A comparison of conventional microscopy, immunofluorescence microscopy and flow cytometry in the detection of Giardia lamblia cysts in beaver fecal samples

Parenteau, Monique; Martineau, Charles; Fournier, Jocelyn

doi:10.1016/s0022-1759(96)00239-6

Cited by 31 publications

(30 citation statements)

References 21 publications

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“…problem in specific detection of the target microorganisms in an accurate, low cost, and speedy fashion (2,4).…”

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confidence: 99%

“…These include both flow cytometry-based techniques and solid-phase image scanning cytometry techniques (3,4,(6)(7)(8). The latter usually uses a planar multielement photodetector, such as a charge-coupled device (CCD) camera, to make images from adjacent elements across the specimen, followed by scene segmentation and use of pattern recognition algorithms to locate the particular targets (9)(10)(11)(12)(13)(14)(15).…”

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confidence: 99%

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Automated detection of rare‐event pathogens through time‐gated luminescence scanning microscopy

Lu¹,

Jin²,

Leif³

et al. 2011

Cytometry Pt A

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Many microorganisms have a very low threshold (\10 cells) to trigger infectious diseases, and, in these cases, it is important to determine the absolute cell count in a lowcost and speedy fashion. Fluorescent microscopy is a routine method; however, one fundamental problem has been associated with the existence in the sample of large numbers of nontarget particles, which are naturally autofluorescent, thereby obscuring the visibility of target organisms. This severely affects both direct visual inspection and the automated microscopy based on computer pattern recognition. We report a novel strategy of time-gated luminescent scanning for accurate counting of rare-event cells, which exploits the large difference in luminescence lifetimes between the lanthanide biolabels, [100 ls, and the autofluorescence backgrounds, \0.1 ls, to render background autofluorescence invisible to the detector. Rather than having to resort to sophisticated imaging analysis, the background-free feature allows a single-element photomultiplier to locate rare-event cells, so that requirements for data storage and analysis are minimized to the level of image confirmation only at the final step. We have evaluated this concept in a prototype instrument using a 2D scanning stage and applied it to rare-event Giardia detection labeled by a europium complex. For a slide area of 225 mm 2 , the time-gated scanning method easily reduced the original 40,000 adjacent elements (0.075 mm 3 0.075 mm) down to a few ''elements of interest'' containing the Giardia cysts. We achieved an averaged signal-to-background ratio of 41.2 (minimum ratio of 12.1). Such high contrasts ensured the accurate mapping of all the potential Giardia cysts free of false positives or negatives. This was confirmed by the automatic retrieving and time-gated luminescence bioimaging of these Giardia cysts. Such automated microscopy based on time-gated scanning can provide novel solutions for quantitative diagnostics in advanced biological, environmental, and medical sciences. ' 2011 International Society for Advancement of Cytometry

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“…problem in specific detection of the target microorganisms in an accurate, low cost, and speedy fashion (2,4).…”

mentioning

confidence: 99%

mentioning

confidence: 99%

Automated detection of rare‐event pathogens through time‐gated luminescence scanning microscopy

Lu¹,

Jin²,

Leif³

et al. 2011

Cytometry Pt A

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“…FCM has found a broad range of applications in bacteriology and parasitology and has been used in studies of various parasitic protozoa such as Plasmodium, Toxoplasma, Eimeria, Leishmania, free living amoeba, Cryptosporidium, Giardia, and microsporidia (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22). It has been used to quantitate and determine the viability of E. cuniculi spores from culture (5), to isolate E. bieneusi spores from stool samples (19), to compare the nucleic acid contents of microsporidian spores isolated from fish (20), and to discriminate between the spores of the three species of Encephalitozoon (22).…”

Section: Discussionmentioning

confidence: 99%

Quantitation of microsporidia in cultured cells by flow cytometry

et al. 2004

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Background: Microsporidia are obligate intracellular protozoan parasites that emerged as major opportunistic pathogens in humans since the onset of the AIDS pandemic. In the present study, we investigated whether FCM is a useful method for the quantitation of intracellular microsporidian spores in cultured cells. Methods: Microsporidia (Encephalitozoon cuniculi) were grown in cell cultures and various cell-lines were coincubated with microsporidian spores at different multiplicities of infection, as well as for different periods of time. After permeabilization of the cells, intracellular spores were stained with a polyclonal anti-E. cuniculi serum and a FITC-labeled secondary antibody. Stained cells were analyzed on a flow cytometer and results were compared with those of fluorescence microscopy.

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“…Many papers reported flow cytometry as a method more sensitive than either conventional or immunofluorescence microscopy for the detection of Giardia sp. cysts in fecal samples (Dixon, 1997;Dixon, 2002;Ferrari, 2003), in detection of Cryptosporidium in SCID mice (Arrowood, 1995), seeded horse feces (Cole, 1999), and seeded human stool specimens (Valdez, 1997). In addition to detection and enumeration, large-scale sorting could also be used in conjunction with flow cytometry to yield partially purified oocysts for research purposes, such as food-spiking and recovery experiments, viability determination, or molecular characterization (Dixon, 2005).…”

Section: Jacobbergermentioning

confidence: 99%