stool specimens
TRANSCRIPT
[Last Modified: 07/20/2009 22:14:57]
Specimen Collection
1. Collect the stool in a dry, clean, leakproof container. Make sure no urine, water, soil or other material gets in the container.
2. This table demonstrates the distribution of protozoa in relation to stool consistency and should be taken into consideration when specimens are received.
3. Fresh stool should be examined, processed, or preserved immediately. An exception is specimens kept under refrigeration when preservatives are not available; these specimens are suitable for antigen testing only.
4. Preserve the specimen as soon as possible. If using a commercial collection kit, follow the kit’s instructions. If kits are not available, the specimen should be divided and stored in two different preservatives, 10% formalin and PVA (polyvinyl-alcohol), using suitable containers. Add one volume of the stool specimen to three volumes of the preservative.
5. Insure that the specimen is mixed well with the preservative. Formed stool needs to be well broken up.
6. Insure that the specimen containers are sealed well. Reinforce with parafilm or other suitable material. Insert the container in a plastic bag.
7. Certain drugs and compounds will render the stool specimens unsatisfactory for examination. The specimens should be collected before these substances are administered, or collection must be delayed until after the effects have passed. Such substances include: antacids, kaolin, mineral oil and other oily materials, non-absorbable antidiarrheal preparations, barium or bismuth (7-10 days needed for clearance of effects), antimicrobial agents (2-3 weeks), and gallbladder dyes (3 weeks).
8. Specimen collection may need to be repeated if the first examination is negative. If possible, three specimens passed at intervals of 2-3 days should be examined.
Preservation of specimens is necessary when stool specimens cannot be examined within the prescribed time interval. Various preservatives are available (see table), with the two most commonly used being 10% aqueous formalin and PVA (polyvinyl-alcohol). If molecular detection (PCR) is required, refer to the molecular diagnosis section to obtain specific information on how to collect, preserve, and ship the specimens.
General Preservatives for Stool Specimens
Preservative Advantages Disadvantages
10% Formalin All purpose fixative
Easy to prepare
Long shelf life
Good preservation of morphology of helminth eggs, larvae, protozoan cysts, and
coccidia
Suitable for concentration procedures and UV
fluorescence microscopy
Suitable for acid-fast, safranin, and chromotrope
stains
Compatible with immunoassay kits and UV
fluorescence microscopy
Not suitable for some permanent smears stained
with trichrome
Inadequate preservation of morphology of protozoan
trophozoites
Can interfere with PCR, especially after extended
fixation time
MIF
(merthiolate-iodine-formaldehyde)
Components both fix and
stain organisms
Easy to prepare
Long shelf life
Useful for field surveys
Suitable for concentration
procedures
Not suitable for some permanent smears stained
with trichrome
Inadequate preservation of morphology of protozoan
trophozoites
Iodine interferes with other
stains and fluorescence
Iodine may cause distortion of
protozoa
LV-PVA
(low viscosity polyvinyl-alcohol)
Good preservation of morphology of protozoan
trophozoites and cysts
Easy preparation of permanent smears stained with such as trichrome (solution both preserves organisms and makes them
Inadequate preservation of morphology of helminth eggs and larvae, coccidia, and
microsporidia
Contains mercuric chloride
Difficult and expensive to
adhere to slides)
Preserved samples remain
stable for several months
dispose of
Difficult to prepare in the
laboratory
Not suitable for concentration
procedures
Cannot be used with
immunoassay kits
Not suitable for acid-fast, safranin and chromotrope
stains
SAF
(sodium acetate-acetic acid-formalin)
Suitable for both concentration procedures and preparation of permanent
stained smears
Easy to prepare
Long shelf life
Suitable for acid-fast, safranin, and chromotrope
stains
Compatible with
immunoassay kits
Requires additive (e.g., albumin-glycerin) for adhesion of specimens to
slides
Permanent stains not as good as with PVA or Schaudinn's
fixative
Schaudinn’s Fixative Good preservation of morphology of protozoan
trophozoites and cysts
Easy preparation of
permanent stained smears
Less suitable for
concentration procedures
Contains mercuric chloride
Inadequate preservation of morphology of helminth eggs and larvae, coccidia, and
microsporidia
Poor adhesion of liquid or
mucoid specimens to slides
Modified PVA
copper or zinc
Permanent smears can be made and stained with
trichrome
Zinc is preferred over copper
No mercuric chloride
Staining not consistent
Organism morphology may be
poor
Copper-morphology of cysts
and trophozoites is poor
Zinc-better morphology but
not comparable to LV-PVA
One-Vial Fixatives
(such as Ecofix, Parasafe, Unifix, Proto-fix, STF, and
others that may be available)
Concentrate and permanent smear can be made out of
one vial
Immunoassays can be done
on most
No mercuric chloride
Certain one-vial fixatives
must use certain stains
Color difference of stain
Staining not always
consistent
Sometimes more expensive
than formalin and LV-PVA
Because 10% formalin and PVA have complementary advantages (see table), it is recommended that the specimen be divided and preserved in both types of preservatives (add one volume of stool to three volumes of the preservative). Commercial two-vials kits are available for this purpose. Preserved specimens can be stored for several months.
Specimen Processing
Stool specimens can be examined fresh or preserved.
Examination of fresh specimens permits the observation of motile trophozoites, but this must be carried out without delay. Liquid (diarrheic) specimens (which are more likely to contain trophozoites) should be examined within 30 minutes of passage (not within 30 minutes of arrival in the laboratory!), and soft specimens (which may contain both trophozoites and cysts) should be examined within one hour of passage. If delays cannot be avoided, the specimen should be preserved to avoid disintegration of the trophozoites. Formed specimens (less likely to contain trophozoites) can be kept for up to one day, with overnight refrigeration if needed, prior to examination.
The following flow chart shows how specimens preserved in formalin and PVA are processed and tested at CDC:
Click on the image for a larger view. (17 Kb)
Specimens preserved in formalin can be tested directly (wet mount, immunoassay, chromotrope stain, UV fluorescence) or can be concentrated prior to further testing.
Concentration procedure separate parasites from fecal debris and increase the chances of detecting parasitic organisms when these are in small numbers. They are divided into flotation techniques and sedimentation techniques.
Flotation techniques (most frequently used: zinc sulfate or Sheather's sugar) use solutions which have higher specific gravity than the organisms to be floated so that the organisms rise to the top and the debris sinks to the bottom. The main advantage of this technique is to produce a cleaner material than the sedimentation technique. The disadvantages of most flotation techniques are that the walls of eggs and cysts will often collapse, thus hindering identification. Also, some parasite eggs do not float.
Sedimentation techniques use solutions of lower specific gravity than the parasitic organisms, thus concentrating the latter in the sediment. Sedimentation techniques are recommended for general diagnostic laboratories because they are easier to perform and less prone to technical errors. The sedimentation technique used at CDC is the formalin-ethyl acetate technique, a diphasic sedimentation technique that avoids the problems of flammability of ether, and which can be used with specimens preserved in formalin, MIF or SAF.
Formalin-Ethyl Acetate Sedimentation Concentration
1. Mix the specimen well. 2. Strain 5ml of the fecal suspension (more or less depending on its consistency)
through wetted cheesecloth-type gauze placed over a disposable paper funnel into a
15 ml conical centrifuge tube. (Conical paper cups with the tips cut off are sufficient).
3. Add 0.85% saline or 10% formalin through the debris on the gauze to bring the volume in the centrifuge tube to 15 ml. Distilled water may be used; however, Blastocystis hominis may be deformed or destroyed.
4. Centrifuge at 500 × g for 10 minutes.
5. Decant supernatant. Add 10 ml of 10% formalin to the sediment and mix thoroughly with wooden applicator sticks.
6. Add 4 ml of ethyl acetate, stopper the tube, and shake vigorously in an inverted position for 30 seconds. Carefully remove the stopper.
7. Centrifuge at 500 × g for 10 minutes.
8. Free the plug of debris from the top of the tube by ringing the sides with an applicator stick. Click here to view a video clip for proper removal of the plug (Adobe Flash). Decant the top layers of supernatant.
9. Use a cotton-tipped applicator to remove debris from sides of the centrifuge tube.
10. Add several drops of 10% formalin to resuspend the concentrated specimen. Proceed with applicable testing.
*Commercial fecal concentration tubes are available that decrease processing time and supplies needed for concentrating specimens (e.g., Fecal Parasite Concentrator, Evergreen Scientific).
Specimens preserved in PVA are mostly used for permanent staining with trichrome. Prior to staining, they are processed as follows:
1. Insure that the specimen is well mixed. 2. Prepare a smear using 2 to 3 drops of the specimen depending on density.
3. Heat fix on slide warmer set at 60°C for 5 minutes or air dry completely at room temperature.
Slides may be trichrome stained or kept for several months in a protective slide tray or box for future staining.
Staining Procedures
Permanently stained smears provide laboratories with a permanent record and a specimen that can be reexamined as needed. In addition, when organisms with unusual morphology are encountered, or when identification is difficult, the slides can be sent to a reference laboratory. For the above reasons, the permanent stained smear is recommended for use with every stool specimen submitted for a routine parasite examination. Below is a list of staining techniques that are being used at CDC.
Modified Acid-Fast Staining ProcedureThis technique is useful for the identification of oocysts of the coccidian species (Cryptosporidium, Cystoisospora, and Cyclospora), which may be difficult to detect with routine stains such as trichrome. Unlike the Ziehl-Neelsen Modified Acid-Fast Stain, this stain does not require the heating of reagents for staining.
Specimen:Concentrated sediment of fresh or formalin-preserved stool may be used. Other types of clinical specimens such as duodenal fluid, bile, pulmonary samples (induced sputum, bronchial wash, biopsies) may also be stained.
Reagents:There are four steps to this procedure requiring the following solutions:
1. Absolute Methanol 2. Acid Alcohol: 10 ml Sulfuric Acid + 90 ml Absolute ethanol. Store at room
temperature.
3. Kinyoun's Carbol fuchsin: may be purchased commercially.
4. 3% Malachite green: dissolve 3 g of malachite green in 100 ml of distilled water. Store at room temperature.
Procedure:
1. Prepare a smear with 1 to 2 drops of specimen on the slide and dry on a slide warmer at 60°C until dry. Do not make the smears too thick!
2. Fix with absolute methanol for 30 seconds.
3. Stain with Kinyoun’s carbol fuchsin for one minute. Rinse briefly with distilled water and drain.
4. Destain with acid alcohol for 2 minutes. Rinse with distilled water and drain.
5. Counterstain with Malachite green for 2 minutes. Rinse briefly with distilled water and drain.
6. Dry on a slide warmer at 60°C for about 5 minutes. Mount with a coverslip using desired mounting media.
7. Examine 200 to 300 fields using 40× or higher objectives. To confirm internal morphology, use 100× oil immersion objective.
Quality Control:A control slide of Cryptosporidium spp. from a 10% formalin preserved specimen should be included with each staining run. Cryptosporidium spp. stains a pinkish-red color. The background should stain uniformly green.
Chromotrope Staining ProcedureThis staining method was developed at CDC using various components of the trichrome staining method to differentiate microsporidia spores from background fecal elements.
Specimen:Prepare a thin smear using approximately 10 µl of 10% formalin fixed stool suspension
(unconcentrated) on a glass slide. Formalin concentrates may also be used but the number of organisms will be essentially the same as before concentration. Heat fix on a slide warmer at 60°C until completely dry (5-10 minutes).
Reagents:There are six steps to this procedure requiring the following solutions:
1. Absolute methanol 2. Chromotrope Stain:
Chromotrope 2R 6.00 gFast green 0.15 gPhosphotungstic acid 0.70 gGlacial acetic acid 3.00 mlMix ingredients and allow to stand for 30 minutes. Then add 100 ml of distilled water. Prepare fresh for use every month.
3. Acid alcohol:
90% ethanol 995.5 mlGlacial acetic acid 4.5 ml
4. 95% ethanol
5. 100% ethanol
6. Xylene or xylene substitute
Procedure:
1. Fix smear in absolute methanol for 5 minutes. 2. Place in chromotrope stain for 90 minutes.
3. Destain in acid alcohol for only 1 to 3 seconds.
4. Rinse in 95% ethanol by dipping.
5. Place in two changes of 100% ethanol for 3 minutes each.
6. Place in two changes of xylene or xylene substitute for 10 minutes each.
7. Drain slide and mount with coverslip using mounting media (e.g., permount). Examine smear after drying using at least 100× objective oil immersion or higher. Examine at least 200 to 300 oil immersion fields.
Quality Control:A control slide of microsporidia from a 10% formalin preserved specimen should be included with each staining run. Spore walls of microsporidia stain a pinkish-red color and measure about 1 µm. Change all solutions subsequent to chromotrope stain after every 10 slides to obtain proper rinsing and dehydration. A microscope with good optics is recommended for accuracy. Use at least 100× objective oil immersion magnification to detect organisms; higher magnifications are better. Because of the difficulty of identification of these small spores, it is recommended that a second reader confirm a positive diagnosis.
Quick-Hot Gram-Chromotrope Staining ProcedureThis is an alternative stain to the chromotrope procedure that is a fast, reliable, and simple method of staining smears to demonstrate microsporidian spores in fecal and other clinical specimens.
Specimen:Prepare a thin smear of the material to be stained (such as feces, urine, sputum, saliva, and cell culture supernatant) and air dry.
For formalin-fixed, paraffin-embedded tissue sections, deparaffinize as usual, hydrate in a series of alcohols, and bring the slides to water before performing the Gram’s stain.
Reagents:
1. Gram Stain Kit 2. Chromotrope Stain:
Chromotrope 2R 1.0 gFast green 0.15 gPhosphotungstic acid 0.25 gGlacial acetic acid 3.0 mlMix dry ingredients then add acetic acid. Let stand for 30 minutes and then add 100 ml distilled water. Prepare fresh for use every month.
3. Acid alcohol:
90% ethanol 995.5 mlGlacial acetic acid 4.5 ml
4. 95% ethanol
5. 100% ethanol
Note: In addition to the listed reagents, a method for heating reagents and maintaining a specific temperature is required. Since only the chromotrope stain needs to be warmed, a hot plate will suffice.
Procedure:
1. Heat-fix smear (3 times for 1 second each over a low flame or 5 minutes on a slide warmer set at 60°C). Cool to room temperature.
2. Perform Gram’s stain omitting the safranin step as follows:
a. Flood slides into gentian violet solution and let stand for 30 seconds. For tissue sections, extend time to 1 minute.
b. Rinse off excess stain gently with water.
c. Flood slides into Gram’s iodine solution and allow to remain on the slide for 30 seconds. For tissue sections, extend time to 1 minute.
d. Remove Gram’s iodine solution by gently rinsing with decolorizer solution. Hold the slide at an angle and add the decolorizer solution dropwise until it flows off the slide colorless. Take extra care during this step to achieve correct staining of spores.
e. Wash the slide gently with cold water to remove excess decolorizer solution.
3. Perform chromotrope stain as follows:
a. Place the slide in warmed (50° to 55°C) chromotrope stain for at least 1 minute. For tissue sections, extend time 30 seconds.
b. Rinse in 90% acid-alcohol for 1 to 3 seconds. Take extra care during this step to achieve correct staining of spores.
c. Rinse in 95% ethanol for 30 seconds.
d. Rinse twice, 30 seconds each time, in 100% ethanol (two separate containers are required for this step). Let dry then mount with Cytoseal60 (Stephens Scientific) or other suitable sealer, following instructions. For tissue sections, we recommend that the slides be washed briefly in a solution of 50% ethyl alcohol/50% xylene for 15 seconds, before mounting.
Quality Control:A control slide of microsporidia spores from a 10% formalin-preserved specimen should be included for each staining run.
In fecal samples, microsporidia spores should appear as dark staining violet ovoid structures against a pale green background. Yeast cells, if present, should stain either dark violet or pinkish-red and are easily differentiated from microsporidia spores.
In cytologic preparations, microsporidia spores should stain deep violet to pink violet and may contain gram-positive granules. Occasionally, spores will demonstrate a prominent equatorial belt-like stripe.
Reference: Moura H, Schwartz DA, Bornay-Llinares F, et al 1997. A new and Improved “Quick-Hot Gram-Chromotrope” Technique That Differentially Stains Microsporidian Spores in Clinical Samples, Including Paraffin-Embedded Tissue Sections. Arch Pathol Lab Med.121:888-893.
Modified Safranin Technique (Hot Method) Staining ProcedureFor Cyclospora, Cryptosporidia, and Cystoisospora species:Oocysts of Cyclospora in clinical specimens are routinely demonstrated using modified acid-fast stain (cold). However, with that technique, the oocysts stain variably from nonstaining to full staining leading to possible misidentification. The modified safranin technique produces a more uniform staining of these oocysts. The stain needs to be heated to boiling using either a hot plate or microwave.
Specimen:Concentrated sediment of fresh or formalin-preserved stool may be used. Other types of clinical specimens such as duodenal fluid may also be stained.
Reagents:There are three steps to this procedure, requiring the following solutions:
1. Acid Alcohol (3%HCL/Methanol): slowly add 3 ml of hydrochloric acid to 97 ml of absolute methanol. Store at room temperature in a tightly closed container.
2. Safranin stain (Available in Gram stain kits)
3. 3% Malachite Green: dissolve 3 g of malachite green in 100 ml of distilled water. Store at room temperature.
Procedure:
1. Prepare a thin smear on the slide and dry on slide warmer. 2. Fix in acid alcohol for 5 minutes.
3. Rinse with distilled water.
4. Place in boiling safranin for 1 minute.
5. Rinse with distilled water.
6. Counterstain with malachite green for 1 minute.
7. Rinse briefly with distilled water.
8. Dry the slide and mount with a coverslip using desired mounting media.
Quality Control:A control slide of Cyclospora spp. from a 10% formalin preserved specimen should be included with each staining run. Cyclospora spp. oocysts will stain reddish-orange. The background should stain uniformly green.
Trichrome Staining ProcedureIt is generally recognized that stained fecal films are the single most productive means of stool examination for intestinal protozoa. The permanent stained smear facilitates detection and identification of cysts and trophozoites and affords a permanent record of the protozoa encountered. Small protozoa, missed by wet mount examinations (of either unconcentrated or concentrated samples) are often seen on the stained smear. The Wheatley Trichrome technique for fecal specimens is a modification of Gomori’s original staining procedure for tissue. It is a rapid, simple procedure, which produces uniformly well-stained smears of the intestinal protozoa, human cells, yeast, and artifact material.
Specimen:The specimens usually consist of fresh stool or stool fixed in polyvinyl alcohol (PVA) smeared on microscope slides and allowed to air dry or dry on a slide warmer at 60°C. Stool preserved in sodium acetate-acetic acid-formalin (SAF) or some of the one-vial fixatives can also be used.
Reagents:There are seven steps to this procedure, requiring the following solutions:
1. 70% Ethanol plus iodine: prepare a stock solution by adding iodine crystals to 70% alcohol until you obtain a dark solution. To use, dilute the stock with 70% alcohol until a dark reddish brown color or strong tea color is obtained.
2. 70% Ethanol (twice)
3. Trichrome Stain: may be purchased commercially
4. 90% Acid Ethanol
90% ethanol 99.5 ml Acetic acid (glacial) 0.5 ml
5. 95% ethanol
6. 100% ethanol (twice)
7. Xylene or xylene substitute (twice)
Procedure:
1. For PVA smears, place the slide in 70% ethanol plus iodine for 10 minutes. For other fixatives, follow the manufacturer's instructions. Omit the iodine step for preservatives that do not contain mercuric chloride.
2. Place slide in 70% Ethanol for 5 minutes.
3. Place in second 70% Ethanol for 3 minutes
4. Place in Trichrome stain for 10 minutes.
5. Destain in 90% ethanol plus acetic acid for 1 to 3 seconds.
6. Rinse several times in 100% ethanol.
7. Place in two changes of 100% ethanol for 3 minutes each.
8. Place in two changes of xylene or xylene substitute for 10 minutes.
9. Mount with coverslip using mounting medium (e.g., permount).
10. Examine the smear microscopically utilizing the 100× objective. Examine at least 200 to 300 oil immersion fields.
Quality Control:A control slide of a known protozoan such as Giardia spp. from a PVA preserved specimen should be included with each staining run. When the smear is thoroughly fixed and the stain is performed correctly, the cytoplasm of protozoan trophozoites will have a blue green color sometimes with a tinge of purple. Cysts tend to be slightly more purple. Nuclei and inclusions (chromatoid bodies, red blood cells, bacteria) and Charcot-Leyden crystals have a red color sometimes tinged with purple. Glycogen is dissolved by the solvents and appears as a clear area in the organism. The background material usually stains green providing a nice color contrast with the protozoa.
Calcofluor White Staining ProcedureThis chemofluorescent technique is useful for the detection of microsporidia, Acanthamoeba spp., Pneumocystis jiroveci, and Dirofilaria spp. Chemofluorescent agents, such as Calcofluor, Fungi-Fluor or Uvitex 2B, also known as optical brightening agents, are rapid and inexpensive screening agents. These reagents are sensitive but nonspecific since many objects and organisms other than parasites may fluoresce. This test should be used as a quick screening tool and not for species identification.
Specimen:Prepare a thin smear using approximately 10 µl of fresh or preserved specimens on a glass slide. Specimens may include stool, urine, culture or other types of samples. Heat fix on a slide warmer at 60°C until completely dry (5-10 minutes).
ReagentsThere are two steps to this procedure requiring the following solutions:
1. Absolute methanol 2. 0.01% Calcofluor white reagent: Prepare a 0.01% solution in 0.1M Tris-buffered
saline, pH 7.2
Procedure
1. Prepare a thin smear of fecal, culture, or other sample material. 2. Fix the smear in methanol for 30 seconds.
3. Stain with 0.01% calcofluor white reagent for 1 minute.
4. Rinse with distilled water and let the smear dry.
5. Mount with a #1 thickness cover slip.
6. Examine with an UV fluorescence microscope equipped with a blue violet filter cube with a wavelength of 400 nm or less.
7. To screen for microsporidia, examine the smear with a 50× or 100× oil immersion objective. The microsporidia spores will fluoresce a brilliant blue-white color on a black background.
Quality ControlA control slide of microsporidia preserved in 10% formalin prepared from culture or from a stool specimen should be included with each staining run.
Microscopic Examination
Calibration of Microscopes Using an Ocular Micrometer:A correctly calibrated microscope is crucial because size is an important characteristic for identification of parasites. This section assumes that an ocular micrometer disk has been installed in one of the oculars and that a stage micrometer is available for calibrating the ocular micrometer. This calibration should be done for each of the microscope's objectives.
Place the stage micrometer on the microscope stage and focus on the micrometer scale, until you can distinguish between the large (0.1 mm) and the small (0.01 mm) divisions of the scale. Adjust the stage micrometer so that the "0" line on the ocular micrometer is superimposed with the "0" line on the stage micrometer. Without changing the stage adjustment, find a point as distant as possible from the two superimposed "0" lines where two other lines are also exactly superimposed. Determine the number of ocular micrometer spaces, as well as the number of millimeters on the stage micrometer, between the two points of superimposition.
For example: Suppose 48 ocular micrometer spaces (units) equal 0.6 mm. Calculate the number of mm/ocular micrometer space.
0.6 mm x 48 ocular micrometer spaces = 0.0125 mm/ocular micrometer space
Since most measurements of microorganisms are given in µm rather than mm, the value calculated above must be converted to µm by multiplying it by 1000 µm/mm.
For example:
0.125 mm ocular space × 1000 µm/mm = 12.5 µm/ocular micrometer space
Thus in this case, 1 ocular micrometer space (unit) is the equivalent of 12.5 µm.
Follow the above steps for each objective. Calibration readings should be posted on each microscope and the microscope should be recalibrated after every cleaning or changing of objectives or oculars.
Wet Mount Preparation:Before preparing a wet mount slide, the microscope should be calibrated. The objectives and oculars used for the calibration procedure should be used for all measurements on the microscope. The calibration factors should always be posted on the side of the microscope.
Protozoan trophozoites, cysts, oocysts, and helminth eggs and larvae may be seen and identified using a wet mount identification technique. To prepare a wet mount, obtain a microscope slide and the stool specimen. Take a small amount of the specimen and place it on a microscope slide. If the stool specimen is still somewhat solid, add a drop or two of saline to the specimen and mix. Ideally, two smears can be prepared on one slide, of which one can be stained with iodine. Thickness of the wet mount should be as the image below illustrates.
If desired the coverslip(s) can be sealed. A preparation of petroleum jelly and paraffin in a 1:1 ratio can be applied with a cotton tip swab as illustrated. It must be heated to approximately 70°C to both mix and use. Sealing the coverslip keeps organisms from moving when using oil immersion objectives and prevents the preparation from drying out. To seal, secure the four corners by placing a drop of hot sealant to anchor the coverslip. Spread a thin layer around the edges. Other suitable sealing preparations can be used if desired.
Systematically scan the entire coverslip area using the 10× objective as illustrated. If something suspicious is seen, a higher magnification may be necessary.
CAUTION: Bringing high power objectives too near the edge of the slide will result in the sealant smearing the objective and interfering with the optors.
Stained Slide Preparation:Permanent stained slides are used for identification of protozoan trophozoites and cysts and for confirmation of species. It also permits consultation reference and diagnosis when needed as well as providing a permanent record of organism(s) observed. The microscope should be calibrated before examination begins. Positive microscope slides as well as reference material (plates, photographs, digital images) should be available by the workstation to compare morphological details and organisms. Refer to the staining section of stools for additional information regarding which stains to use.
Normally 3 × 1 slides are used to prepare permanent stained slides. If the specimen is unpreserved, prepare a thin even smear of the material by streaking the material back and forth on the slide with an applicator stick. If necessary dilute feces with saline. For PVA fixed specimens, apply two or three drops of the specimen to the slide and with a rolling motion or an up and down dabbing motion spread the specimen evenly to cover an area roughly the size of a 22 by 22 mm coverslip. For other fixatives, check manufacturers instructions.
After the staining process is complete, systematically examine the smear microscopically utilizing the 100× oil objective. Examine at least 200 to 300 oil immersion fields. Report protozoa seen as either trophozoites and/or cysts as applicable.
UV Fluorescence Microscopy Procedure:The demonstration of Cyclospora oocysts in wet preparations is greatly enhanced by using
UV fluorescence microscopy. Despite the age of the specimen or sample, Cyclospora oocysts exhibit intense blue color when observed under a fluorescence microscope (UV excitation filter set at 330-365 nm). If this filter set is not available, a less intense green fluorescence can be obtained with blue excitation (450-490 nm). Under bright-field (differential interference contrast or DIC) microscopy, Cyclospora oocysts appear as refractile spheres (8-10 µm) with a distinct oocyst wall. The utilization of both bright-field (DIC) and fluorescence microscopy provides an efficient and reliable approach to diagnosis. However, it does not provide a permanent stained slide that can be archived.
Detection of Parasite Antigens
The diagnosis of human intestinal protozoa depends on microscopic detection of the various parasite stages in feces, duodenal fluid, or small intestine biopsy specimens. Since fecal examination is very labor-intensive and requires a skilled microscopist, antigen detection tests have been developed as alternatives using direct fluorescent antibody (DFA), enzyme immunoassay (EIA), and rapid, dipstick-like tests. Antigen detection methods can be performed quickly and do not require an experienced and skilled morphologist. Much work has been accomplished on the development of antigen detection tests, resulting in commercially available reagents for the intestinal parasites Cryptosporidium spp., Entamoeba histolytica, Giardia intestinalis (lamblia), and Trichomonas vaginalis. In addition, antigen detection tests using blood or serum are available for Plasmodium and Wuchereria bancrofti.
Specimens for antigen detectionFresh or preserved stool samples are the appropriate specimen for antigen detection testing with most kits, but refer to the recommended collection procedures included with each specific kit.
AmebiasisEIA kits are commercially available for detection of fecal antigens for the diagnosis of intestinal amebiasis. Organisms of both the pathogenic E. histolytica and the nonpathogenic Entamoeba dispar strains are morphologically identical. These assays use monoclonal antibodies that detect the galactose-inhibitable adherence protein in the pathogenic E. histolytica. The primary drawback of these assays is the requirement for fresh, unpreserved stool specimens. Several EIA kits for antigen detection of the E. histolytica/E. dispar group are available in the U.S., but only the TechLab kit is specific for E. histolytica.
CryptosporidiosisImmunodetection of antigens on the surface of organisms in stool specimens, using monoclonal antibody-based DFA assays, is the current test of choice for diagnosis of cryptosporidiosis and provides increased sensitivity over modified acid-fast staining techniques. There are commercial products (DFA, IFA, EIA, and rapid tests) available in the United States for the diagnosis of cryptosporidiosis. Several kits are combined tests for Cryptosporidium, Giardia, and E. histolytica. Factors such as ease of use, technical skill and time, single versus batch testing, and test cost must be considered when determining the test of choice for individual laboratories. The most sensitive (99%) and specific (100%) method is reported to be the DFA test, which identifies oocysts in concentrated or
unconcentrated fecal samples by using a fluorescein isothiocyanate (FITC)-labeled monoclonal antibody. A combined DFA test for the simultaneous detection of Cryptosporidium oocysts and Giardia cysts is available.
Some commercial EIA tests are available in the microplate format for the detection of Cryptosporidium antigens in fresh or frozen stool samples and also in stool specimens preserved in formalin, or sodium acetate-acetic acid-formalin (SAF) fixed stool specimens. Concentrated or polyvinyl alcohol-treated (PVA) samples are unsuitable for testing with available antigen detection EIA kits. The kits are reportedly superior to microscopy, especially acid-fast staining, and show good correlation with the DFA test. Kit sensitivities and specificities reportedly range from 93 to 100% when used in a clinical setting. Laboratories which use these EIA kits need to be aware of potential problems with false-positive results and take steps to monitor kit performance.
Rapid immunochromatographic assays are available for the combined antigen detection of either Cryptosporidium and Giardia or Cryptosporidium, Giardia, and E. histolytica. These offer the advantage of short test time and multiple results in one reaction device. Initial evaluations indicate comparable sensitivity and specificity to previously available tests.
The Meridian Merifluor DFA Kit for Cryptosporidium/Giardia, modified acid-fast stain for Cryptosporidium spp., or Wheatley's trichrome stain for Giardia spp. are used at CDC for routine identification of these parasites. These techniques can be used to confirm suspicious or discrepant diagnostic results.
GiardiasisDetection of antigens on the surface of organisms in stool specimens is the current test of choice for diagnosis of giardiasis and provides increased sensitivity over more common microscopy techniques. Commercial products (DFA, EIAs, and rapid tests) are available in the United States for the immunodiagnosis of giardiasis. DFA assays may be purchased that employ FITC-labeled monoclonal antibody for detection of Giardia cysts alone or in a combined kit for the simultaneous detection of Giardia cysts and Cryptosporidium oocysts. The sensitivity and specificity of these kits were both 100% compared to those of microscopy. They may be used for quantitation of cysts and oocysts, and thus may be useful for epidemiologic and control studies.
Some commercial EIA tests are available in the microplate format for the detection of Giardia antigen in fresh or frozen stool samples and also in stool specimens preserved in formalin, MIF, or SAF fixatives. Concentrated or PVA samples are not suitable for testing with EIA kits. EIA kit sensitivity rates were recently reported as ranging from 94-100% while specificity rates were all 100%.
Rapid immunochromatographic assays are available for the combined antigen detection of either Cryptosporidium and Giardia or Cryptosporidium, Giardia, and E. histolytica. These offer the advantage of short test time and multiple results in one reaction device. Initial evaluations indicate comparable sensitivity and specificity to previously available tests.
The Meridian Merifluor DFA Kit for Cryptosporidium/Giardia, modified acid-fast stain for Cryptosporidium spp., or Wheatley's trichrome stain for Giardia spp. are used at CDC for routine identification of these parasites. These techniques can be used to confirm suspicious or discrepant diagnostic results.
TrichomoniasisTrichomoniasis, an infection caused by Trichomonas vaginalis, is a common sexually transmitted disease. Diagnosis is made by detection of trophozoites in vaginal secretions or
urethral specimens by wet mount microscopic examination, DFA staining of specimens, or culture. Sensitivity of the assays were reported as 60% for wet mounts and 86% for DFA when compared to cultures. A kit which employs FITC- or enzyme-labeled monoclonal antibodies for use in a DFA or EIA procedure is available for detection of whole parasites in fluids. A latex agglutination test for antigen detection in vaginal swab specimens is available; the manufacturer's evaluation indicated good sensitivity and specificity.
Organism Kit name Manufacturer - distributora
Type of Testb
Cryptosporidium spp.
Crypto CELISA Cellabs EIA
PARA-TECT™ Cryptosporidium Antigen 96
Medical Chemical Corporation
EIA
ProSpecT Rapid Remel EIA
ProSpecT Remel EIA
Cryptosporidium TechLab EIA
Cryptosporidium Wampole EIA
Crypto CEL Cellabs IFA
XPect Crypto Remel Rapid
Cryptosporidium spp./Giardia lamblia
PARA-TECT™ Cryptosporidium/Giardia DFA 75
Medical Chemical Corporation
DFA
Merifluor Meridian DFA
ProSpecT Remel EIA
Crypto/Giardia CEL Cellabs IFA
ColorPAC* Becton Dickinson Rapid
ImmunoCard STAT!* Meridian Rapid
XPect Remel Rapid
Cryptosporidium spp./Giardia lamblia/Entamoeba histolytica/dispar
Triage BioSite Rapid
Entamoeba histolytica
Entamoeba CELISA Cellabs EIA
E. histolytica Wampole EIA
E. histolytica II TechLab EIA
Entamoeba histolytica/E. dispar
ProSpecT Remel EIA
Giardia lamblia Giardia CELISA Cellabs EIA
PARA-TECT™ Giardia Antigen 96
Medical Chemical Corporation
EIA
ProSpecT Remel EIA
Giardia II TechLab EIA
Giardia Wampole EIA
GiardiaEIA Antibodies, Inc. EIA
Giardia CEL Cellabs IFA
ProSpecT Remel Rapid
Simple-Read Giardia Medical Chemical Corporation
Rapid
Wuchereria bancrofti
Filariasis CELISA Cellabs EIA
a Antibodies, Inc., P O Box 1560, Davis, CA 95617-1560Becton Dickinson, 1 Becton Dr., Franklin Lakes, NJ 07417BioSite, 11030 Roselle St., San Diego, CA 92121Cellabs, P O Box 421, Brookvale, NSW 2100, AustraliaGenzyme Virotech, Gmbh, Lowenplatz 5, 66248, Russelheim, GermanyMedical Chemical Corporation, 19430 Van Ness Avenue, Torrance, CA 90501Meridian Bioscience, Inc., 3471 River Hills Dr., Cincinnati, OH 45244Novocastra, 30 Ingold Rd., Burlingame, CA 94010Remel, 12076 Santa Fe Drive, Lenexa, KS 66215TechLab, VPI Research Park, 1861 Pratt Dr., Blacksburg, VA 24060Wampole Laboratories, P O Box 1001, Cranbury, NJ 08512
b EIA = enzyme immunoassay; Rapid = immunochromatographic cartridge; DFA = direct immunofluorescence assay; IFA = indirect immunofluorescence assay.
Molecular Diagnosis
Microscopic examination is still considered the “gold standard” for the diagnosis of parasitic diseases. If an unequivocal identification of the parasite can not be made, the stool specimen can be analyzed using molecular techniques such as polymerase chain reaction (PCR). PCR amplified fragments can be analyzed by using restriction fragment length polymorphisms (RFLP) or DNA sequencing if further characterization is needed.
Specimen CollectionIf PCR is being requested on a stool specimen, the specimen must be collected in absence of
preservatives kept and shipped either refrigerated (4°C) or frozen (shipped with dry ice). Alternatively stool specimens can also be mixed in potassium dichromate 2.5% (1:1 dilution) or in absolute ethanol (1:1 dilution) and shipped refrigerated. Trichrome stained smears (for G. lamblia or E. histolytica/E. dispar) or acid-fast smears (for C. parvum or C. cayetanensis) should accompany the stool specimen when requesting PCR for any of these protozoa. All stained smears will be read first and if an identification of the parasite can be made, PCR will not be performed. PCR results take approximately 24 hours for completion. Click here for more information about shipping stool specimens to CDC.
DNA ExtractionIt is necessary to extract DNA from the stool specimens for PCR detection. Click to view the DNA extraction protocols recommended for molecular diagnosis of intestinal parasites.
Extraction of parasite DNA from fecal specimens using FastDNA® kitNote 1: Divide fecal specimens into multiple aliquots and store at -80°C without preservatives. Optionally, samples can be preserved in potassium dichromate (1:1 dilution with 5% w/v) or in absolute ethanol (1:1 dilution) and stored at 4°C.
Note 2: This protocol is a summary of the procedure included in the FastDNA® kit manual provided by the manufacturer. Please refer to the manual for detailed product information and protocols.
Special equipment needed:FastPrep FP120 Disrupter (available from Q-Biogene, Carlsbad, Calif.) or similar product.
List of reagents:
1. Phosphate buffered saline solution, 0.01M, pH 7.2 2. EDTA solution, 0.5M, pH 8.0
3. Selected reagents from the FastDNA® kit available from MP Biochemicals or similar product: CLS-VF (Cell Lysis/DNA Solubilizing Solution for Vegetation, Cat. No. 6540-402) PPS (Protein Precipitation Solution, Cat. No. 6540-403) Lysing Matrix Multi Mix E (Cat. No. 6914-050/100) Binding Matrix (Cat. No. 6540-408) SEWS-M (Salt/Ethanol Wash Solution) (Cat. No. 6540-405) DES (DNA Elution Solution) (Cat. No. 6540-406)
4. PVP (Polyvinylpyrrolidone - Cat. No. 85, 645-2, Aldrich Chemical Company, Inc., Milwaukee, WI) or equivalent product.QIAquick PCR purification kit (Cat. No. 28106, Qiagen Inc., Valencia, CA, http://www.qiagen.com) or similar product.
Procedure:
1. Select and label enough unused 1.5 ml siliconized tubes to perform the extractions. 2. Centrifuge an aliquot of 300 to 500 μl of each stool specimen at 14,000 × g at 4°C for
5 minutes.
3. Suspend the pellet obtained in the previous centrifugation in 1 ml of PBS-EDTA. Repeat this procedure two more times using the same centrifugation conditions.
4. Resuspend the pellet in PBS EDTA after the final centrifugation to obtain a total volume of approximately 300 µl of solubilized sample.
5. To the tubes containing the lysing matrix Multi Mix E Matrix, add 300 µl of the washed stool sample, 400 µl of CLS-VF, 200 µl of PPS, and PVP to a final concentration ranging from 0.1% to 1%.
6. Mix by vortexing. Close tubes tightly and place in the FP120 disrupter.
7. Run in the FP120 at 5.0-5.5 speed for 10 seconds.
8. Spin 5 minutes at 14,000 × g at room temperature in a centrifuge designed to hold 1.5 ml tubes.
9. Transfer 600 µl supernatant to new tube. Discard the tubes containing the debris and Multi Mix E Matrix.
10. Add 600 µl of Binding Matrix and mix gently by inverting the tubes.
11. Incubate 5 minutes at room temperature.
12. Spin at 14,000 × g for 1 minute at room temperature. Pour out the supernatant.
13. Resuspend the pellet in 500 µl of Sews-M. Resuspend it thoroughly by pipetting up and down.
14. Spin 1 minute at 14,000 × g at room temperature. Discard the supernatant.
15. Spin 10 seconds and remove residual liquid from the top of the matrix.
16. Resuspend the matrix in 100 µl of DES. Mix with the tip and pipette up and down as in step 13.
17. Incubate 2 to 3 minutes at room temperature.
18. Spin 2 minutes at 14,000 × g.
19. Transfer the supernatant to a clean, labeled tube and store the DNA sample at 4°C until PCR amplification.
20. OPTIONAL STEP (necessary for food samples):Further purification of the eluted DNA may be required in some samples because PCR inhibitors may not be completely removed using the described procedure. In this case, purify DNA using a QIAquick spin column, following the manufacturer's instructions. Use high quality deionized water to elute the DNA from the QIAquick spin column.
21. Store the purified DNA at 4°C until PCR amplification.
For additional information on molecular diagnosis using stool specimens, call the Division of Parasitic Diseases at (770) 488-4072
Reference:
da Silva AJ, Bornay-Llinares FJ, Moura INS, Slemenda SB, Tuttle TL, Pieniazek NJ. Fast and reliable extraction of protozoan parasite DNA from fecal specimens. Mol Diagn 1999;4:57-63.
PCR AnalysisMolecular detection of Cryptosporidium sp. , Cyclospora cayetanensis, Entamoeba histolytica, and E. dispar is performed at CDC by both conventional PCR and real-time PCR. Conventional PCR is available for Giardia lamblia and microsporidia. Click on the links above to learn more about the specific tests and to view analysis of PCR results for the respective parasites listed.
Cryptosporidiosis
Molecular diagnosis
Conventional PCR
A
A: Agarose gel (2%) analysis of a PCR diagnostic test for detection of Cryptosporidium parvum DNA. PCR was performed using primers CPBDIAGF and CPBDIAGR.1
Lane S: Molecular base pair standard (100-bp ladder). Black arrows show the size of standard bands.
Lane 1: C. parvum positive fecal specimen. The red arrow shows the diagnostic band of 435 bp for zoonotic Cryptosporidium parvum.
Real-Time PCR
A TaqMan-based real-time PCR assay for detection and speciation of Cryptosporidium parvum (bovine genotype) and Cryptosporidium hominis (human genotype) has been developed and validated at CDC.2 The assay combines the detection of two genomic targets: the 18S rRNA gene to achieve a sensitive detection of Cryptosporidium spp. and a gene with unknown function to provide species differentiation. Each DNA sample is run in two parallel reactions. The first consists of the highly sensitive detection of the Cryptosporidium 18S rRNA gene and the species-specific detection of C. parvum in a duplex format. The other reaction detects C. hominis on the species level.
Cyclosporiasis
Molecular diagnosisA nested PCR assay targeting the small-subunit ribosomal RNA has been developed.1 Preliminary results indicate that the Cyclospora-specific PCR primers cross react with some Eimeria species and the sensitivity of the assay may be low (62%).2
A
A: Agarose gel (2%) analysis of a PCR diagnostic test for detection of Cyclospora DNA. PCR was performed using nested primers CYCF1E/CYCR2B (first round) and CYCF3E/CYCR4B (second round).1,2
Lane S: Molecular base pair standard (100-bp ladder). Black arrows show the size of standard bands.
Lane 1: Cyclospora positive fecal specimen. The red arrow shows the diagnostic band for Cyclospora cayetanensis (size: 308 bp).
References:
1. Relman DA, Schmidt TM, Gajadhar A, Sogin M, Cross J, Yoder K, et al. Molecular phylogenetic analysis of Cyclospora, the human intestinal pathogen, suggests that it is closely related to Eimeria species. J Infect Dis 1996;173:440-45.
2. Pieniazek NJ, Slemenda SB, da Silva AJ, Alfano EM, Arrowood MJ. PCR confirmation of infection with Cyclospora cayetanensis. Emerg Infect Dis 1996;2:357-9.
Amoebiasis
Molecular diagnosis
Conventional PCR
In reference diagnosis laboratories, molecular analysis by PCR-based assays is the method of choice for discriminating between the pathogenic species (E. histolytica) and the nonpathogenic species (E. dispar).
A
A: Agarose gel (2%) analysis of a PCR diagnostic test for differentiation between E. histolytica and E. dispar.
Lanes 1 - 4: Amplification with the Psp3/Psp51 PCR primer pair specific for E. histolytica. Diagnostic band size - 876 bp.
Lanes 6 - 9: Amplification with the NPsp3/NPsp51 PCR primer pair specific for E. dispar. Diagnostic band size - 876 bp.
Lanes 1 and 6: E. histolytica 200:NIH, zymodeme II (positive control for E. histolytica).
Lanes 2 and 7: E. dispar 351:IMMiT, zymodeme I (positive control for E. dispar).
Lanes 3 and 8: Specimen from a patient with a liver abscess (positive with E. histolytica primers and negative with E. dispar primers). E. histolytica 333:IMMiT, zymodeme XIV.
Lanes 4 and 9: Specimen from an asymptomatic patient (positive with E. dispar primers and negative with E. histolytica primers). E. dispar 389:IMMiT, zymodeme I.
Lane 5: Molecular base pair standard, 100-bp ladder (from 600 to 1,000 bp).
Figure A contributed by Assist. Prof. Przemyslaw Myjak, Ph.D., Institute of Maritime and Tropical Medicine, Gdynia, Poland.
Real-Time PCR
A TaqMan real-time PCR approach has been validated at CDC and is used for differential laboratory diagnosis of amebiasis.2 The assay targets the 18S rRNA gene with species-specific TaqMan probes in a duplex format, making it possible to detect both species in the same reaction vessel.
References
1. Clark CG, Diamond LS. Differentiation of pathogenic Entamoeba histolytica from other intestinal protozoa by riboprinting. Arch Med Res 1992;23:15-16.
2. Qvarnstrom Y, James C, Xayavong M, Holloway B, Moura I, Visvesvara GS, et al. Comparison of real-time PCR rationales for differential laboratory diagnosis of amebiasis. J Clin Microbiol 2005;43:5491-5497.
Giardiasis
Molecular Diagnosis
A
A: 2% agarose gel analysis of a PCR diagnostic test for detection of Giardia DNA. PCR was performed using primers JW1/JW2.1
Lane S: Molecular base pair standard (100-bp ladder). Black arrows show the size of standard bands.
Lane 1: Giardia intestinalis positive fecal specimen. The red arrow shows the diagnostic band for G. intestinalis (size: 183 bp).
Reference:
Weiss JB, van Keulen H, Nash TE. Classification of subgroups of Giardia lamblia based upon ribosomal RNA gene sequences using the polymerase chain reaction. Mol Biochem Parasitol 1992;54:73-86
Microsporidiosis
Molecular methodsThe genus Encephalitozoon contains three species related to human infections: E. hellem, E. cuniculi, and E. intestinalis (syn. Septata
intestinalis). The differentiation between E. hellem and E. cuniculi cannot be achieved by morphological analysis either using light or electron microscopy. Two additional microsporidian species found in humans (Enterocytozoon bieneusi and Vittaforma corneae [syn. Nosema corneum]) can be identified by experienced microscopists only. Thus, molecular diagnosis with species-specific PCR primers is commonly believed to be the gold standard for identification of microsporidian species. Our PCR methods do not cross-amplify about 20 other microsporidian species tested at the CDC laboratory.
A
A: Agarose gel (2%) showing the diagnostic bands for species-specific PCR diagnostic primers designed for microsporidia that infect humans.
Lane S: Molecular base pair standard (100-bp ladder). Black arrows show the size of standard bands.
Lane 1: Encephalitozoon cuniculi positive specimen amplified with PCR primers ECUNF/ECUNR2, diagnostic band size: 549 bp.
Lane 2: Encephalitozoon hellem template amplified with PCR primers EHELF/EHELR1, diagnostic band size: 547 bp.
Lane 3: Encephalitozoon intestinalis template amplified with PCR primers SINTF1/SINTR3,4, diagnostic band size: 528 bp.
Lane 4: Enterocytozoon bieneusi template amplified with PCR primers EBIEF1/EBIER15, diagnostic band size: 607 bp.
Lane 5: Vittaforma cornae template amplified with PCR primers NCORF1/NCORR16, diagnostic band size: 375 bp.
References:
1. Visvesvara GS, Leitch GJ, da Silva AJ, Croppo GP, Moura H, Wallace S, et al. Polyclonal and monoclonal antibody and PCR-amplified small-subunit rRNA identification of a microsporidian, Encephalitozoon hellem, isolated from an AIDS patient with disseminated infection. J Clin Microbiol 1994;32:2760-2768.
2. De Groote MA, Visvesvara GS, Wilson ML, Pieniazek NJ, Slemenda SB, da Silva AJ, et al. Polymerase chain reaction and culture confirmation of disseminated Encephalitozoon cuniculi in patient with AIDS: Successful therapy with albendazole. J Infect Dis 1995;171:1375-1378.
3. Visvesvara GS, da Silva AJ, Croppo GP, Pieniazek NJ, Leitch GJ, Ferguson D, et al. In vitro culture and serologic and molecular identification of Septata intestinalis isolated from urine of a patient with AIDS. J Clin Microbiol 1995;33:930-936.
4. da Silva AJ, Slemenda SB, Visvesvara GS, Schwartz DA, Wilcox CM, Wallace S, Pieniazek NJ. Diagnosis of infections caused by the opportunistic microsporidian Septata intestinalis Cali et al. 1993 using PCR primers targeting the region coding for small subunit rRNA. Mol Diagn 1997 Mar;2:47-52.
5. da Silva AJ, Bornay-Llinares FJ, del Aguila de la Puente C, Moura H, Peralta JM, Sobottka I, et al. Diagnosis of Enterocytozoon bieneusi (Microsporidia) infections by polymerase chain reaction in stool samples using primers based on the region coding for small-subunit ribosomal RNA. Arch Pathol Lab Med 1997;121:874-879.
6. Pieniazek, NJ. unpublished.
Conventional PCR:DNA preparations extracted from fecal samples are tested by PCR with diagnostic primers. Amplified DNA fragments are electrophoretically resolved on an agarose gel for analysis of results.
Real-Time PCR:In real-time PCR, the DNA amplification is monitored by measuring the fluorescence signal generated in the reaction vessel. The fluorescence signal is measured every cycle and is proportional to the amount of accumulated PCR product.
The real-time PCR assays for parasite detection at CDC use either the DNA-binding dye SYBR Green or sequence-specific TaqMan probes as fluorescence detection mechanisms. Probe-based assays have the advantages of high specificity and the possibility to detect multiple targets in the same vessel by combining probes labeled with dyes with different fluorescent spectra. Assays using SYBR Green can be easier and less expensive, but caution should be exercised since all double-stranded DNA is detected, including primer-dimers and other PCR artifacts. The specificity of these assays can be improved by including a dissociation (melting) curve analysis. This helps to distinguish the correct product from possible artifacts, thus avoiding false-positive results. See illustrations for details about the fluorescence detection mechanisms using SYBR Green and TaqMan probes.
SYBR Green
A
A: The principle of SYBR Green detection in real-time PCR is outlined in the figure above.1 The fluorescent dye SYBR Green is added to the PCR mixture (1). SYBR Green is a DNA binding dye that fluoresces strongly when bound to double-stranded DNA. At the start of the reaction, very little double stranded DNA is present, and so the fluorescent signal detected by the thermocycler is low (3). As the reaction proceeds and PCR product accumulates, the amount of double-stranded DNA increases and with it the fluorescence signal (4-5). The signal is only detectable during annealing and extension, since the denaturation step contains predominantly single-stranded DNA (6).
TaqMan
B
B: The principle of TaqMan real-time PCR is depicted in the schematic above.1 The TaqMan probe is designed to be complementary to a specific sequence spanned by the PCR primers. The TaqMan probe has a reporter dye at its 5̀ end and a quencher dye at its 3̀ end. As long as the probe is intact and the reporter and the quencher dyes are in close proximity, no fluorescence signal is emitted due to the quenching effect (black arrow in 1, 2, and 3) (1). After the annealing of the TaqMan probe (2) and the primers (3), the primers are extended by the DNA polymerase. As the polymerase reaches the TaqMan probe, it uses its exonuclease activity to remove the probe one nucleotide at the time (4). This releases the reporter from the proximity of the quencher and allows for the release of a fluorescence signal from the reporter (5).
References
1. da Silva A, Pieniazek N. Latest Advances and Trends in PCR-based Diagnostic Methods. In: Dionisio D, editor. Textbook-Atlas of Intestinal Infections in AIDS. Springer; 2003. p. 397-412.
TaqMan
B
B: The principle of TaqMan real-time PCR is depicted in the schematic above.1 The TaqMan probe is designed to be complementary to a specific sequence spanned by the PCR primers. The TaqMan probe has a reporter dye at its 5̀ end and a quencher dye at its 3̀ end. As long as the probe is intact and the reporter and the quencher dyes are in close proximity, no fluorescence signal is emitted due to the quenching effect (black arrow in 1, 2, and 3) (1). After the annealing of the TaqMan probe (2) and the primers (3), the primers are extended by the DNA polymerase. As the polymerase reaches the TaqMan probe, it uses its exonuclease activity to remove the probe one nucleotide at the time (4). This releases the reporter from the proximity of the quencher and allows for the release of a fluorescence signal from the reporter (5).
References
1. da Silva A, Pieniazek N. Latest Advances and Trends in PCR-based Diagnostic Methods. In: Dionisio D, editor. Textbook-Atlas of Intestinal Infections in AIDS. Springer; 2003. p. 397-412.