scholarly journals Comparison of Serological and Molecular Assays for Bartonella Species in Dogs with Hemangiosarcoma

Pathogens ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 794
Author(s):  
Erin Lashnits ◽  
Pradeep Neupane ◽  
Julie M. Bradley ◽  
Toni Richardson ◽  
Ricardo G. Maggi ◽  
...  
Keyword(s):  
Diagnostic Tests ◽  
Latent Class ◽  
Fluorescent Antibody ◽  
Molecular Diagnostic ◽  
Infection Prevalence ◽  
Diagnostic Assays ◽  
Molecular Assays ◽  
Tissue Biopsy ◽  
Frozen Tissue ◽  
Fresh Frozen

Currently, a gold standard diagnostic test for Bartonella infection in dogs is lacking. This represents a critical limitation for the development and evaluation of new diagnostic tests, as well as for the diagnosis of, and research on, bartonellosis in dogs. This retrospective observational study aims to compare the results of commonly performed and newly-reported Bartonella spp. diagnostic tests in banked clinical specimens from 90 dogs with hemangiosarcoma (HSA) using composite reference standard (CRS) and random effects latent class analysis (RE-LCA) techniques. Samples from each dog were tested using six serological or molecular diagnostic assays, including indirect fluorescent antibody (IFA) and Western blot (WB) for the detection of antibodies in serum, and qPCR and droplet digital PCR (ddPCR) in blood and fresh frozen tissue biopsy samples (mainly splenic HSA tumors and histopathologically normal spleen or skin/adipose tissue). Bartonella infection prevalence was estimated to be 78% based on the CRS (parallel testing with all six assays), and 64% based on the RE-LCA model. The assay with the highest diagnostic accuracy was qPCR performed on fresh frozen tissue biopsy samples (sensitivity: 94% by RE-LCA and 80% by CRS; specificity: 100%). When comparing newly-reported to traditional Bartonella diagnostic assays, ddPCR was more sensitive for the detection of Bartonella DNA than qPCR when testing blood samples (36% vs. 0%, p < 0.0001). Dogs that were positive on serological assays alone with negative molecular assays were highly unlikely (<3%) to be classified as infected by the RE-LCA model. These data indicate that Bartonella spp. DNA can be PCR amplified from fresh frozen tissues from a majority of dogs with HSA using both qPCR and ddPCR, supporting the use of these methods for future controlled studies comparing the prevalence of Bartonella spp. DNA in the tissue of dogs with HSA to that of unaffected controls.

Quantification of Chlamydia trachomatis in cervical and urine specimens from women attending a genitourinary medicine clinic: implications for screening strategies

1998 ◽  
Vol 9 (8) ◽  
pp. 448-451 ◽  
Author(s):  
B J Thomas ◽  
T Pierpoint ◽  
D Taylor Robinson ◽  
A M Renton
Keyword(s):  
Chlamydia Trachomatis ◽  
Diagnostic Tests ◽  
Fluorescent Antibody ◽  
Dna Amplification ◽  
Population Screening ◽  
Urine Samples ◽  
Molecular Diagnostic ◽  
Antibody Staining ◽  
Direct Fluorescent Antibody ◽  
Genitourinary Medicine

Population screening and intervention programmes can reduce the prevalence and incidence of infection with Chlamydia trachomatis , especially if sensitive molecular diagnostic tests are used. However, diagnostic tests that perform well on genitourinary medicine GUM clinic populations may be less useful for screening, particularly if the majority of infected subjects are asymptomatic and their samples contain fewer organisms. We have compared the extent of low organism load in cervical and urine samples from symptomatic and asymptomatic chlamydia positive women, by using a direct fluorescent antibody staining method and counting the chlamydial elementary bodies EBs . We have investigated the ability of an enzyme immunoassay EIA; MicroTrak and a DNA amplification ligase chain reaction; LCR assay to detect low numbers of organisms in cervical samples and the ability of the LCR assay to detect low numbers of organisms in urine. A low organism load 10 EBs was seen by direct fluorescent antibody DFA staining in about 30 of cervical samples and in about 75 of urines from chlamydia positive women; the proportions in symptomatic women were not significantly different from those in asymptomatic women. The EIA identified only 16 of cervical samples that contained 10 EBs by DFA staining; the LCR identified 100 of cervical samples and 93 of urine samples that contained 10 EBs by DFA staining. The findings suggest that the ability of chlamydial diagnostic tests to identify positive women should be similar among patients attending a GUM clinic and those taking part in a population screening programme, and that sensitive molecular assays such as the LCR should identify subjects with a low organism load in both groups.

Laboratory Methods for the Diagnosis of SARS-Cov-2

2022 ◽  
pp. 58-77
Author(s):  
Mohamed Echchakery ◽  
Samia Boussaa ◽  
Souad El Mouahid ◽  
Maryam Mountassir ◽  
Said El Hizazi ◽  
...  
Keyword(s):  
Genome Sequence ◽  
Diagnostic Tests ◽  
Epidemiological Studies ◽  
Virus Antigen ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Laboratory Methods ◽  
Virus Surface ◽  
Diagnostic Assays ◽  
Human Endeavor

The coronavirus disease 2019 (COVID-19) which has become the pandemic par excellence of our time places pressure on various aspects of human endeavor and as such requires detailed study to better combat it. However, diagnostic tests were used to provide data on the incidence of COVID-19 and to assess the immune status of infected individuals. The objective of this chapter is to describe the diagnostic methods currently used to identify SARS-CoV-2 infection. Obtaining the first SARS-CoV-2 genome sequence was decisive for the development of molecular diagnostic assays that currently make it possible to diagnose and screen for the Sars-CoV-2 infection. Their uses depend on the target to be detected. Antigenic tests detect the presence of a virus antigen, which usually makes a proteinaceous part of the virus surface. The serology tests detect the presence of antibodies generated against SARS-CoV-2 and are also a relevant tool for epidemiological studies.

Molecular Biology of PCR Testing for COVID-19 Diagnostics

2021 ◽  
Author(s):  
Vinita Chittoor-Vinod
Keyword(s):  
Molecular Biology ◽  
Respiratory Disease ◽  
Diagnostic Tests ◽  
Genetic Material ◽  
Molecular Diagnostic ◽  
Basic Principles ◽  
Asymptomatic Carriers ◽  
Diagnostic Assays ◽  
Global Pandemic ◽  
Asymptomatic Individuals

COVID-19 cases were first reported in December 2019, and since then it has spread quickly to create a global pandemic. This respiratory disease is caused by the SARS-CoV-2 virus. A major contributing factor for the fast spread of this virus is that the infectivity by the asymptomatic carriers is similar to symptomatic patients. Thus, to identify the asymptomatic individuals and to provide the essential treatment and care to COVID-19 patients, we rely heavily on diagnostic assays. Efficient, reproducible and accessible diagnostic tests are crucial in combatting a pandemic. Currently, there are few key detection tests which have been successfully employed to field-use. However, there are constant efforts to enhance their efficacy and accessibility. This chapter aims at explaining the basic principles of the current molecular diagnostic tests, which determine the presence of the virus through the detection of its genetic material. This chapter will aid the readers in understanding the basic workings of these molecular diagnostic tests.

scholarly journals Test Verification and Validation for Molecular Diagnostic Assays

2012 ◽  
Vol 136 (1) ◽  
pp. 11-13 ◽  
Author(s):  
Kevin C Halling ◽  
Iris Schrijver ◽  
Diane L Persons
Keyword(s):  
Clinical Laboratory ◽  
Molecular Diagnostic ◽  
Molecular Assay ◽  
Validation And Verification ◽  
Diagnostic Assays ◽  
Molecular Assays ◽  
Clinical Laboratories ◽  
Hereditary Disorders ◽  
Test Verification

With our ever-increasing understanding of the molecular basis of disease, clinical laboratories are implementing a variety of molecular diagnostic tests to aid in the diagnosis of hereditary disorders, detection and monitoring of cancer, determination of prognosis and guidance for cancer therapy, and detection and monitoring of infectious diseases. Before introducing any new test into the clinical laboratory, the performance characteristics of the assay must be “verified,” if it is a US Food and Drug Administration (FDA)–approved or FDA-cleared test, or “validated,” if it is a laboratory-developed test. Although guidelines exist for how validation and verification studies may be addressed for molecular assays, the specific details of the approach used by individual laboratories is rarely published. Many laboratories, especially those introducing new types of molecular assays, would welcome additional guidance, especially in the form of specific examples, on the process of preparing a new molecular assay for clinical use.

scholarly journals Clinical Performance of the cobas Liat SARS-CoV-2 & Influenza AB for the Detection of SARS-CoV-2 in Nasal Samples

2022 ◽  
Author(s):  
Yusaku Akashi ◽  
Michiko Horie ◽  
Junichi Kiyotaki ◽  
Yuto Takeuchi ◽  
Kenichi Togashi ◽  
...  
Keyword(s):  
Infectious Diseases ◽  
Diagnostic Tests ◽  
Clinical Utility ◽  
Clinical Performance ◽  
Point Of Care ◽  
Concordance Rate ◽  
Molecular Diagnostic ◽  
Molecular Assays ◽  
High Concordance

Background and Objective: Point-of-care type molecular diagnostic tests have been used for detecting SARS-CoV-2, although their clinical utility with nasal samples has yet to be established. This study evaluated the clinical performance of the cobas Liat SARS-CoV-2 & Influenza AB (Liat) in nasal samples. Methods: Nasal and nasopharyngeal samples were collected and were tested using the Liat, the cobas 6800 system and the cobas SARS-CoV-2 & Influenza AB (cobas), and a method developed by National Institute of Infectious Diseases, Japan (NIID). Results: A total of 814 nasal samples were collected. The Liat assay was positive for SARS-CoV-2 in 113 (13.9%). The total, positive, and negative concordance rate between the Liat and cobas/NIID assays were 99.3%/98.4%, 99.1%/100%, and 99.3%/98.2%, respectively. Five samples were positive only using the Liat assay. Their Ct values ranged from 31.9 to 37.2. The Ct values of the Liat assay were significantly lower (p < 0.001) but were correlated (p < 0.001) with those of other molecular assays. In the participants who tested positive for SARS-CoV-2 on the Liat assay using nasopharyngeal samples, 88.2% of their nasal samples also tested positive using the Liat assay. Conclusion: The Liat assay showed high concordance with other molecular assays in nasal samples. Some discordance occurred in samples with Ct values > 30 on the Liat assay.

scholarly journals Comparison of nine different commercially available molecular assays for detection of SARS-CoV-2 RNA

2021 ◽  
Author(s):  
Ute Eberle ◽  
◽  
Clara Wimmer ◽  
Ingrid Huber ◽  
Antonie Neubauer-Juric ◽  
...  
Keyword(s):  
Real Time ◽  
Rt Pcr ◽  
Diagnostic Assays ◽  
Molecular Assays ◽  
Pcr Assays ◽  
Laboratory Experiences

AbstractTo face the COVID-19 pandemic, the need for fast and reliable diagnostic assays for the detection of SARS-CoV-2 is immense. We describe our laboratory experiences evaluating nine commercially available real-time RT-PCR assays. We found that assays differed considerably in performance and validation before routine use is mandatory.

scholarly journals Implementation of Rapid Molecular Diagnostic Tests and Antimicrobial Stewardship Involvement in Acute-Care Hospitals

2020 ◽  
Vol 41 (S1) ◽  
pp. s278-s279
Author(s):  
Maiko Kondo ◽  
Matthew Simon ◽  
Esther Babady ◽  
Angela Loo ◽  
David Calfee
Keyword(s):  
Antimicrobial Stewardship ◽  
Diagnostic Tests ◽  
New Technology ◽  
Clinical Care ◽  
Bloodstream Infections ◽  
Clinical Decision ◽  
Research Network ◽  
Molecular Diagnostic ◽  
Financial Outcomes ◽  
Stewardship Program

Background: In recent years, several rapid molecular diagnostic tests (RMDTs) for infectious diseases diagnostics, such as bloodstream infections (BSIs), have become available for clinical use. The extent to which RMDTs have been adopted and how the results of these tests have been incorporated into clinical care are currently unknown. Methods: We surveyed members of the Society for Healthcare Epidemiology of America Research Network to characterize utilization of RMDT in hospitals and antimicrobial stewardship program (ASP) involvement in result communication and interpretation. The survey was administered using Qualtrics software, and data were analyzed using Stata and Excel software. Results: Overall, 57 responses were received (response rate, 59%), and 72% were from academic hospitals; 50 hospitals (88%) used at least 1 RMDT for BSI (Fig. 1). The factors most commonly reported to have been important in the decision to adopt RMDT were improvements in antimicrobial usage (82%), clinical outcomes (74%), and laboratory efficiency (52%). Among 7 hospitals that did not use RMDT for BSI, the most common reason was cost of new technology. In 50 hospitals with RMDT for BSI, 54% provided written guidelines for optimization or de-escalation of antimicrobials based upon RMDT results. In 40 hospitals (80%), microbiology laboratories directly notified a healthcare worker of the RMDT results: 70% provided results to a physician, nurse practitioner, or physician assistant; 48% to the ASP team; and 33% to a nurse. Furthermore, 11 hospitals (22%) had neither guidelines nor ASP intervention. In addition, 24 hospitals (48%) reported performing postimplementation evaluation of RMDT impact. Reported findings included reduction in time to antibiotic de-escalation (75%), reduction in length of stay (25%), improved laboratory efficiency (20%), and reduction in mortality and overall costs (12%). Among the 47 hospitals with both RMDT and ASP, 79% reported that the ASP team routinely reviewed blood culture RMDT results, and 53.2% used clinical decision support software to do so. Finally, 53 hospitals (93%) used 1 or more RMDT for non–bloodstream infections (Fig. 1). Fewer than half of hospitals provided written guidelines to assist clinicians in interpreting these RMDT results. Conclusions: RMDTs have been widely adopted by participating hospitals and are associated with positive self-reported clinical, logistic, and financial outcomes. However, nearly 1 in 4 hospitals did not have guidelines or ASP interventions to assist clinicians with optimization of antimicrobial prescribing based on RMDT results for BSI. Also, most hospitals did not have guidelines for RMDT results for non-BSI. These findings suggest that opportunities exist to further enhance the potential benefits of RMDT.Funding: NoneDisclosures: None

scholarly journals Analysis and phylogenetic comparisons of full-length VP2 genes of the 24 bluetongue virus serotypes

2007 ◽  
Vol 88 (2) ◽  
pp. 621-630 ◽  
Author(s):  
S. Maan ◽  
N. S. Maan ◽  
A. R. Samuel ◽  
S. Rao ◽  
H. Attoui ◽  
...  
Keyword(s):  
Bluetongue Virus ◽  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
Full Length ◽  
Molecular Diagnostic ◽  
Mammalian Host ◽  
Reading Frame ◽  
Diagnostic Assays ◽  
Primary Determinant ◽  
Serotype Identification

The outer capsid protein VP2 of Bluetongue virus (BTV) is a target for the protective immune response generated by the mammalian host. VP2 contains the majority of epitopes that are recognized by neutralizing antibodies and is therefore also the primary determinant of BTV serotype. Full-length cDNA copies of genome segment 2 (Seg-2, which encodes VP2) from the reference strains of each of the 24 BTV serotypes were synthesized, cloned and sequenced. This represents the first complete set of full-length BTV VP2 genes (from the 24 serotypes) that has been analysed. Each Seg-2 has a single open reading frame, with short inverted repeats adjacent to conserved terminal hexanucleotide sequences. These data demonstrated overall inter-serotype variations in Seg-2 of 29 % (BTV-8 and BTV-18) to 59 % (BTV-16 and BTV-22), while the deduced amino acid sequence of VP2 varied from 22.4 % (BTV-4 and BTV-20) to 73 % (BTV-6 and BTV-22). Ten distinct Seg-2 lineages (nucleotypes) were detected, with greatest sequence similarities between those serotypes that had previously been reported as serologically ‘related’. Fewer similarities were observed between different serotypes in regions of VP2 that have been reported as antigenically important, suggesting that they may play a role in the neutralizing antibody response. The data presented form an initial basis for BTV serotype identification by sequence analyses and comparison of Seg-2, and for development of molecular diagnostic assays for individual BTV serotypes (by RT-PCR).

scholarly journals Use of Quantitative Molecular Diagnostic Assays to Investigate Fusarium Dry Rot in Potato Stocks and Soil

2005 ◽  
Vol 95 (12) ◽  
pp. 1462-1471 ◽  
Author(s):  
D. W. Cullen ◽  
I. K. Toth ◽  
Y. Pitkin ◽  
N. Boonham ◽  
K. Walsh ◽  
...  
Keyword(s):  
Control Strategies ◽  
Disease Incidence ◽  
Harvest Date ◽  
Molecular Diagnostic ◽  
Enzyme Linked Immunosorbent Assay ◽  
Potato Seed ◽  
Fusarium Spp ◽  
Dry Rot ◽  
Diagnostic Assays ◽  
Final Harvest

Specific and sensitive quantitative diagnostics, based on real-time (TaqMan) polymerase chain reaction (PCR) and PCR enzyme-linked immunosorbent assay, were developed to detect dry-rot-causing Fusarium spp. (F. avenaceum, F. coeruleum, F. culmorum, and F. sulphureum). Each assay detected Fusarium spp. on potato seed stocks with equal efficiency. Four potato stocks, sampled over two seed generations from Scottish stores, were contaminated with F. avenaceum, F. sulphureum, F. culmorum, F. coeruleum or a combination of species, and there was a general trend towards increased Fusarium spp. contamination in the second generation of seed sampled. F. sulphureum and F. coeruleum caused significantly (P < 0.05) more disease in storage than the other species when disease-free tubers of potato cvs. Spunta and Morene were inoculated at a range of inoculum concentrations (0, 104, 105, and 106 conidia/ml). Increased DNA levels were correlated with increased disease severity between 8 and 12 weeks of storage. The threshold inoculum levels resulting in significant disease development on both cultivars were estimated to be 104 conidia/ml for F. sulphureum and 105 conidia/ml for F. coeruleum. To study the effect of soil infestation and harvest date on disease incidence, seed tubers of cvs. Morene and Spunta were planted in a field plot artificially infested with the four Fusarium spp. F. culmorum and F. sulphureum were detected in soil taken from these plots at harvest, and F. sulphureum DNA levels increased significantly (P < 0.05) at the final harvest. All four Fusarium spp. were detected in progeny tubers. There was a trend toward higher levels of F. culmorum detected in progeny tubers at the earliest harvest date, and higher levels of F. sulphureum at the final harvest. The use of diagnostic assays to detect fungal storage rot pathogens and implications for disease control strategies are discussed.

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