Parasitic Infections Lab Diagnosis: Introduction, Importance and application

Parasitic Infections Lab Diagnosis: Introduction, Importance and Applications

Introduction and importance of parasitic infections lab diagnosis

Parasitic infections have also emerged as a public health issue, especially in developing countries. Correct laboratory diagnosis of such parasitic infections (Parasitic Infections Lab Diagnosis) not only helps in the early treatment of the diseases but also helps to decrease hospital stay and economic burden. It includes the selection of specimens and preparations for the patients (If required, collection of the specimen, transportation, and preservation, and processing too). Some parasites and their related diseases-

Entamoeba histolytica: Amoebic dysentery

Giradia lamblia: Diarrhoea

Leishmania donovani: Kala-azar

Plasmodium : Malaria

Trypanosoma cruzi: Chagas disease

Toxoplasma gondii: Encephalomyelitis

Taenia solium: Cysticercosis

Echinococcus granulosus : Hydatid disease

Ascaris lumbricoides: Diarrhora

Ancyclostoma : diarrhoea

Strongyloides: Diarrhoea

Trichuris trichiura : Diarrhoea

Enterobius vermicularis: Diarrhoea

Wucherechia bancrofti: Filaria

Trichomonas vaginalis : Vaginal infection

Parasite suspected and Choice of specimen for Parasitic Infections Lab Diagnosis

For most of the intestinal Parasites e.g. Entamoeba histolytica, Giardia lamblia, Ancyclostoma duodenale, Hymenolepsis nana, Enterobiuss vermicularis, Ascaris lumbricoids, Taenia species, and coccidian parasites :
Freshly passed or preserved stool
Haemoparasites e.g. Plasmodium spp., Babesia spp., Leishmania spp., T. cruzi, Wuchererchia bancrofti, Brugiya malayi, B. timori : Peripheral Blood (nocturnal periodicity especially for filarial worms)
T. Vaginalis, eggs of S. haematobium, W. bancrofti etc.: Urine( first voided or 24 hours urine) Or genital samples
Eggs of Paragonimus westermani, A. duodenale, Nector americans, S. stercoralis during migratory phase: Sputum
T. gondi, E. histolytica, L. donavani, C. parvum, Microsporidia, etc: Biopsy or aspirated samples from deeper tissues.
G. lamblia: Duodenal aspirates
Trophozoites of Nagleria fowleri, Acanthamoeba spp. : CSF
Microfilaria of Onchocerca volvulus : Skin snips

Preparation of Patients for parasitic infections lab diagnosis

Avoid antibiotics or antidiarrhoeal agents, barium enema (at least 1 week ), mineral oil, bismuth, etc. prior to fecal collection. Washing of mouth during sputum collection. Use of laxatives like magnesium sulfate ( Mgso4) if required.

Collection of specimens for parasitic infections lab diagnosis

Clean, dry, wide-mouthed container(minimum 3 specimens )
Scrappings from ulcers in the rectum or the sigmoid colon (sigmoidoscope)
Expectorated sputum, bone marrow, splenic puncture, lumbar puncture aspirates were collected by a trained person in strict aseptic precaution.

Specimen preservation for parasitic infections lab diagnosis

For better diagnosis loose stool for half an hour, semisolid an hour, and formed specimen can be kept for up to one day, with overnight refrigeration at 4°C but not recommended, as it may alter the morphology of parasites. Each preservative has specific limitations and no single solution enables all techniques to be performed with optimal results. The choice of preservative is also important during concentration technique and preparation of permanent stained smear.
Preservatives and their uses for Parasitic Infections Lab Diagnosis
10 % Formalin
Advantages
Good preservation of helminths eggs, larvae, protozoan cysts, and coccidia.
It is suitable for concentration procedures, UV fluorescence microscopy.
disadvantages
Not suitable for trichome stain.
Inadequate preservation of trophozoites.
Merthiolate –Iodine- formaldehyde ( MIF)

Advantages
Both fix and stain organisms.
Useful for field surveys.

disadvantages
Not suitable for trichome stain.
Inadequate preservation of trophozoites.
Iodine interfere with fluorescence microscopy

Low viscosity polyvinyl alcohol (LV-PVA)

Advantages
Good preservation for protozoan trophozoites and cysts.
Easy to prepare permanent smear.
suitable for trichome stain (both preserves organisms and make them adhere to slides).

Disadvantages

Inadequate preservation of helminths, coccidia, and microsporidia.
Expensive
Not suitable for concentration procedure, acid-fast, safranine, and chromotrope stains.
Sodium acetate-acetic acid-formalin (SAF)

Advantages
Suitable for both concentration procedures and preparation of permanent stained smears, acid-fast, safranine, and chromotrope stains

Disadvantages
Requires additives (e.g. albumin-glycerine) for adhesion.
Permanent stains are not as good as with PVA or Schaudin’s fixative.

Schaudinn’s fixative

Advantages
Good preservation of morphology of protozoan trophozoites and cysts.
Easy preparation of permanent stained smears.

Disadvantages
Good preservation of morphology of protozoan trophozoites and cysts.
Easy preparation of permanent stained smears.

Modified PVA (copper or Zinc)

Advantages
Permanent smears can be made and stained with trichomes.
No mercuric chloride

Disadvantages
Staining is not consistent.
Poor morphology.

2% Potassium dichromate

Advantages
Better preservative for coccidian Parasites.

Quality Control for Parasitic Infections Lab Diagnosis

A freshly prepared buffy coat blood sample is mixed with almost 2 grams of the soft fresh fecal specimen. Prepare several fecal smears and fix them immediately in Schaudinn fixative. Mix remaining feces – buffy coat mixture in 10 ml of PVA or SAF, allow 30 minutes for fixation and prepare several fecal smears. Stain slides by using a normal staining procedure. After staining if WBCs appear well fixed and display typical morphology it will ensure that any intestinal parasites placed in the same lot no. of preservative would also be fixed.

Transportation (Shipment) for Parasitic Infections Lab Diagnosis

In general, only preserved specimens should be shipped. The primary container of preserved fecal material is placed in a secondary container and sealed. It is then sent to the referral center by keeping it into a mailing container again after sealing and labeling. Glass slides – wrapped in shock absorbent material or placed in a sturdy slide container. In some cases, unpreserved specimens require e.g. for the culture of microsporidium and PCR. In these cases, the specimens must be placed in clean containers quickly as possible and should be sent in cold –Packs.
Stool for Parasitic Infections Lab Diagnosis
Macroscopic examination:
Aid in determining what types of organisms may be present. Described as formed, semiformed, soft, loose, or watery. Loose or watery- trophozoites, semi-loose –cyst stages. Blood or mucus if present may also indicate amoebic dysentery, Intestinal schistosomiasis, severe trichuriasis, etc. Body fragments of adult helminths can be seen on the surface of the stool specimen by the naked eye.
Microscopy
Direct smear preparation
It helps to assess the worm burden of a patient.

It provides a quick diagnosis of a heavily infected specimen.
It also checks organism motility.
Unstained preparation demonstrates actively motile forms of parasites (mainly trophozoites of E. histolytica, G.lamblia, larvae of Strongyloides stercoralis, etc. ) and helminthic eggs.

Saline Preparation
A minute portion of the feces is homogeneously mixed with 0.85 % sodium chloride (NaCl), on a clean and dry slide, a coverslip is placed over it.
Stained preparation for Parasitic Infections Lab Diagnosis
It is useful for the identification of cysts or dead trophozoites.

It also provides nuclear and cytoplasmic details.

Iodine preparation
It is useful, especially for protozoal cysts.
A minute portion of feces is mixed with a drop of Lugol’s Iodine homogeneously on a glass slide, cover with a coverslip.
Nuclei: pale retractile
Cytoplasm: Yellowish
Glycogen material: brown color

Nair’s buffered methylene blue preparation
It demonstrates nuclei of the trophozoites of protozoa.
Reporting
In direct smear preparation protozoan trophozoites and cysts, similarly, helminth eggs and larvae may be seen which should be reported. Some artifacts or other important structures can be seen which should be reported. e.g. Charcot-Leyden crystals, RBCs, macrophages, undigested food particles, etc. Note: Formalin preserved specimens can be used in place of saline however motility can’t be observed.

Quality control

Iodine solution or any stain(e.g. buffered methylene blue) should be checked before use or periodically (once a week). Iodine should be free of any signs of bacterial or fungal contamination. Color should be that of strong tea.
Human WBCs mixed with negative stool can be used as QC specimens, as the human cells stain with the same color as that seen in the protozoa.
Calibration of microscope for Parasitic Infections Lab Diagnosis
Correct calibration of ocular and stage micrometer of the microscope is very important because the size is an important characteristic for the identification of parasites. The use of UV fluorescence microscopy, phase contrast microscopy increases the better findings of some protozoans. e.g. coccidian parasites.

Smear after concentration for Parasitic Infections Lab Diagnosis
It increases the chances of detecting parasites even in small numbers.
It attempts to separate parasites from the bulk of fecal debris through differences in Specific gravity. Various methods applied are-

Sedimentation technique
Flotation technique

Sedimentation technique for Parasitic Infections Lab Diagnosis
Eggs and cysts heavier than the suspending liquid, become concentrated in the bottom of a tube. The most commonly used techniques are: Formalin-Ethyl-acetate sedimentation concentration: (Ritchie’s concentration method)

Procedure

Emulsify approximately 1 ml feces in 10-12 ml of normal saline.
Filter through two layers of moist gauge.
Centrifuge for 1 minute at 900 g.
Pour off supernatant and re-suspend in fresh saline.
Centrifuge again and add 10 ml of 10% formalin to sediment.
Allow standing for 5 minutes.
Add 3 ml of ethyl acetate or ether; shake vigorously (ethyl acetate extract debris and fat, remaining eggs and parasites sediment on bottom).
Centrifuge 1 minute at 900 g.
Plug between formalin and ethyl acetate or ether layer, decant supernatant, and examine sediment.

Modified Ritchie’s concentration method
(for Cryptosporidium)
To the sediment, produced by standard Ritchie’s concentration, re-suspend in 5 ml deionized water and layer over 5 ml of saturated sodium chloride. Again centrifuge at 500 g for 10 minutes, oocyst concentrated in a layer just above sodium chloride while most fecal debris at the bottom. Remove 3.5 to 4 ml of the top layer. Take the remainder of the upper layer and approximately. 0.5 ml saline layer,re-suspend in 13 ml of deionized water. Centrifuge at 500 g for 10 minutes .Decant the supernatant and prepare a smear from the sediment.

KOH concentration method
(for Cyclospora)

Place 2 ml well-mixed specimen in 15 ml of 10% KOH and mix well. Allow standing for 5 minutes at room temperature. Add 8-10 ml saline and mix. Filter through 4 layers of the gauge into a clean centrifuge tube. Centrifuge at 2000 rpm for 2 minutes, decant and discard the supernatant. Re-suspend sediment in approximately 10 ml saline centrifuge at 2000 g for 2 minutes. Decant and discard the supernatant and finally examine sediment.

Flotation technique for Parasitic Infections Lab Diagnosis -Use of a solution having higher specific gravity than the organism where the parasitic cyst and eggs having low specific gravity float on the surface.

Solutions used are:
Zinc sulfate (ZnSO4), Sheather’s sugar, etc.
Zinc sulfate flotation technique
Freshly collected stool or preserved stool is mixed with 0.85 % NaCl ( i.e. isotonic solution), centrifuge and re-suspend (if required), and again centrifuge.
Decant supernatant fluid and re-suspend with 1-2 ml of ZnSO4.
Centrifuge for 1 minute at 5000 g .
2 layers will appear, from the surface take a drop of egg or cyst-rich fluid and examine.
Modified Sheather’s sugar flotation
(for Cyptosporidium)
Fecal suspension + Sheather’s sugar flotation solution. Stir vigorously. Centrifuge and examine the smear from the surface and observe with a phase-contrast microscope. Not float on a saturated salt solution: eggs of Ascaris, Taenia spp., operculated eggs of trematodes, Larvae of Strongyloides, etc. Stains used for direct smears
Modified D’Antoni’s Iodine
Distilled water (D/W): 100 ml
Potassium iodide (KI): 1 gm
Powdered crystals of iodine (I): 1.5 gm
Lugol’s Iodine
D/W: 100 ml
KI: 10 gm
Iodine: 5 gm
Gram’s Iodine
Lugol’s Iodine: 1 part
D/W: 14 part

MIF stain
Lugol’s solution: 0.1 ml
Formaldehyde solution: 0.125 ml
tincture of Merthiolate: 0.775 ml

Permanent Stained smear for Parasitic Infections Lab Diagnosis
It facilitates the detection and identification of cysts and trophozoites. It gives better cytological detail and confirms the findings from direct smear and smear examined after concentration. It provides laboratories to maintain a permanent record. Useful for study purposes and can be sent to a consultant for the confirmation of doubt or unusual identification. A permanent smear is recommended for use with every stool specimen submitted to routine parasite examination, but the question arises how practical is it? Preparation of slides for permanent staining: a small number of faces is transferred to a clean slide with an applicator stick. The material is then streaked out in a thin, uniform film. Note: stool that does not stick properly on the slide, requires some adhesives on the slide (e.g. serum, egg albumin). Smears from fresh specimens should be placed immediately in the preservative of choice before drying.

Staining procedures most commonly used for Parasitic Infections Lab Diagnosis are-

Modified Acid-fast staining Procedure
useful for the identification of oocysts of the coccidian parasites
Reagents
Absolute methanol
Acid alcohol
Kinyoun’s carbol fuchsin
3 % malachite green

Procedure
Prepare smear and fix it with absolute methanol for 30 seconds. Stain with Kinyoun’s carbol fuchsin for one minute. Rinse in distilled water and drain. Destain with acid alcohol for 2 minutes. Rinse with distilled water and drain. Counterstain with malachite green for 2 minutes. Rinse in distilled water, air dry, and examine under a microscope.

Quality control
A control slide of Cryptosporidium spp. from a 10 % formalin preserved specimen should be included in each staining run. Cryptosporidium spp. stains a pinkish-red color against a uniformly green background.

Gomori’s trichome stain for Parasitic Infections Lab Diagnosis
It is a rapid, simple method that uniformly stains intestinal protozoa, human cells, yeast, and artifact material.
Specimen: Fresh or PVA/SAF preserved stool
If fresh, then fix in Schaudinn’s fluid for 30 minutes.
Wash in 70 % alcohol for 15 minutes.
Wash in 70 % alcohol and add sufficient Iodine.
(start for mercury preserved specimen)

Wash in 70 % alcohol, 2 changes-each every 2 minutes.
( Start for nonmercury contained PVA, SAF preserved)

Stain in Gomori’s Trichome for 8 to 15 minutes.

Rinse 90 % alcohol with 1 % acetic acid for 1-2 seconds (s).

Dip twice in 100 % alcohol.

Dehydrate in second change, clear with xylene, and mount.

Result
The cytoplasm of trophozoites: blue-green with a purple tinge.
Cyst: slightly purple
Nuclei and inclusions( RBCs, chromatoid bodies, bacteria): red
Glycogen dissolved: appear as a clear area
Background: green.

Quality control

Control slide of known protozoa such as Giardia spp. from PVA preserved specimen should be included with each staining run.

Ryan’s trichome blue stain
(for Microsporidial spore)
Difficult to identify due to their small size (1-1.8 um)
The technique stains well as pinkish-red spore walls having a polar tube against the bluish background. Bacteria, yeast, and various debris also stain red.

Thus known positive control should be run for comparison.
Calcofluor white staining procedure
Chemifluorescent technique- It is useful for the detection of microsporidia and Acanthamoeba spp. It is used only for screening because It is nonspecific that other material may also fluoresce.
Quality control: A control slide of microsporidia preserved in 10 % formalin –included with each staining run.

Special procedures for recovery of Helminths larvae and eggs for Parasitic Infections Lab Diagnosis

Baermann’s technique is useful for the concentration of Strongyloides stercoralis larvae.

The technique exploits the tendency of Strongyloides larvae to migrate from solid into surrounding liquid medium when stimulated by slightly elevated temperature and then to settle to the bottom. Larvae migrated towards the bottom of the funnel and settle down on the underneath tube.

Filter paper strip procedure for recovery of Strongyloides larvae
Agar Plate method
( for Strongyloides larvae)
More sensitive than Baermann and filter paper strip method.
Procedure
Take approximately 2 ml of stool and place it in the center of the culture plate.

Seal it with adhesive tape and incubate at room temperature for 48 hours.
Larvae migrate over the surface of agar which can be seen as tracks (Larvae of Strongyloides exhibit whip-like movement while hookworm larvae glide like snakes).

Confirmation is done by a microscopic study from the washing material of the agar surface.
Cellophane Tape swab or NIH swab (for Pin-worm egg)

Schistosomal Hatching test
Feces containing viable schistosomes eggs hatched within a few hours in presence of water to miracidia larvae that migrate toward light can be observed by magnification lens on the illuminated area of the side armor water in the neck of the flask examined.

Methods for the examination of worm burden for Parasitic Infections Lab Diagnosis
Quantitatively estimates ova which are then utilized to estimate the no. of worms infecting the patient – helpful for the treatment and follow up.

Stoll’s egg counting technique
The entire 24 hours stool specimen is measured in grams.
Place feces in a calibrated bottle or large test tube and add sufficient N/10 NaOH to bring volume to 60 ml.
Add a few glass beads and shake vigorously to make a uniform suspension.
With a pipette remove immediately 0.15 ml suspension and drain onto a slide and examine all the eggs on the slide.
Multiply egg counted by 100 to obtain the eggs per gram of feces.
Kato’s thick-smear technique: Pseudo-parasites and pitfalls during microscopy: Pseudo-parasites is an object that resembles a parasite or its eggs but is neither a parasite nor parasitic in the host under consideration. Pseudo-parasites and other artifacts sometimes may lead to misinterpretation as a false-positive result that has to be kept in mind by every examiner. Some pitfalls are as follows: Oil droplets in the stool may be confused with cyst-which has to be distinguished by seeing internal details. Trichuris egg-like objects from the stool of a person taking an “Australian pollen” dietary supplement may confuse with Trichuris. Polymorphs, leucocytes, macrophages should be differentiated from cysts. Certain pollen grains with equal size may confuse with Cyclospora, Cryptosporidium, etc.

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