scholarly journals Persistence of Leptospira borgpetersenii Serovar Hardjo in Refrigerated Raw Milk: A Transmission Risk of Leptospirosis to Humans

Pathogens ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 291
Author(s):  
Elisa Mazzotta ◽  
Letizia Ceglie ◽  
Isabella Giurisato ◽  
Laura Bellinati ◽  
Laura Lucchese ◽  
...  
Keyword(s):  
Bacterial Culture ◽  
Limit Of Detection ◽  
Infectious Agent ◽  
Raw Milk ◽  
Storage Conditions ◽  
Transmission Risk ◽  
Time Range ◽  
Analytical Sensitivity ◽  
Zoonotic Risk ◽  
Leptospira Borgpetersenii

Leptospira borgpetersenii serovar Hardjo (LH) is an important infectious agent of reproduction pathologies and lactation decline in cattle, with a possible zoonotic role. To figure out the potential zoonotic risk for human raw-milk consumption, the present study aims at assessing the persistence and viability of LH in refrigerated raw milk over a 10-day period, which is set as the maximum time range for raw-milk domestic consumption. A negative sample of fresh raw milk was contaminated with an LH strain (2 × 108 Leptospires/mL) and analyzed by a rrs (16S) gene targeting real-time PCR (rPCR) protocol for LH DNA at days 1, 2, 3, 6, 7, 9, and 10. Seven aliquots of the same sampling time were inoculated into a semisolid EMJH media for bacterial culture. All aliquots tested positive in both rPCR and culture, which demonstrates that raw milk does not alter the detectability and viability of LH, respectively. The analytical sensitivity (LoD, limit of detection) determined for the rPCR (103 Leptospires/mL) was repeatable during the study, whereas it gradually decreased when it came to the bacterial culture. This study demonstrates that bovine raw milk might be a potential vehicle of infection by LH, even when storage conditions are strictly respected.

scholarly journals Development of a Genus-Specific Brucella Real-Time PCR Assay Targeting the 16S-23S rDNA Internal Transcribed Spacer from Different Specimen Types

2020 ◽  
Vol 7 (4) ◽  
pp. 175
Author(s):  
Rejoice Nyarku ◽  
Ayesha Hassim ◽  
Annelize Jonker ◽  
Melvyn Quan
Keyword(s):  
Sensitivity And Specificity ◽  
Internal Transcribed Spacer ◽  
Bacterial Culture ◽  
Limit Of Detection ◽  
Quantitative Polymerase Chain Reaction ◽  
Surface Protein ◽  
Qpcr Assay ◽  
Rapid Screening ◽  
Analytical Sensitivity ◽  
Tissue Samples

The aim of this study was to develop a 16S-23S ribosomal deoxyribonucleic acid internal transcribed spacer (ITS) quantitative polymerase chain reaction (qPCR) assay for the early diagnosis and rapid screening of brucellosis. Blood, milk, and tissue samples were spiked with B. abortus biovar 1 (B01988-18 strain) to determine the analytical sensitivity and specificity of the assay. The 95% limit of detection of the ITS qPCR assay was highest in tissue, followed by blood, then milk, i.e., 0.48, 4.43, and 15.18 bacteria/PCR reaction, respectively. The diagnostic performance of the assay was compared to the Brucella cell surface protein (BCSP) 31 qPCR assay and bacterial culture. Out of 56 aborted foetal tissue samples from bovine, ovine, and caprine, 33% (19/56) were positive for Brucella spp. The sensitivity and specificity of the ITS qPCR assay was 87% and 95% respectively, compared to 92% and 89% for the BCSP31 qPCR assay and 47% and 55% for bacterial culture, respectively. The assay was efficient, sensitive, and specific, making it a valuable tool in the early detection of the Brucella pathogen.

Detectability of Plasma Proteins in SRM Measurements

2018 ◽  
Vol 16 (1) ◽  
pp. 74-81 ◽  
Author(s):  
Olga I. Kiseleva ◽  
Elena A. Ponomarenko ◽  
Yulia A. Romashova ◽  
Ekaterina V. Poverennaya ◽  
Andrey V. Lisitsa
Keyword(s):  
Blood Plasma ◽  
Human Plasma ◽  
Limit Of Detection ◽  
Human Blood Plasma ◽  
Analytical Sensitivity ◽  
Plasma Proteome ◽  
Protein Targets ◽  
Blood Plasma Sample ◽  
Specificity And Sensitivity ◽  
Human Plasma Proteome

Background: Liquid chromatography coupled with targeted mass spectrometry underwent rapid technical evolution during last years and has become widely used technology in clinical laboratories. It offers confident specificity and sensitivity superior to those of traditional immunoassays. However, due to controversial reports on reproducibility of SRM measurements, the prospects of clinical appliance of the method are worth discussing. </P><P> Objective: The study was aimed at assessment of capabilities of SRM to achieve a thorough assembly of the human plasma proteome. </P><P> Method: We examined set of 19 human blood plasma samples to measure 100 proteins, including FDA-approved biomarkers, via SRM-assay. </P><P> Results: Out of 100 target proteins 43 proteins were confidently detected in at least two blood plasma sample runs, 36 and 21 proteins were either not detected in any run or inconsistently detected, respectively. Empiric dependences on protein detectability were derived to predict the number of biological samples required to detect with certainty a diagnostically relevant quantum of the human plasma proteome. </P><P> Conclusion: The number of samples exponentially increases with an increase in the number of protein targets, while proportionally decreasing to the logarithm of the limit of detection. Analytical sensitivity and enormous proteome heterogeneity are major bottlenecks of the human proteome exploration.

scholarly journals Validation of a Novel Diagnostic Approach Combining the VersaTREK™ System for Recovery and Real-Time PCR for the Identification of Mycobacterium chimaera in Water Samples

2021 ◽  
Vol 9 (5) ◽  
pp. 1031
Author(s):  
Roberto Zoccola ◽  
Alessia Di Blasio ◽  
Tiziana Bossotto ◽  
Angela Pontei ◽  
Maria Angelillo ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Detection System ◽  
Bacterial Load ◽  
Microbial Control ◽  
Hospital Setting ◽  
Sample Volume ◽  
Analytical Sensitivity ◽  
Initial Water

Mycobacterium chimaera is an emerging pathogen associated with endocarditis and vasculitis following cardiac surgery. Although it can take up to 6–8 weeks to culture on selective solid media, culture-based detection remains the gold standard for diagnosis, so more rapid methods are urgently needed. For the present study, we processed environmental M. chimaera infected simulates at volumes defined in international guidelines. Each preparation underwent real-time PCR; inoculates were placed in a VersaTREK™ automated microbial detection system and onto selective Middlebrook 7H11 agar plates. The validation tests showed that real-time PCR detected DNA up to a concentration of 10 ng/µL. A comparison of the isolation tests showed that the PCR method detected DNA in a dilution of ×102 CFU/mL in the bacterial suspensions, whereas the limit of detection in the VersaTREK™ was <10 CFU/mL. Within less than 3 days, the VersaTREK™ detected an initial bacterial load of 100 CFU. The detection limit did not seem to be influenced by NaOH decontamination or the initial water sample volume; analytical sensitivity was 1.5 × 102 CFU/mL; positivity was determined in under 15 days. VersaTREK™ can expedite mycobacterial growth in a culture. When combined with PCR, it can increase the overall recovery of mycobacteria in environmental samples, making it potentially applicable for microbial control in the hospital setting and also in environments with low levels of contamination by viable mycobacteria.

scholarly journals PERFORMANCE CHARACTERISTICS OF A FRET-BASED IMMUNOASSAY FOR QUANTITATION OF INFLIXIMAB ON A POINT-OF-CARE INSTRUMENT SYSTEM

2021 ◽  
Vol 27 (Supplement_1) ◽  
pp. S57-S57
Author(s):  
Edgar Ong ◽  
Ruo Huang ◽  
Richard Kirkland ◽  
Michael Hale ◽  
Larry Mimms
Keyword(s):  
Whole Blood ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Quantitative Detection ◽  
Therapeutic Drug ◽  
Analytical Sensitivity ◽  
Sample Type ◽  
Hook Effect ◽  
Limit Of Quantitation ◽  
Assay Precision

Abstract Introduction A fast (&lt;5 min), time-resolved fluorescence resonance energy transfer (FRET)-based immunoassay was developed for the quantitative detection of infliximab (IFX) and biosimilars for use in therapeutic drug monitoring using only 20 µL of fingerstick whole blood or serum at the point-of-care. The Procise IFX assay and ProciseDx analyzer are CE-marked. Studies were performed to characterize analytical performance of the Procise IFX assay on the ProciseDx analyzer. Methods Analytical testing was performed by spiking known amounts of IFX into negative serum and whole blood specimens. Analytical sensitivity was determined using limiting concentrations of IFX. Linearity was determined by testing IFX across the assay range. Hook effect was assessed at IFX concentrations beyond levels expected to be found within a patient. Testing of assay precision, cross-reactivity and potential interfering substances, and biosimilars was performed. The Procise IFX assay was also compared head-to-head with another CE-marked assay: LISA-TRACKER infliximab ELISA test (Theradiag, France). The accuracy of the Procise IFX assay is established through calibrators and controls traceable to the WHO 1st International Standard for Infliximab (NIBSC code: 16/170). Results The Procise IFX assay shows a Limit of Blank, Limit of Detection, and Lower Limit of Quantitation (LLoQ) of 0.1, 0.2, and 1.1 µg/mL in serum and 0.6, 1.1, and 1.7 µg/mL in whole blood, respectively. The linear assay range was determined to be 1.7 to 77.2 µg/mL in serum and whole blood. No hook effect was observed at an IFX concentration of 200 µg/mL as the value reported as “&gt;ULoQ”. Assay precision testing across 20 days with multiple runs and reagent lots showed an intra-assay coefficient of variation (CV) of 2.7%, an inter-assay CV of &lt;2%, and a total CV of 3.4%. The presence of potentially interfering/cross-reacting substances showed minimal impact on assay specificity with %bias within ±8% of control. Testing of biosimilars (infliximab-dyyb and infliximab-abda) showed good recovery. A good correlation to the Theradiag infliximab ELISA was obtained for both serum (slope=1.01; r=0.99) and whole blood (slope=1.01; r=0.98) samples (Figure 1). Conclusion Results indicate that the Procise IFX assay is sensitive, specific, and precise yielding results within 5 minutes from both whole blood and serum without the operator needing to specify sample type. Additionally, it shows very good correlation to a comparator assay that takes several hours and sample manipulation to yield results. This makes the Procise IFX assay ideal for obtaining fast and accurate IFX quantitation, thus allowing for immediate drug level dosing decisions to be made by the physician during patient treatment.

scholarly journals Quantitative Detection of Clostridium perfringens in the Broiler Fowl Gastrointestinal Tract by Real-Time PCR

2005 ◽  
Vol 71 (7) ◽  
pp. 3911-3916 ◽  
Author(s):  
Mark G. Wise ◽  
Gregory R. Siragusa
Keyword(s):  
Gastrointestinal Tract ◽  
Real Time ◽  
Clostridium Perfringens ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Quantitative Detection ◽  
Necrotic Enteritis ◽  
Analytical Sensitivity ◽  
Quantitative Real Time Pcr ◽  
Pcr Inhibitor

ABSTRACT Strains of Clostridium perfringens are a frequent cause of food-borne disease and gas gangrene and are also associated with necrotic enteritis in chickens. To detect and quantify the levels of C. perfringens in the chicken gastrointestinal tract, a quantitative real-time PCR assay utilizing a fluorogenic, hydrolysis-type probe was developed and utilized to assay material retrieved from the broiler chicken cecum and ileum. Primers and probe were selected following an alignment of 16S rDNA sequences from members of cluster I of the genus Clostridium, and proved to be specific for C. perfringens. The assay could detect approximately 50 fg of C. perfringens genomic DNA and approximately 20 cells in pure culture. Measurements of the analytical sensitivity determined with spiked intestinal contents indicated that the consistent limit of detection with ileal samples was approximately 102 CFU/g of ileal material, but only about 104 CFU/g of cecal samples. The decreased sensitivity with the cecal samples was due to the presence of an unidentified chemical PCR inhibitor(s) in the cecal DNA purifications. The assay was utilized to rapidly detect and quantify C. perfringens levels in the gut tract of broiler chickens reared without supplementary growth-promoting antibiotics that manifested symptoms of necrotic enteritis. The results illustrated that quantitative real-time PCR correlates well with quantification via standard plate counts in samples taken from the ileal region of the gastrointestinal tract.

scholarly journals Negligible risk of zoonotic anisakid nematodes in farmed fish from European mariculture, 2016 to 2018

2021 ◽  
Vol 26 (2) ◽  
Author(s):  
Maria Letizia Fioravanti ◽  
Andrea Gustinelli ◽  
George Rigos ◽  
Kurt Buchmann ◽  
Monica Caffara ◽  
...  
Keyword(s):  
Atlantic Salmon ◽  
Transmission Risk ◽  
Fish Parasites ◽  
Gilthead Seabream ◽  
Farmed Fish ◽  
Eu Regulation ◽  
Zoonotic Risk ◽  
Freezing Treatment ◽  
Zoonotic Parasites ◽  
Anisakid Larvae

Background The increasing demand for raw or undercooked fish products, supplied by both aquaculture and fisheries, raises concerns about the transmission risk to humans of zoonotic fish parasites. This has led to the current European Union (EU) Regulation No 1276/2011 amending Annex III of Regulation (EC) No 853/2004 and mandating a freezing treatment of such products. Zoonotic parasites, particularly anisakid larvae, have been well documented in wild fish. Data on their presence in European aquaculture products, however, are still scarce, except for Atlantic salmon (Salmo salar), where the zoonotic risk was assessed as negligible, exempting it from freezing treatment. Aim To evaluate the zoonotic Anisakidae parasite risk in European farmed marine fish other than Atlantic salmon. Methods From 2016 to 2018 an observational parasitological survey was undertaken on 6,549 farmed fish including 2,753 gilthead seabream (Sparus aurata), 2,761 European seabass (Dicentrarchus labrax) and 1,035 turbot (Scophthalmus maximus) from 14 farms in Italy, Spain and Greece. Furthermore, 200 rainbow trout (Oncorhynchus mykiss) sea-caged in Denmark, as well as 352 seabream and 290 seabass imported in Italy and Spain from other countries were examined. Fish were subjected to visual inspection and candling. Fresh visceral organs/fillet samples were artificially digested or UV pressed and visually examined for zoonotic anisakid larvae. Results No zoonotic parasites were found in any of the fish investigated. Conclusions The risk linked to zoonotic Anisakidae in the examined fish species from European mariculture appears negligible. This study laid the groundwork for considerations to amend the current EU regulation.

Preservation and Storage Mechanisms for Raw Milk Samples for Use in Milk-Recording Schemes

1996 ◽  
Vol 59 (2) ◽  
pp. 151-154 ◽  
Author(s):  
HUMBERTO G. MONARDES ◽  
ROBERT K. MOORE ◽  
BRIAN CORRIGAN ◽  
YVON RIOUX
Keyword(s):  
Cell Count ◽  
Somatic Cell Count ◽  
Potassium Dichromate ◽  
Dairy Herd ◽  
Raw Milk ◽  
Storage Conditions ◽  
Sample Storage ◽  
Milk Samples ◽  
Whole Process ◽  
And Storage

This study, carried out by the Quebec Dairy Herd Analysis Service, compares (during summer conditions in Quebec) the performance of three types of preservatives for raw milk under four different systems of sample storage: no refrigeration, refrigeration at the laboratory only, refrigeration during transport and at the lab, and complete refrigeration from sampling at the farm to analysis. The objective was to determine the best preservative and storage conditions for protecting milk components during transportation and storage of raw milk samples collected at the farm and sent to a central testing lab for analysis. Milk samples were analyzed at day 3 and at day 7 after sampling to observe the effect of aging. A total of 12,480 samples were collected during the trial. The components studied were percentage of fat and protein and somatic cell count (SCC). In general, samples preserved with bronopol (2-bromo-2-nitropropane-1,3-diol and 2-bromo-2-nitropropanol) in liquid or in microtab tended to give higher readings for fat and protein contents than samples preserved with potassium dichromate. Significantly lower fat values were observed in 7-day-old samples compared to 3-day-old samples. Fat depression was more accentuated in nonrefrigerated samples. Under current methods of handling raw milk samples, refrigeration during the whole process of sampling, transportation, and until analysis, seems an ideal to attain to avoid significant reductions of fat values.

Clinical Validation of the Lymph3Cx Assay to Distinguish Primary Mediastinal Large B-Cell Lymphoma From Diffuse Large B-Cell Lymphoma

2019 ◽  
Vol 152 (Supplement_1) ◽  
pp. S136-S136
Author(s):  
Colleen Ramsower ◽  
Tameson Yip ◽  
Betty Glinsmann-Gibson ◽  
Ryan Robetorye ◽  
Lisa Rimsza
Keyword(s):  
B Cell ◽  
Cell Lymphoma ◽  
Limit Of Detection ◽  
Gene Expression Signature ◽  
B Cell Lymphoma ◽  
Clinical Diagnostics ◽  
Diagnostic Methods ◽  
Analytical Sensitivity ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Abstract Introduction Primary mediastinal large B-cell lymphoma (PMLBCL) is recognized as a distinct lymphoid neoplasm. However, differentiation from diffuse large B-cell lymphoma (DLBCL) can be difficult due to the need for close clinicopathologic correlation and variability in morphology and immunophenotype. In addition, a recent study identified lymphomas that share molecular and morphological features with PMBCL, yet do not involve the mediastinum. Correct classification of PMBCL may impact therapeutic decision making, increasing the demand for more accurate diagnostic methods. Recent research identified a new gene expression signature that robustly differentiated PMLBCL from DLBCL in both training and independent validation cohorts using RNA isolated from routinely available formalin-fixed, paraffin-embedded (FFPE) tissues. Methods This assay, known as the “Lymph3Cx,” utilizes the nCounter platform (NanoString Technologies, Seattle, WA) and consists of probes for 58 target genes (including both discriminatory and housekeeping genes) that can distinguish PMLBCL cases from DLBCL as well as the “cell-of-origin” subtypes of DLBCL. In the current study reported here, the Molecular Diagnostics–Arizona Laboratory (MDAZL) performed a full validation of the Lymph3Cx assay for use in our clinical diagnostic laboratory. Results Assay performance included accuracy (92.9%), precision (100%), analytical sensitivity (100 ng RNA limit of detection), analytical specificity (measures are in place to prevent interfering substances), reproducibility between technologists and technical replicates (100%), specimen stability (18 months for unstained tissue sections), and RNA Isolation Kit Utility (two kits with equal performance) in order to become the first CLIA-certified, CAP-accredited clinical diagnostics laboratory to offer a molecular assay for distinction of PMLBCL from DLBCL. Conclusion The Lymph3Cx assay will be prospectively used for evaluation of Mayo Clinic patients and for use as an integrated biomarker in clinical trials.

scholarly journals Optimization and Comparison of Information-Dependent Acquisition (IDA) to Sequential Window Acquisition of All Theoretical Fragment Ion Spectra (SWATH) for High-Resolution Mass Spectrometry in Clinical Toxicology

2019 ◽  
Vol 65 (7) ◽  
pp. 862-870 ◽  
Author(s):  
Jeffrey D Whitman ◽  
Kara L Lynch
Keyword(s):  
Mass Spectrometry ◽  
High Resolution ◽  
Data Acquisition ◽  
High Resolution Mass Spectrometry ◽  
Limit Of Detection ◽  
Clinical Toxicology ◽  
Urine Samples ◽  
Analytical Sensitivity ◽  
High Resolution Mass ◽  
Resolution Mass

Abstract BACKGROUND Untargeted data acquisition on high-resolution mass spectrometers (HRMSs) has been used in clinical toxicology for screening and identifying unknown compounds in patient samples. A common modality for untargeted HRMS data acquisition is information-dependent acquisition (IDA), which analyzes the most abundant small molecules within an acquisition cycle. This process can potentially lead to false negatives of clinically relevant compounds at low concentrations. Sequential window acquisition of all theoretical fragment ion spectra (SWATH) has emerged as a method of unbiased, untargeted HRMS data acquisition in which no spectral data are lost. SWATH has yet to be optimized and assessed for use in clinical toxicology. METHOD We developed a variable-window SWATH method (vSWATH) and compared it to IDA by limit of detection studies in drug-supplemented urine (81 compounds) and against a retrospective cohort of 50 clinical urine samples characterized by LC-MS/MS. RESULTS vSWATH had a lower limit of detection than IDA for 33 (41%) drugs and metabolites added into urine samples. Both IDA and vSWATH were equivalent in discovering compounds from clinical urine samples and confirmed 26 additional compounds not previously discovered by targeted LC-MS/MS. Lastly, the unbiased acquisition of spectra in vSWATH allowed for identification of 5 low-abundance compounds missed by IDA. CONCLUSIONS This vSWATH method for clinical toxicology demonstrated equivalent analytical sensitivity and specificity for untargeted drug screening and identification in urine samples. vSWATH provided the additional benefit of collecting all tandem mass spectrometry spectra in a sample, which could be useful in discovering low-abundance compounds not discovered by IDA.

scholarly journals Development and Evaluation of an iiPCR Assay for Salmonella and Shigella Detection on a Field-Deployable PCR System

2020 ◽  
Vol 2020 ◽  
pp. 1-5
Author(s):  
Tian Du ◽  
Ji-hong Lin ◽  
Jun-hua Zhao ◽  
Hai-bo Wang ◽  
Qiu-hua Mo
Keyword(s):  
Predictive Value ◽  
Large Scale ◽  
Limit Of Detection ◽  
Analytical Sensitivity ◽  
Pcr Assay ◽  
Preliminary Screening ◽  
Oral Transmission ◽  
Resource Limited ◽  
Clinical Accuracy ◽  
Insulated Isothermal Pcr

Background. Salmonella and Shigella are often associated with fecal-oral transmission and cause large-scale outbreaks in centralized catering units and, therefore, should be frequently and strictly monitored, especially among food handlers. However, no specific and sensitive on-site detection method is available until now. Methods. In this study, an insulated isothermal PCR assay for the detection of Salmonella and Shigella on a field-deployable PCR system was developed. Specificity, sensitivity, reproducibility, and clinical accuracy of the assay were characterized and evaluated. Results. The insulated isothermal PCR assay could be completed within 58 minutes with minimal pretreatment needed. The assay was specific and with good reproducibility. The limit of detection was 103 CFU/mL and 101 CFU/mL for Salmonella and Shigella, respectively, which was comparable to multiplex real-time PCR. Mock on-site clinical evaluation results showed that the analytical sensitivity and specificity of the insulated isothermal PCR assay were 100% and 96.6%, while the positive predictive value and negative predictive value were 94.1% and 100%, respectively. Conclusion. Based on our results, we believe that the assay developed herein could serve as an alternative method for preliminary screening and provide a valuable platform for the on-site detection of Salmonella and Shigella, especially in resource-limited and developing countries.

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