Combination of cell culture and quantitative PCR (cc–qPCR) to assess disinfectants efficacy on Cryptosporidium oocysts under standardized conditions

Shahiduzzaman, M.; Dyachenko, Viktor; Keidel, J.; Schmäschke, Ronald; Daugschies, Arwid

doi:10.1016/j.vetpar.2009.09.042

Cited by 28 publications

(10 citation statements)

References 26 publications

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“…As sensitivity and specificity may be difficult to interpret, further information about the test, such as the posttest probability of infection, may help in the interpretation of the results vis-à-vis an animal's disease status (34). In this study, qPCR detected infection in all of the calves, including recently infected asymptomatic animals, and it may be the ideal test for on-farm control programs and epidemiological investigations, as demonstrated in previous studies (14,21,27). The disadvantages to using this procedure are that expensive equipment and reagents and trained personnel are required to carry it out.…”

Section: Discussionsupporting

confidence: 61%

“…In conclusion, this study has shown that the sensitivity and [3] ϳ dbeta(1,1)I(0.9,1) th1 [4] ϳ dbeta(1,1)I(0.4,0.8) th1 [5] ϳ dbeta(1,1)I(0.4,0.7) th1 [6] ϳ dbeta(1,1)I(0.7,0.9) th1 [7] ϳ dbeta(1,1)I(0.3,0.6) th1 [8] ϳ dbeta(1,1)I(0,0.5) th1 [9] ϳ dbeta(1,1)I(0,0.5) th1 [10] ϳ dbeta(1,1)I(0,0.5) th1 [11] ϳ dbeta(1,1)I(0,0.5) th1 [12] ϳ dbeta(1,1)I(0.7,1) th1 [13] ϳ dbeta(1,1)I(0.7,1) th1 [14] ϳ dbeta(1,1)I(0.7,1) th1 [15] ϳ dbeta(1,1)I(0.7,1) th1 [16] ϳ dbeta(1,1)I(0,0.1) th1 [17] ϳ dbeta(1,1)I(0,0.1) th1 [18] ϳ dbeta(1,1)I(0,0.1) th1 [19] ϳ dbeta(1,1)I(0,0.1) th1 [20] ϳ dbeta(1,1)I(0,0.1) th1 [21] ϳ dbeta(1,1)I(0,0.1) th1 [22] ϳ dbeta(1,1)I(0,0.1) th1 [23] ϳ dbeta(1,1)I(0,0.1) th1 [24] ϳ dbeta(1,1)I(0.7,1) th1 [25] ϳ dbeta(1,1)I(0.7,1) th1 [26] ϳ dbeta(1,1)I(0.7,1) th1 [27] ϳ dbeta(1,1)I(0.7,1) th1 [28] ϳ dbeta(1,1) th1 [29]…”

Section: Discussionmentioning

confidence: 99%

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Age-Stratified Bayesian Analysis To Estimate Sensitivity and Specificity of Four Diagnostic Tests for Detection of Cryptosporidium Oocysts in Neonatal Calves

Waele¹,

Berzano²,

Berkvens³

et al. 2011

J Clin Microbiol

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There is no gold standard diagnostic test for the detection of bovine cryptosporidiosis. Infection is usually highest in 2-week-old calves, and these calves also excrete high numbers of oocysts. These factors may give rise to variations in the sensitivity and specificity of the various diagnostic tests used to detect infection in calves of various ages. An age-stratified Bayesian analysis was carried out to determine the optimum diagnostic test to identify asymptomatic and clinical Cryptosporidium sp. infection in neonatal calves. Fecal samples collected from 82 calves at 1 week, 2 weeks, 3 weeks, and 4 weeks of age were subjected to the following tests: microscopic examination of smears stained with either phenol-auramine O or fluorescein isothiocyanate (FITC)-conjugated anti-Cryptosporidium monoclonal antibody, nested-PCR, and quantitative real-time PCR. The results confirmed a high prevalence of Cryptosporidium sp. infection, as well as a high level of oocyst excretion, in 2-week-old calves. The sensitivities of all the tests varied with the age of the calves. Quantitative real-time PCR proved to be the most sensitive and specific test for detecting infection irrespective of the age of the calf. The microscopic techniques were the least sensitive and exhibited only moderate efficiency with 2-week-old calves excreting large numbers of oocysts, the majority of which were diarrheic. It was concluded that, when interpreting the results of routine tests for bovine cryptosporidiosis, cognizance should be taken of the sensitivity of the tests in relation to the age of the calves and stage of infection.

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Section: Discussionsupporting

confidence: 61%

Section: Discussionmentioning

confidence: 99%

Age-Stratified Bayesian Analysis To Estimate Sensitivity and Specificity of Four Diagnostic Tests for Detection of Cryptosporidium Oocysts in Neonatal Calves

Waele¹,

Berzano²,

Berkvens³

et al. 2011

J Clin Microbiol

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“…The parasite has been detected in surface water bodies used for recreation, commercially harvested shellfish generally consumed raw or undercooked (see review 40,50) and for drinking water that could infect humans. Moreover, contrary to running water, using filter- However, although cell culture combined with qPCR has been largely described for C. parvum oocysts (38,39,42,43,51), only a few studies have described cell culture from T. with biliary salt suspension. We tested different protocols to break the oocyst wall while keeping the sporocyst wall intact (44,45,53).…”

Section: Discussionmentioning

confidence: 99%

“…In vitro cell culture assays are alternative approaches to bioassays. They have been widely applied associated with qPCR or RT-qPCR methods to detect infectious viruses (36,37), and Cryptosporidium parvum (38)(39)(40)(41)(42)(43) and T. gondii oocysts (44,45) following sporozoite excystation. In these latter studies, excystation relied on a mechanical treatment to obtain free sporocysts, followed by their incubation with bile salts to release the sporozoites.…”

Section: Introductionmentioning

confidence: 99%

Toxoplasma gondii Oocyst Infectivity Assessed Using a Sporocyst-Based Cell Culture Assay Combined with Quantitative PCR for Environmental Applications

Rousseau

Escotte-Binet

Carbona

et al. 2019

Appl Environ Microbiol

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Toxoplasma gondii is a ubiquitous foodborne protozoan that can infect humans at low dose and displays different prevalences among countries in the world. Ingestion of food or water contaminated with small amounts of T. gondii oocysts may result in human infection. However, there are no regulations for monitoring oocysts in food, mainly because of a lack of standardized methods to detect them. The objectives of this study were (i) to develop a reliable method, applicable in biomonitoring, for the rapid detection of infectious oocysts by cell culture of their sporocysts combined with quantitative PCR (sporocyst-CC-qPCR) and (ii) to adapt this method to blue and zebra mussels experimentally contaminated by oocysts with the objective to use these organisms as sentinels of aquatic environments. Combining mechanical treatment and bead beating leads to the release of 84% ± 14% of free sporocysts. The sporocyst-CC-qPCR detected fewer than ten infectious oocysts in water within 4 days (1 day of contact and 3 days of cell culture) compared to detection after 4 weeks by mouse bioassay. For both mussel matrices, oocysts were prepurified using a 30% Percoll gradient and treated with sodium hypochlorite before cell culture of their sporocysts. This assay was able to detect as few as ten infective oocysts. This sporocyst-based CC-qPCR appears to be a good alternative to mouse bioassay for monitoring infectious T. gondii oocysts directly in water and also using biological sentinel mussel species. This method offers a new perspective to assess the environmental risk for human health associated with this parasite. IMPORTANCE The ubiquitous protozoan Toxoplasma gondii is the subject of renewed interest due to the spread of oocysts in water and food causing endemic and epidemic outbreaks of toxoplasmosis in humans and animals worldwide. Displaying a sensitivity close to animal models, cell culture represents a real alternative to assess the infectivity of oocysts in water and in biological sentinel mussels. This method opens interesting perspectives for evaluating human exposure to infectious T. gondii oocysts in the environment, where oocyst amounts are considered to be very small.

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“…Methods employed to make controls that are used as templates to establish standard curves for Cryptosporidium qPCR quantification, vary from serial dilutions of template DNA extracted from a known stock concentration of oocysts [21–23], serial dilution of a known stock concentration of oocysts and subsequent extraction [24, 25], template prepared from inoculation of serial diluted oocyst suspensions into cell culture [26–28], the serial dilution of extracted template DNA from a known quantity of oocysts inoculated onto cell culture [27], or serial dilution of cloned amplification targets in plasmids [27, 29].…”

Section: Introductionmentioning

confidence: 99%

Assessment of differences between DNA content of cell-cultured and freely suspended oocysts of Cryptosporidium parvum and their suitability as DNA standards in qPCR

et al. 2019

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BackgroundAlthough more modern methods are available, quantitative PCR (qPCR) is reproducible, sensitive and specific with instruments and expertise readily available in many laboratories. As such, the use of qPCR in Cryptosporidium research is well established and still widely used by researchers globally. This method depends upon the generation of standards at different concentrations to generate standard curves subsequently used for the quantification of DNA.MethodsWe assessed four types of DNA template used to generate standard curves in drug screening studies involving Cryptosporidium spp.: (i) serially diluted Cryptosporidium parvum oocysts (106–1); (ii) diluted template DNA from pure oocysts (×10–×106 dilution of 106 oocyst DNA template); (iii) oocysts incubated in human ileocecal adenocarcinoma (HCT-8) cells (105–1 and 5 × 104–50); and (iv) diluted DNA template (5 × 104) from cell culture incubated parasites (×10–×1000).ResultsSerial dilutions of both cell culture and pure oocyst suspension DNA template yielded better linearity than cell culture derived standards, with dilutions of 106 oocysts exhibiting similar quantification cycle (Cq) values to those obtained from DNA template dilutions of 106 oocysts. In contrast, cell culture incubated oocysts demonstrated significantly higher DNA content than equivalent freely suspended oocysts and diluted DNA template from both cell culture derived and freely suspended oocysts across numerous concentrations.ConclusionsFor many studies involving Cryptosporidium, only relative DNA content is required and as such, the superior linearity afforded by freely suspended oocysts and diluted DNA template (from either cell culture derived standards or freely suspended oocysts) will allow for more accurate relative quantification in each assay. Parasite division in the cell culture standards likely explains the higher DNA content found. These standards, therefore, have the potential to more accurately reflect DNA content in cell culture assays, and despite more modern methods available for absolute quantification, i.e. droplet digital PCR (ddPCR), the ubiquity of qPCR for the foreseeable future encourages further investigation into the reduced linearity observed in these standards such as varying oocyst seeding density, non-linear growth rates and assay efficiency.

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Combination of cell culture and quantitative PCR (cc–qPCR) to assess disinfectants efficacy on Cryptosporidium oocysts under standardized conditions

Cited by 28 publications

References 26 publications

Age-Stratified Bayesian Analysis To Estimate Sensitivity and Specificity of Four Diagnostic Tests for Detection of Cryptosporidium Oocysts in Neonatal Calves

Age-Stratified Bayesian Analysis To Estimate Sensitivity and Specificity of Four Diagnostic Tests for Detection of Cryptosporidium Oocysts in Neonatal Calves

Toxoplasma gondii Oocyst Infectivity Assessed Using a Sporocyst-Based Cell Culture Assay Combined with Quantitative PCR for Environmental Applications

Assessment of differences between DNA content of cell-cultured and freely suspended oocysts of Cryptosporidium parvum and their suitability as DNA standards in qPCR

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