1.         General Procedure For Submission Of Specimen

Parasitic protozoan and helminthic infections can be diagnosed from various type of specimen depending on the sites of infection in the host.  Blood parasites are usually detected in the blood, intestinal parasites in the faeces and tissue-inhabiting parasites in aspirates, biopsy specimens, sputum or urine. The tables at the end of this chapter provide an aid to the finding of appropriate specimens for the diagnosis of various parasitic infections.

Every specimen must be labelled with the patient's name and registration number, as well as the date of collection.  It must also be accompanied by a completed request form giving full particulars of the  patient, the specimen and the required examinations.  Send all specimens to the Parasitology Unit of Infectious Disease Research Centre immediately after collection, preferably not later than 24 h.

2.         Specimens For Examination Of Intestinal Protozoan And Helminthic Infections: Collection, Preparation And Transport

2.1.       Faeces

i.          For examination of helminth ova and protozoons:

Collect 5 gram or thumb size for formed stool or 5ml for liquid stool in a clean, dry receptacle, e.g., wide mouthed, screw capped plastic container. If a specimen is to be sent to a distant place, fix specimens in PVA (polyvinyl- alcohol) and 10% formalin or SAF (sodium acetate-acetic acid-formalin). The specimen should be free of urine, antiseptic or soil.

ii.          Liquid specimens, especially those with blood and mucus, should be examined within 30 minutes of voiding, and semi-liquid specimen examined  within 1 hour of voiding so that motile forms of the protozoans can be examined. Also, this will permit the culture of low infections that may escape detection by direct examination techniques. If delay is unavoidable, preserve the specimens in PVA and 10% formalin or SAF.

For the preservation of trophozoites and cysts of intestinal protozoans for subsequent staining and identification, use PVA. With the aid of an applicator, mix thoroughly 1 part of faecal specimen with 3 parts of  PVA in a vial with a plastic cap. DO NOT USE VIALS WITH ALLUMINIUM CAPS. Cap the vial securely before sending it to the laboratory. Smears may be prepared immediately or months later for permanent staining.

For the diagnosis of cryptosporidiosis, send at least 5 ml of fresh stool to the laboratory immediately after collection. If delay is unavoidable, send stool in 10ml formol saline or SAF in the proportion of 1 part sample to 5 parts fixative.

When quantitation of helminth infection is required, send at least 5 ml of stool for egg counting. The method used in this laboratory is the Stoll's dilution technique.

Note:  Faeces from patients receiving magnesia, powdered aluminium salts, barium, bismuth, oil, or antibiotics may be unsatisfactory for identification of protozoans.

iii.         For examination of nematode larvae:

When the number of ova is small, especially in infections due to hookworm, Trichostrongylus sp., and Strongyloides sp., culture is necessary for identification. If stool is to be sent from a distant hospital, it must be collected by the modified Harada-Mori technique. The materials required are a narrow strip of filter paper (3 cm x 16 cm) and a plastic bag (4 cm x 18 cm).

Smear about 0.5 ml of faeces on one side of the filter paper leaving about 4 cm at one end and 2 cm at the other end unsmeared. Place the filter paper in the plastic bag with 4 cm unsmeared end towards the bottom. Label with patient's identification number at the 2 cm unsmeared end. Moisten the filter with a few drops of water before sealing the bag. Pack the bag with enough absorbent material in a strong box and send to the laboratory.

iv.         For examination of helminthic worms:

Live nematodes for identification should be fixed in hot (50-70^oC) alcohol followed by preservation in 70% alcohol containing 5% glycerine. Similarly, intestinal trematodes and cestodes should be fixed in the following solutions:

a)   70% alcohol,

b)   5 % formalin, or normal saline containing 5% formalin.

Note: The fixative must be heated up before use.

2.2.       Perianal Secretion and Faecal Debris

For the diagnosis of pinworm infection, a perianal swab should be taken. Helminth's eggs that resistant to drying, e.g. eggs of Ascaris, Trichuris and Taenia, may also be diagnosed from the swab. Perianal swabs should be taken in the morning before defaecation or bathing.

Place a strip of Scotch tape, about 10 cm x 2 cm, over a tongue depressor with the sticky side out. Fix it in place by folding the sticky surface of both ends of the tape inward towards the wood. Next, spread out the thighs of the subject to expose the outer anal canal. Press the tape against the right and left  perianal folds taking care to cover both wet and dry areas. Remove the tape and, with the sticky side down, spread it smoothly on a microscopic slide. At the same time, place a label bearing the subject's identification data between the slide and tape at one end. Send the specimen to the laboratory as soon as possible.  

2.3        Serum

For the serological diagnosis of amoebiasis, send 2 ml of serum in a sterile bottle. The sample must be stored at 4^oC should there be any delay in transport. If centrifugation is not available, collect 5 ml of blood into a sterile plain tube and send it to the laboratory immediately.

2.4        Sputum

Eggs of  Paragonimus and larvae of various helminths may, occasionally be found in sputum. Collect as much sputum as possible in plain bottles. In the case of very light infections, it is advisable to collect sputum over a 24-h or, when indicated, a 48-h period.

2.5.       Tissue materials

i.          Aspirates, fluids and punctures:

Tissue materials that can be used for diagnosis of Entamoeba histolytica include the following; punctures of spleen, liver or glands, pleural fluid, cerebrospinal fluid and aspirates from liver abscesses.  These specimens must be placed in sterile normal saline contained in a vial. The specimens must reach the laboratory within 30 minutes of collection. If delay is unavoidable, preserve the specimens in formol-saline or SAF and PVA.

Giardia trophozoites, eggs of liver flukes, and larvae of various helminths may sometimes be detected in duodenal or biliary aspirates. Specimens should reach the laboratory within 30 minutes of collection, otherwise they should be preserved in formol-saline or SAF and PVA.

ii.          Biopsy specimen:

Immerse biopsy materials, e.g., tissue from the appendix, in warm (approximately 37^oC) physiological saline contained in a vial. Send to the laboratory immediately. If delay is unavoidable, fix the tissue in formol-saline.

The presence of Trichinella spiralis larvae may be demonstrated in muscle  tissue obtained by biopsy or at autopsy. Send at least 0.5 cm cube of fresh muscle tissue to the laboratory immediately after collection. If delay is unavoidable, fix the tissue in formol-saline.

3.         Specimens For Examination Of Schistosomiasis: Collection, Preparation And Transport

3.1.       Blood

Aseptically collect 5 ml of venous blood into a sterile plain bottle.  Send the specimen to the laboratory immediately.  To avoid hemolysis, it is advisable to send 2 ml of serum in a sterile bottle.  The serum specimen must be kept at 4^oC should there be any delay in transport.  Seal the bottle and put it in a polythene bag.  Place the bag in a thermos flask packed with ice and send it to the laboratory.

3.2.       Stool

Schistosomiasis may be diagnosed from faeces.  Send at  least 5 ml of fresh stool to the laboratory immediately after collection.  If delay is unavoidable, send 1 ml of stool preserved in 5 ml formol saline or SAF.

3.3.       Urine

Collect at least 100 ml of urine in a screw-capped bottle.  Send it to the laboratory within 24 h of collection.

4.         Specimens For Examination Of Vaginal Trichomoniasis: Collection, Preparation And Transport

4.1.       Swab

Using a sterile speculum lubricated with sterile physiological saline, collect vaginal exudate from the lateral wall of the vagina.  Place the swab immediately in 1 ml sterile physiological saline.  Send to the laboratory within 24 h of collection so that active forms of the parasites can be examined or cultured.

4.2.       Urine

Urine may also be used for the diagnosis of Trichomonas vaginalis infection. Collect at least 100 ml of urine in a screw-capped bottle.  Send it to the laboratory within 24 h of collection.

5.         Specimens For Examination Of Filariasis: Collection, Preparation And Transport

Diagnosis of filarial infections may be established by the detection of microfilariae in blood, chylous urine, or other body fluid. It may also be established serologically by the detection of antibody using the indirect fluorescent antibody test (IFAT).  The serologic method is especially useful in cases of suspected filarial infections, e.g., tropical pulmonary eosinophilla.

5.1.       Blood

i.          For concentration techniques: 

Collect 5 ml of venous blood, preferably after 9.00 p.m. into a sterile bottle containing heparin and send it to the laboratory as soon as possible.

ii.          For thick film: 

Collect 20 or 60ul of blood using a pipette after pricking a finger or ear lobe.   Blood collection must be done at night after 9.00 p.m.  Make an oval thick blood film on a clean glass slide.  Dry it in a horizontal position, taking care to protect it from dust and pests.  Send it to the laboratory as soon as possible.

iii.         For serological diagnosis: 

Collect 5 ml of blood by venipuncture.  Allow it to clot, separate the serum and send the latter in frozen packing to the laboratory as soon as possible.  If centrifugation is not available, send 5 ml of blood in a sterile bottle.

6.         Specimens For Examination Of Malaria: Collection, Preparation And Transport

6.1        Blood Film

i.          Thick:

Clean the pulp of a finger or in the case of infants,  a big toe with 70% alcohol using a piece of gauze or a pledget of cotton wool.  Wipe the finger dry with a fresh piece of gauze or cotton wool.  Prick the finger with a lancet or sterile needle, and squeeze it between a finger and a thumb until a large globule of blood exudes.  If the finger has been dried properly, the blood will remain as a globule, if not the blood will run over the surface.  Should the blood run, wipe it away and squeeze out a fresh drop.

Place the under surface of a clean slide against the globule of  blood and with a quick circular movement in the plane of the surface, make a film the size of a ten-cent coin.  Label the slide with the patient's name, registration number and the date of  collection.  Keep the film in a horizontal position and protect it from dust and insects while it dries.  Drying may be hastened by placing the slide on a microscope lamp or by rapidly passing it a few times over a spirit flame.

Note:  The film must be of such thickness that the hands of a watch can be seen through it when it is dry.  Make sure that it is dry before staining it, otherwise it will be washed away.

ii.          Thin film:

Clean the pulp of a finger or in the case of infants,  a big toe with 70% alcohol using a piece of gauze or a pledget of cotton wool. Prick the finger with a lancet or sterile needle and squeeze out a small globule of blood. Touch the blood with the end edge of a slide or a cover- lip spreader.  Apply the spreader to a slide at an angle of about 45^o and allow the blood to extend along the edge of the spreader to about two-thirds the width of the slide, then, keeping even contact, push the spreader forward along the slide.  Dry the film quickly by waving in the air. Label the slide with the patient's name, registration number and the date of collection.

Unstained blood films, thick or thin smear, for later staining should be kept in covered cardboard slide trays or boxes to protect them from dust and insects. For despatch, wrap the dry blood films (thin films may be preliminary fixed with methanol) with long strips of paper about 8 cm wide, folding it so that there will be a layer between each slide.  Secure the bundle with rubber bands or adhesive tape.  Pack the bundle with sufficient absorbent material in a suitable tin or box.  Mark the package with the words ‘FRAGILE – HANDLE WITH CARE’.  Send the specimens to the laboratory as soon as possible, preferably within 24 h of collection.

iii.         Serological diagnosis:

Collect 5 ml of blood by venipuncture.  Allow it to clot, separate the serum and send in frozen packing to the laboratory as soon as possible.  If centrifugation is not available, send 5 ml of blood in a sterile bottle. Label the bottle with the patient's name, registration number, and the date of collection.

6.2.       Brain Smear

To establish the diagnosis of cerebral malaria at autopsy when a full post- mortem examination is not being performed, it is necessary to obtain, by needling, brain materials for smears.  This is done as follows.

Pass a stout exploring hypodermic needle, at least 7.5 cm in length, through the conjunctiva under the cover of the upper eyelid and into the brain through the orbital place of the frontal bone. The brain tissue will usually be forced into the needle but suction with a syringe may be needed.  Expel the brain tissue, about the size of the head of a pin, onto the centre of a clean side and then apply another slide crosswise.  Gently press the two slides together until the brain tissue spreads thinly to the edge, then, abruptly pull the slides apart in the plane of their surfaces thus making two thin smears.  Label the slide with the patient's name, registration number, and the date of collection of specimen.

Wrap the dry brain smears with long strips of paper, about 8 cm wide, folding it so that there is a layer of paper between each slide.  Secure the bundle with rubber bands or adhesive tape.  Pack the bundle with sufficient absorbent material in a suitable tin or box.  Mark the package ‘FRAGILE – HANDLE WITH CARE’.  Send the specimens to the laboratory as soon as possible, preferably within 24 h of collection.

7.         Specimens For Examination Of Toxoplasmosis: Collection, Preparation And Transport

Toxoplasma antibodies can be detected by indirect fluorescent antibody Test (IFAT).  Serum or blood collected on filter paper is used.

The organisms, Toxoplasma gondii, can also be detected in ventricular aspirates, biopsy specimens from lymph node, liver or spleen, and at necropsy.  It can also be detected by intra- peritoneal inoculation into mice.

7.1        Aspirates and Biopsies for Mice Inoculation Diagnosis:

Collect the appropriate aspirates and/or biopsies and immerse them in normal saline.  Despatch the specimens in frozen packing to the Parasitology Unit.

7.2.       Blood:

i.          For separation of serum:

Collect 2 ml of venous blood, separate the serum, pack it in ice, and send it to the laboratory as soon as possible.

ii.          On Filter Paper: 

In the case of infants, collect blood from a toe after pricking it with a lancet or sterile needle. Squeeze out the blood and spread it, to the size of a twenty-cent coin, on a filter paper (No. 4). Allow it to dry in air and then place it in a polythene bag.  Seal the bag with Scotch tape and despatch it in an envelope to the Division.

Table 1:           SPECIMENS FOR EXAMINATION OF CESTODE INFECTIONS

Table 2:           SPECIMENS FOR EXAMINATION OF NEMATODE INFECTIONS

Table 3:           SPECIMENS FOR EXAMINATION OF TREMATODE INFECTIONS

Table 4:           SPECIMENS FOR EXAMINATION OF PROTOZOAN INFECTIONS