43-1.60). Conclusions and Relevance: Mailing HPV kits to underscreened women increased screening uptake compared with usual care alone, with no significant differences in precancer detection or treatment. Results support the feasibility of mailing HPV kits to women who are overdue for screening as an outreach strategy to increase screening uptake in US health care systems. Efforts to increase kit uptake and follow-up of positive results are warranted to maximize detection and treatment of CIN2+. Trial Registration: ClinicalTrials.gov identifier: NCT02005510.

Clinical Effect of Expedited Pathogen Identification and Susceptibility Testing for Gram-Negative Bacteremia and Candidemia by Use of the Accelerate PhenoTM System.

BACKGROUND: Fast diagnostic tests providing earlier identification (ID) of pathogens, and antimicrobial susceptibility testing (AST) may reduce time to appropriate antimicrobial therapy (AAT), decrease mortality, and facilitate antimicrobial deescalation (ADE). Our objective was to determine the theoretical reduction in time to AAT and opportunities for ADE with Accelerate PhenoTM System (AXDX). METHODS: The prospective cohort (April 14, 2016 through June 1, 2017) was from the Barnes-Jewish Hospital, a 1250-bed academic center. Emergency department (ED) or intensive care unit (ICU) blood cultures Gram-stain positive for gram-negative bacilli (GNB) or yeast. AXDX was used in parallel with standard-of-care (SOC) diagnostics to determine differences in time to pathogen ID and AST. Theoretical opportunities for ADE from AXDX results were determined. RESULTS: In total, 429 blood cultures were screened, 153 meeting inclusion criteria: 110 on-panel GNB, 10 Candida glabrata, and 5 Candida albicans. For GNB SOC, median time from blood culture positivity to ID and AST were 28.2 and 52.1 h. Median time to ID and AST after AXDX initiation was 1.37 and 6.7 h for on-panel organisms. For on-panel Candida, time to ID was approximately 21 h faster with AXDX. ADE or AAT was theoretically possible with AXDX in 48.4%. Of on-panel organisms, 24.0% did not receive initial AAT. In-hospital mortality was 46.7% without initial AAT, and 11.6% with AAT. Coverage of AXDX was 75.3%, specificity 99.7%, positive predictive value (PPV) 96.0%, and negative predictive value (NPV) 97.6%. On-panel sensitivity was 91.5%, specificity 99.6%, PPV 96.0%, and NPV 99.0%. CONCLUSIONS: AXDX provides more rapid ID and AST for GNB and ID for yeast than SOC. AXDX could potentially reduce time to AAT and facilitate ADE.

Management of Mycobacterium avium subsp. paratuberculosis in dairy farms: Selection and evaluation of different DNA extraction methods from bovine and buffaloes milk and colostrum for the establishment of a safe colostrum farm bank.

The aim of this study was to develop and validate different innovative DNA extraction methods to detect Mycobacterium avium subsp. paratuberculosis (MAP) DNA from bovine and buffalo colostrum. Paratuberculosis is a chronic inflammatory infection of domestic and wild animals, especially ruminants, caused by MAP. The primary route of disease transmission is feces, but MAP can also be excreted in milk and colostrum. In 2015, the Italian Ministry of Health has issued a voluntary control plan of MAP in order to allow risk-based certification of bovine and buffaloes farms. In addition to the annual diagnostic screening and to the clinical surveillance of animals the plan includes the adoption of biosecurity and management measures to progressively mitigate the incidence of MAP. To achieve this goal it is crucial to ensure the accuracy of the methods used to detect the presence of MAP in bovine and buffaloes milk and colostrum, in order to: (1) support a "safe colostrum farm-bank" set-up and thus prevent the main within-farm MAP transmission route and (2) to allow the MAP-free certification of milk products for export purposes. To achieve these goals, seven different DNA extraction protocols were identified from bibliography, out of which three methods were finally selected after the adoption of an evaluation procedure aimed at assessing the efficiency of extraction of DNA, the purity of DNA and the adaptability of the DNA amplification: NucleoSpin® Food Kit (Macherey-Nagel), NucleoSpin® Food Kit (Macherey-Nagel) combined with the magnetic beads, and QIAamp Cador Pathogen Mini kit (QIAGEN). In particular, the NucleoSpin® Food Kit (Macherey-Nagel) and the QIAamp Cador Pathogen Mini kit (QIAGEN) were tested on bovine and buffalo colostrum, showing a LOD between 4 × 104 (2.6 × 106  cfu/ml) and 4.08 (26.7 cfu/ml) IS900 target copies and a LOD between 5.3 × 105 (4.1 × 106  cfu/ml) and 53 (4.1 × 103  cfu/ml) IS900 target copies, respectively.

Performance evaluation of commercial library construction kits for PCR-based targeted sequencing using a unique molecular identifier.

BACKGROUND: Target enrichment is a critical component of targeted deep next-generation sequencing for the cost-effective and sensitive detection of mutations, which is predominantly performed by either hybrid selection or PCR. Despite the advantages of efficient enrichment, PCR-based methods preclude the identification of PCR duplicates and their subsequent removal. Recently, this limitation was overcome by assigning a unique molecular identifier(UMI) to each template molecule. Currently, several commercial library construction kits based on PCR enrichment are available for UMIs, but there have been no systematic studies to compare their performances. In this study, we evaluated and compared the performances of five commercial library kits from four vendors: the Archer® Reveal ctDNA™ 28 Kit, NEBNext Direct® Cancer HotSpot Panel, Nugen Ovation® Custom Target Enrichment System, Qiagen Human Comprehensive Cancer Panel(HCCP), and Qiagen Human Actionable Solid Tumor Panel(HASTP). RESULTS: We evaluated and compared the performances of the five kits using 50 ng of genomic DNA for the library construction in terms of the library complexity, coverage uniformity, and errors in the UMIs. While the duplicate rates for all kits were dramatically decreased by identifying unique molecules with UMIs, the Qiagen HASTP achieved the highest library complexity based on the depth of unique coverage indicating superb library construction efficiency. Regarding the coverage uniformity, the kits from Nugen and NEB performed the best followed by the kits from Qiagen. We also analyzed the UMIs, including errors, which allowed us to adjust the depth of unique coverage and the length required for sufficient complexity. Based on these comparisons, we selected the Qiagen HASTP for further performance evaluations. The targeted deep sequencing method based on PCR target enrichment combined with UMI tagging sensitively detected mutations present at a frequency as low as 1% using 6.25 ng of human genomic DNA as the starting material. CONCLUSION: This study is the first systematic evaluation of commercial library construction kits for PCR-based targeted deep sequencing utilizing UMIs. Because the kits displayed significant variability in different quality metrics, our study offers a practical guideline for researchers to choose appropriate options for PCR-based targeted sequencing and useful benchmark data for evaluating new kits.

Rapid detection of carbapenemases directly from positive blood cultures by the ß-CARBA test.

The rapid detection of blood stream infections (BSI) by carbapenemase-producing Enterobacterales (CPE) is indispensable to early optimize antibiotic treatment and to improve survival. While phenotypic tests are time-consuming and PCR is expensive and not available in many routine laboratories, colorimetric tests (e.g., Carba NP test) can provide rapid results at moderate cost. However, up to now, the detection of CPE-BSI requires a further 3-h incubation in broth supplemented with zinc sulfate and imipenem after a blood culture has become positive, thereby causing delay and additional hands-on time. The purpose of this study was to develop and evaluate a new method for the detection of CPE directly from positive blood culture without the need for incubation in broth, based on the commercially available colorimetric ß-CARBA test. For the evaluation, blood cultures spiked with 140 different Enterobacterales isolates producing diverse beta-lactamases were tested with the new method. Of these, 70 were CPE (OXA-48-like, NDM, KPC, VIM, and GIM). After blood cultures turned positive, blood culture fluid was drawn, and erythrocytes were hemolyzed with SDS, washed, and equilibrated before the ß-CARBA was performed on the bacterial pellet. All carbapenemases were reliably detected, including weak carbapenemases of the OXA-48 group. The sensitivity was 100% (95% CI 94.9-100) and the specificity 94.3% (95% CI 89.2-99.4). The time to result was 20 to 45 min. Carbapenemases can rapidly and reliably be detected directly from blood cultures using the new method, which could help to improve the outcome of these difficult-to-treat infections.

Evaluating the integration of HIV self-testing into low-resource health systems: study protocol for a cluster-randomized control trial from EQUIP Innovations.

BACKGROUND: Throughout sub-Saharan Africa HIV-testing rates remain low. Barriers to testing, such as inconvenient service hours and long wait times, lack of privacy, and fear of unwanted disclosure, continue to impede service utilization. HIV self-testing (HIVST) is one strategy that addresses these barriers and has been shown to increase use of HIV-testing when distributed through community-based settings. However, the scalability of HIVST is limited because it has yet to be fully integrated into existing health systems and routine care. To address this gap, we designed a study to test the effect of offering HIVST to routine outpatient department (OPD) clients on uptake of HIV-testing as compared to standard of care and optimized standard of care. METHODS/DESIGN: This is a non-blinded, multi-site, cluster-randomized control trial. The health facility is the unit of randomization (cluster). Fifteen facilities were randomized to one of three arms: (1) Standard of care using routine provider-initiated testing and counseling (PITC); (2) Optimized standard of care using optimized PITC defined by additional training, job aids, and monitoring of PITC strategies with OPD providers and support staff; and (3) HIVST defined by HIVST demonstrations for OPD clients, HIVST kit distribution, and private spaces for HIVST kit use and/or interpretation. The primary outcome is the proportion of OPD clients tested for HIV on the day that they accessed OPD services. Secondary outcome measures are the proportion of OPD clients newly identified as HIV-positive and antiretroviral therapy (ART) initiation. Costs and cost-effectiveness will be evaluated. Nested studies will determine the acceptability of facility-based HIVST among OPD clients and health care providers, the presence of adverse events, such as coercion to test or unwanted status disclosure, and a process evaluation to determine feasibility and scale-up of facility-based HIVST for the future. DISCUSSION: This study protocol tests whether facility-based HIVST can positively contribute to HIV-testing among OPD clients in resource-limited settings. This will be one of the first studies to test the integration of HIVST into facility-based, primary health services in sub-Saharan Africa. TRIAL REGISTRATION: ClinicalTrials.gov, ID: NCT03271307 . Registered on 31 August 2017. Pan African Clinical Trials: PACTR201711002697316 . Registered on 1 November 2017.

Comparison of Singleplex Specific IgE Detection Immunoassays: ImmunoCAP Phadia 250 and Immulite 2000 3gAllergy.

BACKGROUND: The identification of clinically meaningful specific immunoglobulin E (sIgE) is important for the diagnosis and management of allergic diseases. Various in vitro sIgE detection methods are available worldwide. Depending on the number of antigens that can be tested simultaneously, there are two representative methods: singleplex and multiplex. Singleplex sIgE detection is mainly provided by Thermo Fisher (ImmunoCAP) and Siemens (Immulite). This study aimed to compare the diagnostic agreement of two singleplex sIgE detection assays. METHODS: Sera from 209 Korean patients with allergic disease were used to compare the ImmunoCAP and Immulite assays with respect to the following allergens: inhalant allergens (Dermatophagoides farinae, cat and dog dander, oak, rye grass, mugwort, Alternaria, German cockroach) and food allergens (hen's egg white, cow's milk, wheat, peanut, soybean, and shrimp). Data from 902 paired comparison tests were included for comparisons. Qualitative, semi-quantitative, and quantitative comparisons were performed using statistical analyses. RESULTS: In qualitative comparisons, the positivity and negativity agreements ranged from 75% (wheat, shrimp) to 96% (Alternaria). Class consistency (classes 0-6) was well matched. Spearman's rank correlation coefficients for all allergens except shrimp were over 0.7. In quantitative comparisons, all allergens excluding shrimp showed >0.7 intra-class correlation coefficients. CONCLUSIONS: The ImmunoCAP and Immulite systems showed similar performances. However, clinicians should consider fundamental methodological differences between the assays.

Multicomponent High-throughput Drug Screening via Inkjet Printing to Verify the Effect of Immunosuppressive Drugs on Immune T Lymphocytes.

High-throughput drug screening based on a multi-component array can be used to identify a variety of interaction between cells and drugs for suitable purposes. The signaling of immune cells is affected by specific proteins, diverse drug combinations, and certain immunosuppressive drugs. The effect of a drug on an organism is usually complex and involves interactions at multiple levels. Herein, we developed a multilayer fabricating system through the high-throughput assembly of nanofilms with inkjet printing to investigate the effects of immunosuppressive drugs. Immunosuppressive drugs or agents occasionally cause side effects depending on drug combinations or a patient's condition. By incorporating various drug combinations for understanding interaction between drugs and immune cells, we were able to develop an immunological drug screening kit with immunosuppressive drugs. Moreover, the ability to control the combination of drugs, as well as their potential for high-throughput preparation should be of great benefit to the biomedical and bioanalytical field.

Comparison of Intact PTH and Bio-Intact PTH Assays Among Non-Dialysis Dependent Chronic Kidney Disease Patients.

BACKGROUND: The third-generation bio-intact parathyroid hormone (PTH) (1-84) assay was designed to overcome problems associated with the detection of C-terminal fragments by the second-generation intact PTH assay. The two assays have been compared primarily among dialysis populations. The present study evaluated the correlations and differences between these two PTH assays among patients with chronic kidney disease (CKD) stages 3 to 5 not yet on dialysis. METHODS: Blood samples were collected from 98 patients with CKD stages 3 to 5. PTH concentrations were measured simultaneously by using the second-generation - PTH intact-STAT and third-generation bio-intact 1-84 PTH assays. Other serum biomarkers of bone mineral disorders were also assessed. CKD stage was calculated by using the CKD-Epidemiology Collaboration (EPI) formula. RESULTS: Serum bio-intact PTH concentrations were strongly correlated but significantly lower than the intact PTH concentrations (r=0.963, P<0.0001). This finding was consistent among CKD stages 3 to 5. PTH concentrations by both assays (intact and bio-intact PTH) positively correlated with urea (r=0.523, r=0.504; P=0.002, respectively), phosphorus (r=0.532, r=0.521; P<0.0001, respectively) and negatively correlated with blood calcium (r=-0.435, r=-0.476; P<0.0001, respectively), 25(OH) vitamin D, (r=-0.319, r=-0.353; respectively, P<0.0001) and the estimated glomerular filtration rate (r=-0.717, r=-0.688; P<0.0001, respectively). CONCLUSIONS: Among patients with CKD stages 3 to 5 not on dialysis, the bio-intact PTH assay detected significantly lower PTH concentrations compared with intact PTH assay. Additional studies that correlate the diagnosis and management of CKD mineral and bone disorders with bone histomorphometric findings are needed to determine whether bio-intact PTH assay results are better surrogate markers in these early stages of CKD.

Implementation of National Institute for Health and Care Excellence (NICE) guidance to measure immunoglobulin A with all coeliac screens: can an affordable solution be devised?

There has been a dramatic increase in requests for coeliac disease (CD) serological screening using immunoglobulin (Ig)A tissue transglutaminase antibodies (IgA-tTG). Recently, the UK National Institute for Health and Care Excellence has revised its guidance, recommending that total IgA should also be measured in all samples. This is justified, as false-negative results may occur with IgA deficiency. However, implementation of this guidance will incur considerable expense. Tests that measure IgA-tTG antibodies can detect IgA deficiency, indicated by low background signal. This provides an opportunity to identify samples containing IgA ≤ 0·2g/l, obviating the need for unselected IgA measurement. We investigated the feasibility of this approach in two centres that use the EliA™ Celikey™ assay or QUANTA Lite® enzyme-linked immunosorbent assay to quantify IgA-tTG antibodies. In both cases, total IgA correlated strongly with background IgA-tTG assay signal. Using the Celikey™ assay, a threshold of < 17·5 response units achieved 100% sensitivity (95% confidence intervals 79·4-100%) for detection of IgA ≤ 0·2g/l, circumventing the need for IgA testing in > 99% of sera. A similar principle was demonstrated for the QUANTA Lite® assay, whereby a threshold optical density of < 0·0265 also achieved 100% sensitivity (95% confidence intervals 78·2-100%) for IgA ≤ 0·2 g/l, avoiding unnecessary IgA testing in 67% of cases. These data suggest that CD screening tests can identify samples reliably containing low IgA in a real-life setting, obviating the need for blanket testing. However, this approach requires careful individualized validation, given the divergent efficiency with which assays identify samples containing low IgA.

Comparison of RNA extraction kits for the purification and detection of an enteric virus surrogate on green onions via RT-PCR.

Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) offers a rapid and sensitive molecular method for detection of enteric viruses. Unfortunately, these assays are often hampered by the low virus titre found in foods and PCR inhibition due to matrix carryover during RNA extraction. Four commercial RNA extraction kits (Qiagen's QIAamp Viral RNA Mini and UltraSens Virus kits, MoBio UltraClean Tissue & Cells RNA Isolation kit, and Ambion MagMAX Viral RNA Isolation kit) were evaluated for their ability to extract and purify MS2 bacteriophage RNA, an enteric virus surrogate, from inoculated green onions, a food which has been associated with viral gastroenteritis outbreaks. Inoculated green onion wash concentrates and green onion pieces with and without Qiagen QIAshredder homogenization were assayed in the kit comparison. MS2 detection and PCR inhibition were evaluated using a duplex real-time RT-PCR for MS2 and an exogenous internal amplification control (IAC) assay. Without homogenization, MS2 inoculated at 40pfu/g was detected in at least 4 lots of green onion wash concentrates using the silica-membrane spin-column kits. Inhibition was a factor for the magnetic silica-based MagMAX kit, which resulted in detection of MS2 in 1 of 5. Addition of QIAshredder homogenization prior to extraction did not adversely affect the silica-membrane kit results but improved the MS2 detection by MagMAX to 5 of 5 lots. Use of a 1:10 dilution of primary RNA extracts also improved detection. The QIAamp Viral RNA Mini and MagMAX kits were further compared for detection of MS2 from green onion pieces inoculated at 20 and 5pfu/g. Using homogenization, the MagMAX kit detected 20pfu/g in only 1 of 2 green onion lots, whereas the QIAamp Viral RNA kit detected 2 of 2 lots at 5 pfu/g without homogenization.