scholarly journals Comparison of Assays for Sensitive and Reproducible Detection of Cell Culture-Infectious Cryptosporidium parvum and Cryptosporidium hominis in Drinking Water

2011 ◽  
Vol 78 (1) ◽  
pp. 156-162 ◽  
Author(s):  
Anne M. Johnson ◽  
George D. Di Giovanni ◽  
Paul A. Rochelle
Keyword(s):  
Cell Culture ◽  
Drinking Water ◽  
Cryptosporidium Parvum ◽  
False Positives ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Cryptosporidium Hominis ◽  
Content Type ◽  
Detection Assay ◽  
Pcr Targeting

ABSTRACTThis study compared the three most commonly used assays for detectingCryptosporidiumsp. infections in cell culture: immunofluorescent antibody and microscopy assay (IFA), PCR targetingCryptosporidiumsp.-specific DNA, and reverse transcriptase PCR (RT-PCR) targetingCryptosporidiumsp.-specific mRNA. Monolayers of HCT-8 cells, grown in 8-well chamber slides or 96-well plates, were inoculated with a variety of viable and inactivated oocysts to assess assay performance. All assays detected infection with low doses of flow cytometry-enumeratedCryptosporidium parvumoocysts, including infection with one oocyst and three oocysts. All methods also detected infection withCryptosporidium hominis. The RT-PCR assay, IFA, and PCR assay detected infection in 23%, 25%, and 51% of monolayers inoculated with threeC. parvumoocysts and 10%, 9%, and 16% of monolayers inoculated with one oocyst, respectively. The PCR assay was the most sensitive, but it had the highest frequency of false positives with mock-infected cells and inactivated oocysts. IFA was the only infection detection assay that did not produce false positives with mock-infected monolayers. IFA was also the only assay that detected infections in all experiments with spiked oocysts recovered from Envirochek capsules following filtration of 1,000 liters of treated water. Consequently, cell culture with IFA detection is the most appropriate method for routine and sensitive detection of infectiousCryptosporidium parvumandCryptosporidium hominisin drinking water.

scholarly journals Multicenter Evaluation of BD Max Enteric Parasite Real-Time PCR Assay for Detection of Giardia duodenalis, Cryptosporidium hominis, Cryptosporidium parvum, and Entamoeba histolytica

2016 ◽  
Vol 54 (11) ◽  
pp. 2681-2688 ◽  
Author(s):  
S. Madison-Antenucci ◽  
R. F. Relich ◽  
L. Doyle ◽  
N. Espina ◽  
D. Fuller ◽  
...  
Keyword(s):  
Entamoeba Histolytica ◽  
Cryptosporidium Parvum ◽  
Fluorescent Antibody ◽  
Giardia Duodenalis ◽  
The United States ◽  
Parasitic Infections ◽  
Pcr Assay ◽  
Cryptosporidium Hominis ◽  
Content Type ◽  
Common Causes

Common causes of chronic diarrhea among travelers worldwide include protozoan parasites. The majority of parasitic infections are caused by Giardia duodenalis , Entamoeba histolytica , Cryptosporidium parvum , and Cryptosporidium hominis . Similarly, these species cause the majority of parasitic diarrhea acquired in the United States. Detection of parasites by gold standard microscopic methods is time-consuming and requires considerable expertise; enzyme immunoassays and direct fluorescent-antibody (DFA) stains have lowered hands-on time for testing, but improvements in sensitivity and technical time may be possible with a PCR assay. We performed a clinical evaluation of a multiplex PCR panel, the enteric parasite panel (EPP), for the detection of these common parasites using the BD Max instrument, which performs automated extraction and amplification. A total of 2,495 compliant specimens were enrolled, including 2,104 (84%) specimens collected prospectively and 391 (16%) specimens collected retrospectively. Approximately equal numbers were received in 10% formalin (1,273 specimens) and unpreserved (1,222 specimens). The results from the EPP were compared to those from alternate PCR and bidirectional sequencing (APCR), as well as DFA ( G. duodenalis and C. parvum or C. hominis ) or trichrome stain ( E. histolytica ). The sensitivity and specificity for prospective and retrospective specimens combined were 98.2% and 99.5% for G. duodenalis , 95.5% and 99.6 for C. parvum or C. hominis , and 100% and 100% for E. histolytica , respectively. The performance of the FDA-approved BD Max EPP compared well to the reference methods and may be an appropriate substitute for microscopic examination or immunoassays.

Development of an astrovirus RT–PCR detection assay for use with conventional, real-time, and integrated cell culture/RT–PCR

2004 ◽  
Vol 50 (4) ◽  
pp. 269-278 ◽  
Author(s):  
Ann C Grimm ◽  
Jennifer L Cashdollar ◽  
Frederick P Williams ◽  
G Shay Fout
Keyword(s):  
Cell Culture ◽  
Real Time ◽  
Real Time Pcr ◽  
Environmental Water ◽  
Positive Control ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Detection Assay ◽  
Stool Samples ◽  
Pcr Method

Astrovirus is a common cause of gastroenteritis in humans that has been determined to be responsible for outbreaks of illness in several countries. Since astrovirus can be waterborne, it is important to be able to identify this virus in environmental water. We have developed and optimized a reverse transcription – polymerase chain reaction (RT–PCR) method that was able to amplify all eight astrovirus serotypes in a single reaction. In addition, a positive control construct was designed so that any inhibitors of this astrovirus assay could be detected. The assay was adapted for use in a real-time PCR assay and the sensitivity of these two methods was compared. The real-time assay was then combined with CaCo2 cell culture to produce an integrated cell culture/RT–PCR (ICC/RT–PCR) assay that was able to detect low levels of astrovirus after an incubation of 7 days or less. Also, the sensitivity of the ICC/RT–PCR assay was compared with RT–PCR alone. The methods were used to detect astrovirus in acute phase illness stool samples as well as in a water sample spiked with astrovirus.Key words: astrovirus, RT–PCR, real-time PCR, ICC/RT–PCR, environmental water.

scholarly journals Abilities of the mCP Agar Method and CRENAME Alpha Toxin-Specific Real-Time PCR Assay To Detect Clostridium perfringens Spores in Drinking Water

2013 ◽  
Vol 79 (24) ◽  
pp. 7654-7661 ◽  
Author(s):  
Andrée F. Maheux ◽  
Ève Bérubé ◽  
Dominique K. Boudreau ◽  
Romain Villéger ◽  
Philippe Cantin ◽  
...  
Keyword(s):  
Drinking Water ◽  
Water Sample ◽  
Real Time ◽  
Clostridium Perfringens ◽  
Real Time Pcr ◽  
Potable Water ◽  
Alpha Toxin ◽  
Pcr Assay ◽  
Content Type ◽  
The Public

ABSTRACTWe first determined the analytical specificity and ubiquity (i.e., the ability to detect all or most strains) of aClostridium perfringens-specific real-time PCR (rtPCR) assay based on thecpagene (cpartPCR) by using a bacterial strain panel composed ofC. perfringensand non-C. perfringens Clostridiumstrains. All non-C. perfringens Clostridiumstrains tested negative, whereas allC. perfringensstrains tested positive with thecpartPCR, for an analytical specificity and ubiquity of 100%. ThecpartPCR assay was then used to confirm the identity of 116 putativeC. perfringensisolates recovered after filtration of water samples and culture on mCP agar. Colonies presenting discordant results between the phenotype on mCP agar andcpartPCR were identified by sequencing the 16S rRNA andcpagenes. Four mCP−/rtPCR+colonies were identified asC. perfringens, whereas 3 mCP+/rtPCR−colonies were identified as non-C. perfringens. ThecpartPCR was negative with all 51 non-C. perfringensstrains and positive with 64 of 65C. perfringensstrains. Finally, we compared mCP agar and a CRENAME (concentration andrecovery of microbial particles,extraction ofnucleicacids, andmolecularenrichment) procedure pluscpartPCR (CRENAME +cpartPCR) for their abilities to detectC. perfringensspores in drinking water. CRENAME +cpartPCR detected as few as oneC. perfringensCFU per 100 ml of drinking water sample in less than 5 h, whereas mCP agar took at least 25 h to deliver results. CRENAME +cpartPCR also allows the simultaneous and sensitive detection ofEscherichia coliandC. perfringensfrom the same potable water sample. In itself, it could be used to assess the public health risk posed by drinking water potentially contaminated with pathogens more resistant to disinfection.

scholarly journals The Detection of Enteroviruses in Sewage Using Caco-2 Cells

2013 ◽  
Vol 62 (1) ◽  
pp. 97-100 ◽  
Author(s):  
MAGDALENA WIECZOREK ◽  
ŁUKASZ KURYK ◽  
AGNIESZKA WITEK ◽  
ANNA DIUWE ◽  
BOGUMIŁA LITWIŃSKA
Keyword(s):  
Cell Culture ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Beef Extract

The work presented here demonstrates the utility of Caco-2 cells to detect enteroviruses in sewage. Viruses were concentrated by beef extract elution and organic flocculation prior to analysis by cell culture assays and RT-PCR. Enteroviruses were detected in all sewage samples, but only one sample was positive solely in RT-PCR assay. We proved that Caco-2 cells were more effective than RD and L20B cells in enterovirus isolation, depending on procedures used in the inoculation process.

scholarly journals A New Highly Sensitive and Specific Real-Time PCR Assay Targeting the Malate Dehydrogenase Gene ofKingella kingaeand Application to 201 Pediatric Clinical Specimens

2018 ◽  
Vol 56 (8) ◽  
Author(s):  
Nawal El Houmami ◽  
Guillaume André Durand ◽  
Janek Bzdrenga ◽  
Anne Darmon ◽  
Philippe Minodier ◽  
...  
Keyword(s):  
Real Time ◽  
Malate Dehydrogenase ◽  
Real Time Pcr ◽  
Detection Sensitivity ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Content Type ◽  
Clinical Specimens ◽  
Specificity And Sensitivity ◽  
Pcr Assays

ABSTRACTKingella kingaeis a significant pediatric pathogen responsible for bone and joint infections, occult bacteremia, and endocarditis in early childhood. Past efforts to detect this bacterium using culture and broad-range 16S rRNA gene PCR assays from clinical specimens have proven unsatisfactory; therefore, by the late 2000s, these were gradually phased out to explore the benefits of specific real-time PCR tests targeting thegroELgene and the RTX locus ofK. kingae. However, recent studies showed that real-time PCR (RT-PCR) assays targeting theKingellasp. RTX locus that are currently available for the diagnosis ofK. kingaeinfection lack specificity because they could not distinguish betweenK. kingaeand the recently describedKingella negevensisspecies. Furthermore,in silicoanalysis of thegroELgene from a large collection of 45K. kingaestrains showed that primers and probes fromK. kingaegroEL-based RT-PCR assays display a few mismatches withK. kingae groELvariations that may result in decreased detection sensitivity, especially in paucibacillary clinical specimens. In order to provide an alternative togroEL- and RTX-targeting RT-PCR assays that may suffer from suboptimal specificity and sensitivity, aK. kingae-specific RT-PCR assay targeting the malate dehydrogenase (mdh) gene was developed for predicting no mismatch between primers and probe and 18 variants of theK. kingae mdhgene from 20 distinct sequence types ofK. kingae. This novelK. kingae-specific RT-PCR assay demonstrated high specificity and sensitivity and was successfully used to diagnoseK. kingaeinfections and carriage in 104 clinical specimens from children between 7 months and 7 years old.

scholarly journals Viroselect: A novel SARS-CoV-2 detection assay to resolve inconclusive samples

2021 ◽  
Author(s):  
Ketki Jawade ◽  
Akhauri Yash Sinha ◽  
Sharad Bhagat ◽  
Shilpa Bhowmick ◽  
Bhagyashree Chauhan ◽  
...  
Keyword(s):  
Retrospective Analysis ◽  
High Throughput ◽  
Hypervariable Region ◽  
Structural Protein ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Target Region ◽  
Detection Assay ◽  
High Concordance ◽  
Reporting Delays

ABSTRACTBackgroundIndia bears the second largest burden of SARS-CoV-2 infection. A multitude of RT-PCR detection assays with disparate gene targets including automated high throughput platforms are available. Varying concordance and interpretation of diagnostic results in this setting can result in significant reporting delays leading to suboptimal disease management. Here, we report the development of a novel ORF-1a based SARS-CoV-2 RT-PCR assay, Viroselect, showing high concordance with conventional assays and the ability to resolve inconclusive results generated during the peak of the epidemic in Mumbai, India.MethodsWe identified a unique target region within SARS-CoV-2 ORF1a, non-structural protein (nsp3), that was used to design and develop our assay. This hypervariable region (1933-3956) between SARS-CoV-2, SARS-CoV, and MERS-COV was utilized to design our primers and probe for RT-PCR assay. We further evaluated concordance of our assay with commonly used EUA (USFDA) manual kits as well as an automated high throughput testing platform. Further, a retrospective analysis using Viroselect on samples reported as ‘inconclusive’ during April-October 2020 was carried out.ResultsA total of 701 samples were tested. Concordance analysis of 477 samples demonstrated high overall agreement of Viroselect assay with both manual (87.6%; 95% CI) as well as automated (84.7%; 95% CI) testing assays. Also, in the retrospective analysis of 224 additional samples reported as ‘inconclusive’, Viroselect was able to resolve 100% (19/19) and 93.7% (192/205) samples which were termed inconclusive by manual and automated high throughput platform respectively.ConclusionWe show that Viroselect had high concordance with conventional assays, both manual and automated, as well as highlight its potential in resolving inconclusive samples.

scholarly journals Calcium-Mediated Biophysical Binding ofCryptosporidium parvumOocysts to Surfaces Is Sensitive to Oocyst Age

2019 ◽  
Vol 85 (17) ◽  
Author(s):  
Tooba Sarkhosh ◽  
X. Frank Zhang ◽  
Kristen L. Jellison ◽  
Sabrina S. Jedlicka
Keyword(s):  
Drinking Water ◽  
Carboxylic Acid ◽  
Cryptosporidium Parvum ◽  
Gastrointestinal Disease ◽  
Fate And Transport ◽  
Drinking Water Treatment ◽  
Outer Wall ◽  
Content Type ◽  
Binding Strength ◽  
Binding Forces

ABSTRACTCryptosporidium parvumcauses potentially life-threatening gastrointestinal disease in humans and may not be effectively removed from drinking water via conventional methods. Prior research has shown that environmental biofilms immobilize oocysts from the water column, but the biophysical mechanisms driving this attraction are still under investigation. This study investigates the affinity ofC. parvumoocysts to silanized surfaces. Surfaces were prepared with hydroxyl, amine, and carboxyl moieties. Binding forces between the oocysts and these engineered substrates were analyzed, with and without divalent ions, using atomic force microscopy. Binding forces were measured over several weeks to investigate the influence of age on adhesion.C. parvumoocysts bind most strongly to carboxylic acid functional groups, with rupture forces greater than that required to break noncovalent molecular bonds, regardless of oocyst age. This adhesion is shown to be due to divalent cation bridging mechanisms. In addition, the binding strength increases over a 5-week period as the oocysts age, followed by a decrease in the binding strength, which may be related to structural or biochemical changes in the outer wall-bound glycosylated proteins. This study sheds new light on the biochemical parameters that influenceC. parvumoocyst binding to surfaces. Increased understanding of how age and water chemistry influence the binding strength of oocysts may inform future developments in environmental detection and drinking water treatment, such as with the development of oocyst-specific sensors that allow for more frequent tracking of oocysts in the environment.IMPORTANCEThe mechanisms by which pathogens bind to surfaces are of interest to a wide variety of scientific communities, as these mechanisms drive infectivity, fate, and transport of the pathogenic organisms. This study begins to reveal the mechanism of direct binding ofCryptosporidium parvumto surfaces containing both carboxylic acid and amine moieties, in an attempt to understand how much of the binding ability is due to long-range electrostatic forces versus other mechanisms (specific or nonspecific) of bonding. In addition to improving the scientific understanding of fate and transport of oocysts, an expanded understanding of the binding mechanisms may aid in the development of new tools and sensors designed to detect and track oocysts in waterways. Furthermore, the methods used to examine binding in this study could be translated to other waterborne pathogens of interest.

scholarly journals Comparative Clinical Evaluation of NeoPlex RB-8 with Seeplex PneumoBacter ACE for Simultaneous Detection of Eight Respiratory Bacterial Pathogens

2019 ◽  
Vol 58 (2) ◽  
Author(s):  
Jeong Woo Kim ◽  
Seong Soo Hong ◽  
In Seop Lee ◽  
Hyun Young Chi ◽  
Soo-Ok Kim ◽  
...  
Keyword(s):  
Sensitivity And Specificity ◽  
Legionella Pneumophila ◽  
Simultaneous Detection ◽  
Nasopharyngeal Swab ◽  
Pcr Assay ◽  
Content Type ◽  
Bordetella Parapertussis ◽  
Molecular Assays ◽  
Detection Assay ◽  
Higher Sensitivity

ABSTRACT There are several convenient and accurate molecular assays to detect respiratory bacterial infection. The NeoPlex RB-8 detection kit (NeoPlex RB-8) is a new multiplex real-time PCR assay that simultaneously detects Streptococcus pneumoniae, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila, Haemophilus influenzae, Bordetella pertussis, Bordetella parapertussis, and Moraxella catarrhalis in a single test. This study compared the clinical concordance of NeoPlex RB-8 with another method, Seeplex PneumoBacter ACE detection assay (Seeplex PB ACE), which simultaneously detects S. pneumoniae, M. pneumoniae, C. pneumoniae, L. pneumophila, H. influenzae, and B. pertussis. We tested 2,137 nasopharyngeal swab and sputum specimens using both assays. For discordant Bordetella parapertussis and M. catarrhalis specimens, we also performed bidirectional sequencing. For S. pneumoniae, M. pneumoniae, C. pneumoniae, L. pneumophila, H. influenzae, and B. pertussis, which are detected by both NeoPlex RB-8 and Seeplex PB ACE, the positive and negative agreement between the two assays ranged from 91.7 to 100% (κ = 0.918 to 1). S. pneumoniae and H. influenzae were the most discordant targets and measured with higher sensitivity and specificity by NeoPlex RB-8 than Seeplex PB ACE. For Bordetella parapertussis and M. catarrhalis, which are not detected by Seeplex PB ACE, NeoPlex RB-8 sensitivity and specificity were >99%. Overall, NeoPlex RB-8 was highly comparable to Seeplex PB ACE, but NeoPlex RB-8 was more clinically accurate, with higher throughput and more convenience.

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