DIAGNOSIS OF PARASITIC INFECTIONS

DIAGNOSIS OF PARASITIC INFECTIONS

Several different methods are available for detecting parasitic infections. These include;

a. Serological tests or immunodiagnosisb. Xenodiagnosis or radiologyc. In vitro culture techniqued. Direct microscopic examination of secretions, excretions and tissues of

the host.The choice of a particular technique depends on;

a. Available resourcesb. The type of product to be examinedc. The organism(s) suspected to be presentd. The training and experience of the investigatore. The degree of certainty required by the investigation (e.g presence of

parasite versus intensity of infection)f. The purpose that the test is to serve.

- Diagnostic issues are different depending on the purpose for which they are sought. For example, for public health purposes, diagnostic tools are used to facilitate effective treatment. Whereas an epidemiologist uses diagnostic tools to quantitative transmission or assess the impact of control measures on incidence of infection, prevalence, overall morbidity or transmission.

g. The environment in which the investigation is to be carried out should also be considered in choosing a diagnostic method to use in ones study.

It is often necessary to apply a number of diagnostic techniques together as only one technique does not often give all the information one may be interested in.

Sensitivity and specificity are key issues in diagnostics.

Let us now consider each of these diagnostic techniques- in terms of their merits and their drawbacks.

Serological Test/Immunodiagnosis

This is very sensitive. But much is still desired of its accuracy.

Serological tests are based on the detection of specific antibodies which are called up by specific infections or antigens.

- Mixed infections do occur, so under such situations, serological testing will be most inaccurate.

- Moreover, it has been shown that the mere presence of a specific antibody does not confirm that the host is suffering from a specific infection or disease. Indeed it could be a manifestation of already healed infection. Serological tests make no distinction between the presence of a disease causing organism and the recovered condition f the disease.

- Thirdly, serological tests require specialized techniques and equipments, which make them expensive, and thus limit their use.

Serological methods are however widely used in diagnosis of parasitic infections, especially in human medicine because of the speed of performance of the tests- the tests are usually fast.

Xenodiagnosis or Radiology, Utrasonography e.t.c. belong to the ream of medical physics.

They are sometimes employed in the diagnosis of paragonimiasis and cestode infections where cysts might be located in atopic sites. E.g. Malaria in the brain.

Again this test is fast but expensive equipments are required. A lot of skill is also needed.

In Vitro Culture

This method is also helpful and it is widely used in detecting parasitic infections, in situations were parasite-load is very low. For example, in cases of malaria in the blood, Amoebiasis, Toxoplasmosis. Trypanosomiasis, e.t.c.

Using this technique, all one does is to take a small sample of the specimen (Blood, stool, sputum, e.t.c) and inoculate it into a suitable medium (say blood agar slope) and maintain the inoculate at appropriate temperature (say - for blood 280C) for a number of days. If the material contains parasites, the parasites will grow and possibly multiple in the medium.

As I indicated earlier, this technique is practically useful were parasitemia is very low. The media required could sometimes be expensive.

Direct Microscopic Examination

Diagnosis of parasitic infections based on clinical findings from X-ray examination, ultrsonography, immunodiadnosis and other methods is supported or confirmed by the demonstration of the parasite. This is usually achieved by microscopic examination of

stool, blood, urine, sputum and other secretions, excretions and tissues specimens of the host.

The most important methods of microscopic investigationUnder suitable conditions all parasites which affect man and animals can be demonstrated relatively easily (-presupposing relevant experience) in blood or tissues and in the excreta (urine, stools) with the aid of the light microscope. In everyday practice only a few species require immunodiagnostic detection methods (e.g. species of the genera Toxoplasma and Trichinella). In microscopic investigations of worm infections it must always be remembered that the prepatent period (the time between infection and the formation of demonstrable stages, e.g. eggs, larvae) varies depending on the species of species of parasite. Prior to the elapse of this period there is no prospect of obtaining a positive result, even if specific symptoms of disease are present.Caution should always be exercised when handling material suspected of containing parasites. Samples of blood and stools should basically be considered as infectious with regard to fungi, bacteria and viruses. Cysts of amoebae and Giardia lamblia, sometimes larvae of Strongyloides species and some tapeworm eggs are capable of direct infection. Samples of blood urine and stools not used in the investigation must be adequately disinfected or sterilized and then disposed of..

Investigators must also familiarize themselves with artifacts of a non-parasitic nature present in specimens, so as not to confuse these with parasites. For example, leucocytes in a fresh stool preparation are often taken for amoebic cysts or if observed in the urinary sediment are mistaken for non-flagellated cells of Trichomonas species. Another source of confusion is ciliated cells, which in pathogenic processes are sometimes shed in increase numbers (e.g. from the nasal mucosa or the female genital tract) and which attract attention in fresh preparations because their cilia are still beating. They then give the impression of being ciliates. Pollen grains are sometimes taken for protozoan cysts or worm eggs, and plant and synthetic fibers for worms or worm larvae.

Examination by microscopy requires much patience and care. When using concentration techniques, for example, at least 100 visual fields, or if possible an entire preparation, should be systematically scanned. This applies to both blood and stool investigations.

A special characteristic of the blood microfilariae is that, because of their periodicity, which depends on the species, they only appear in the peripheral blood during the daytime or night. In the event of inadequate morphological knowledge and experience the advice of a specialist laboratory should be sought.

Diagram

Pollen grains that are found in faces and which are often mistaken for parasite eggs. 1 borage; 2 mullein; 3 mallow and marshmallow; 4 linden tree; 5 poppy; 6 fir and other conifers; 7 coltsfoot; 8 artichoke; 9 violet; 10 cabbage; 11 rhododedron; 12 broom. [From BRUMPT et al. (1951).]

Microscopic Examination of blood sample(For malaria parasites, trypanosomes, microfilariae)

When examining the blood for parasites by light microscopy, thin and thick blood films should always be prepared.

The thin blood film (staining method can also be used for organ and biopsy impression smears):

How to Prepare A Thin Blood FilmA small drop of blood is placed at one end of a clean glass slide. A second slide (or cover glass) is placed at an angle on the first slide in contact with the drop of blood. With a steady movement the second slide is drawn toward the other end of the first slide, leaving a blood film behind. The drop of blood should be small enough to allow the film to terminate before it reaches the end of the slide.

The subsequent procedure is then as follows:1. Fix the air-dried smear for 3 min with methanol;2. Dry in air;3. Stain with GIEMSA (5 drops of stock solution to 5ml neutral or buffered

distilled water per preparation); duration of staining 30 min;4. Rinse off the staining solution with water; allow todry.

Examination of the slides for protozoa should be done with an oil immersion lens. If filariae are suspected, the examination is carried out with a high-dry lens, after the stained smear has been covered with a layer of immersion oil and a cover glass. For staining microfilariae DELAFIELD’S haematoxylin procedure is preferred to GIEMSA.

Impression smears are recommended for the demonstration of tissue parasites. For this the smoothly cut surface of an organ (liver, lung) is pressed vertically on a slide and the smear air-dried, and processed like a blood film (see above).

The “Thick film”A relatively small drop of blood is put on a clean, grease-free slide and spread with the corner of another slide, using a circular motion to about the size of a thumb nail. The blood is stirred for at least 30 seconds to improve coagulation and air-dried. Without prior fixation or heating it is placed in a dish (film side down) of tap or distilled water for 5-10 min to remove the haemoglobin, one end of the slide being raised on a support. With freshly prepared preparations haemolysis takes only a few minutes; with preparations older than about 3 weeks it is advisable to add a few drops of acetic acid. However, the acid must be removed before the staining, e.g with tap water (neutralized). After removal of the haemoglobin the film is stained with GIEMSA (without fixation). With fresh preparations haemolysis can also be combined with staining.

For staining, the blood film is covered with GIEMSA solution (stock solution diluted 1:20 with water) for 20-30 min. then the stain is carefully rinsed off with distilled or buffered water, or if necessary rain water. The thick films should be dried in air (not between filter paper).

It is important that water of neutral pH (7.0) be used – either double-distilled water or WELSE’S original buffer mixture should be used in accordance with the instructions.

Microscopic Examination of stool samplesIf there is any suspicion of infection with intestinal parasites, at least three stool samples- obtained on different days, should be examined. In this way the probability of detecting all the parasites present is about 90%, and with five stool samples about 95% (this varies according to the species).

Each stool sample should first be examined macroscopically, looking for roundworms, hookworms, threadworms or tapeworms passed spontaneously or after medication.

Tapeworm segments often still show spontaneous movement. They are therefore often mistaken for whole worms. The use of suitable concentration techniques is recommended for the microscopic examination, utilizing both fresh and fixed still samples.

In a fresh preparation the characteristic movements of protozoa (amoeba, flagellates) and also their typical morphology can be clearly recognized on microscopy (e.g Trichomonas, giardia; oocysts of coccidian). If lively, motile small worm larvae are seen with microscopy.

When stool samples are stained (e.g with lodine solution), the cytoplasmic and nuclei of protozoa can be recognized. Both parts are of importance for species differentiation.

As a general rule, a faecal smear should be so thin that print can be read through it. Effs with colourless shells are more easily recognizable, when the faecal sample is mixed with LUGOL’S solution or 2% eosin solution, instead of water.

Examination of stools for intestinal protozoaCoccidian oocysts which may be found in humans can be demonstrated more reliably using concentration techniques, such as those used for the demonstration of worm eggs, than by simple microscopic examination. The demonstration of the oocysts of cryptosporidia requires special methods (see p. 87).

1. The untreated stool specimen. place a sample of fresh faces (preferably still warm)the size of a lentil, under a cover slip with a drop of physiological saline solution and examine at moderate magnification (+400-500). Vegetative stages of intestinal flagellates and amoebae can be detected easily by their movements.

2. Iodine staining .A small stool sample is placed between two cover slips, which are carefully pressed against each other. The two cover slips are drawn away from each other in opposite directions and the coated side is

immediately placed in prepared drops of 4% iodine solution. After a few seconds the two smears will be saturated and any vegetative cell and cysts present will be stained

1. The stained stool preparation (method of HEIDENHAIN). These smears are prepared either on a slide or on three to four cover slips and are fixed whilst still moist in sublimate alcohol.

The preparations (without being allowed to dry) are then treated as follows:1. 30min in iodine –alcohol (70% ethanol and iodine or LuGoL’s solution, roughly

the colour of cognac).2. At least 1 h in 70% ethanol3. Rinse briefly in water4. 1 h in 4% aqueous ferric ammonium sulfate solution (violet crystals should only

be dissolved in distilled water)5. Rinse briefly in water 6. 1 h in HEIDENHAIN’S haematoxylin( 1 g haematoxylin in 10 ml 96% ethanol

and 90 ml distilled water ; the solution must ripen for at least 4 weeks with the admission of air in a loosely stoppered clear glass bottle)

7. Rinse briefly in water 8. Differentiate by agitation in 2% aqueous ferric ammonium sulfate solution for 1-

4min.9. Rinse for at least 30 min in running (tap) water.10. mount in balsam after passing through an ascending alcohol series and xylene

(particularly important for the differentiation of amoebae species; see under E. histolytic a ,p. 53).

When performing microscopy the most favorable areas in the preparation must be sought; the quality of differentiation of the nuclear structures varies according to the thickness of the smear (see point 8).

Examination of stools for worm eggs, including concentration techniques (In part also suitable for protozoan cysts)1. Direct Examination. A piece of faecal material roughly the size of a lentil (fresh

or fixed ) is mixed on the slide with a little tap water or physiological saline. The resulting suspension should be as thin as possible. The sample is spread out and covered with a cover slip, and examined microscopically at a magnification of +100-200.

When looking for worm eggs, the mucus adherent to the faeces, in particular blood – stained specks of mucus, should always be investigated microscopically as well. Concentration techniques are often used because the eggs are frequently present in small numbers (see under 2-5).

2. Concentration Technique using saturated saline solution (suitable only for the demonstration of nematode eggs, in particular hookworm eggs). A faecel sample of about the size of a hazelnut, roughly- i.g is mixed with 20 times the amount of concentrated saline solution, which is added slowly. Coarse particles (plant residues etc.) are removed through a coarse-meshed filter or skimmed off the surface After 20-

40min the eggs which have risen to the top are taken from the surface of the solution with a round wire loop (about 1 cm in diameter) bent at right angles. Several of the fluid films adherent to the wire loop are placed on a slide. The samples can be examined at a magnification of x 100-200. after use the wire loop should be flamed.

3. Zinc sulphate Concentration Technique (Flotation method). 4-5g faeces is mixed with 0.1 aqueous Triton x-100 solution, passed through a gauze filter in order to remove the coarse plant components, and centrifuged at 2000 rpm for 3 min. the resulting sediment is resuspended in 33% zinc sulphate solution (specific gravity 1.180) and is again centrifuged for 3 min at 2000 rpm. The eggs now on the surface can be removed with a loop and transferred to a slide (the addition of one drop of iodine solution makes the differentiation easier). This procedure is suitable both for worm eggs and larvae and also for amoebic cysts and oocusts of the intestinal coccidian of man and animals.

Instead of the zinc sulphate solution a sucrose solution (56g to q00ml of water) can be used, to which a disinfectant has been added (e.g 1.3 phenol) to prevent fungal growth (from FRENKEL). This method is recommended for the demonstration of coccidian oocysts.

2. TELEMANN Concentration Technique (universal procedure for all worm eggs). A sample of faecal material roughly the size of a bean is suspended in a glass beaker with 7ml semi-dilute hydrochloric acid (16%-18%) An equal amount of ether is added and the mixture stirred until it is a homogeneous emulsion. The mixture is poured through a wire gauze sieve (mesh width 1mm) or a double layer of muslin into a centrifuge tube, with the aid of a funnel, and is centrifuged for 1min. four layers develop; a yellowish zone of ether at the surface, then a plug of detritus, a zone of hyufrochloric acid, and at the bottom a small sediment. The latter contains pieces of muscle and cellulose as well as the worm eggs (caution is required because of the danger of explosion with ether). The sediment is transferred to a slide with a pipettes and after a cover slip has been applied, is examined microscopical

3. Concentration by the MIFC Technique (merthiolate-iodine-formaldehyude concentration; BLAGG et at., 1955). Two stock solutions are required:

A 250 ml distilled water, 200ml Thimerosal (1: 1000 in distilled water). 25ml concentrated formalin, 5ml glycerin (=40ml)

B. Fresh 5% iodine solution (5% iodine in 10% potassium iodide solution in 100ml distilled water) which should not be above 3 weeks old

Both solutions should be stored in brown bottles.

Immediately prior to the processing of a stool sample, 4ml of stock solution A are mixed with 1ml solution B (or a multiple of these).

A stool sample about the size of a hazelnut is mixed with the specified amount of the mixture of solutions a and B. the sample. Fixed and stained in this way, is passed through a double gaze filter and is mixed in a centrifuge tube with 7ml ether 9which should be stored in the refrigerator at 4-0-C). This mixture is vigorously shaken so that no ether is

to be found on the surface. The tube is left standing for 2 min and is then centrifuged for 5 min (approx. 3000 rpm). the plug of detritus between the ether and the MIF zones is gently detached from the sides of the tube with a rod and is poured out with the liquid component. At the bottom of the tube worm eggs, protozoan cysts and their vegetative forms are found in the sediment (caution is required because of the danger of explosion with ether). The sediment should be examined quantitatively under the microscope 9x 400).

For the dispatch of a stool sample, 0.5 g is first mixed with 0.15ml solution B and shortly after with 2,35ml solution A. and then stirred to form a homogeneous suspension. Stool material preserved in this way can still be examined microscopically after a period of months.

When looking for Enterobius vermicularis a stool sample is unsuitable- apart from macroscopic examination- because the eggs adhere to the edge of the anus and must be obtained by washing them off or detaching them with a strip of adhesive cellophane (see p. 218)

MICROSCOPIC EXAMINATION OF URINE AND SPUTUM

The following parasites can be found in the urinary sediment: Trichomonas vaginalis, eggs of Schistosoma haematobium, sometimes (rarely), S. mansoni and S. Intercalatum and also, rarely, microfilariae. Enterobius species eggs have occasionally been found in samples from female subjects. The eggs were probably carried into the vagina and then flushed out in the urine . Involvement of the kidneys and bladder by paragonimus species (eggs) and Echinococcus species (protoscolices) when disseminated is also possible.

Contamination from outside sources must always be considered when dealing with urinary sediments. Pollen grains often give rise to false interpretations and fly larvae are occasionally found in the urine (pseudomyiasis). These have always been introduced accidentally from outside.

To carry out the urine examination in practice a clean urine sample, preferably a morning sample, is centrifuged at about 2500 rpm for 3-5 min and the sediment is examined under the microscope.

When the lung are infected with paragonimus species, eggs are found in the sputum. Cysts of Pneumocystis carinii can also be found in the lungs. For this, the sample of sputum is added to 3 times its volume of physiological saline, is well mixed and then centrifuged. The parasites can be found in the sediment. Protoscolices or individual scolex hooks can also be found in cases of pulmonary echinococcosis.

GENERAL COMMENTS ON SEROLOGICAL DIAGNOSIS

Intracellular parasites or those which live in close contact with an organ (e.g Leishmania, Trichinella) mostly stimulate their host to marked antibody production. The quality of the antibody detection procedure largely depends- (assuming correct technical execution)- on the quality of the antigens, these should be homologous as far as possible. A deficiency in most of the serological method used in practice to date is the absence of comparability of antigens and of internationally recognized standard sera. Apart from the use of polyclonal antibodies for the detection of parasites, the customary method hitherto, attempts are increasingly being made to detect soluble antigens in serum and stools as well.

Finally, new methods are becoming available using monoclonal antibodies, which are expected to give greater specificity.

Monoclonal antibodies are produced by the hybridoma technique - an in vitro procedure based on the idea of creating a combination of the properties of basically different cells through fusion, to form hybrid cells. In the practical procedure for this, mice or rats are first infected with the desired antigen so as to stimulate the formation of specific lymphocytes.

They (the lymphocytes) are predominantly found in the spleen and can be obtained from this organ. The lymphocytes are encouraged to fuse with a certain kind of tumour cell. As is well known, tumour cells have the property of unlimited growth and they therefore introduce this property into any hybrid cells that form. In this way a new uniform cell develops which now has the highly desirable property of multiplying indefinitely in vitro and also releases antibodies into the surrounding nutrient medium. These monoclonal antibodies which have developed from one cell (clone), can now be used for the detection of circulating antigens from parasites which may be found in the serum. It has already been possible to use this procedure for some parasites, e.g for Toxoplasma gondii. It has been able to demonstrate six individual antibodies produced from hybridomas.

OTHER PARASITOLOGICAL TECHNIQUES

EXTRACTION, FIXING AND PRESERVATION PROCEDURES FOR VARIOUS PARASITES

Tremotodes

Trematodes may be found in the nasal chambers, in gills and gill chambers, in the small and large intestines, lungs, and urinary bladders.

The Gills are placed in a dish of physiological saline (84% NaCl solution), with some Choloretone crystals. Trematodes hiding there become relaxed any may be “combed out” of the gills with a dissecting needle. The dish is set aside and examine later under the microscope.

To examine the complete digestive tract:

Remove the entire tract and place it in a dish of saline, then cut it into short sections. Split each section and examine it.

Large trematodes will be quite obvious, whereas small ones will hide among the villi of the intestine and should be located with the dissecting needle. The fingernail or a dull scalpel may be forced against the worm in such a way as to dislodge the sucker without destroying the specimen. Transfer worms to dishes of saline to clean them prior to fixation.

Cut the lungs open in saline and examine under the dissecting microscope.

Likewise, puncture the urinary bladder, draining it of its content, cut open and examine in a dish of saline.

Also examine the urine drained from the bladder.

Finally, look for tramatodes under the skin of the animal and elsewhere, which may harbour adult or larval trematodes.

Preservation

Small specimens obtained may be fixed as follows:

Transfer the specimens in a pipette with a minimum amount of water to the bottom of dry petri dish or a glass plane. The water should be just sufficient to permit the animal to expand. When they are fully expand, drop a coverslip on top of the specimen. The more muscular specimens may be covered with part of a microscope slide. The volume of

water should be small enough so as not to float the slide or coverslip, but rather to create suction which holds the worm in an expanded position.

Add FAA (Formalin – acetic – alcohol) to one end of the coverslip and pull this under and around the specimen by absorbing water, with a piece of filter paper, at the opposite end. Again, be careful not to introduce enough liquid to float the coverslip and allow the animal to contract. Specimens should be treated up to 1 hour with additional quantities of FAA, adding when necessary.

Finally, lift the coverslip and slide the specimen into a beaker containing 50% Alcohol. Leave the specimen in this medium for 10 min. and wash in 70% alcohol, dehydrate in successive wash of alcohol – 75% 85%, 95 – up to absolute alcohol. Clear in xylene and mount in DPX or Canada Balsam on microscope slides.

Another method for dealing with very large trematodes is to place them between two microscope slide which were subsequently bound with rubber bands. This preparation is then placed in a container with fixative for 2 – 3 hours and then transferred to a 50% alcohol and as above.

It might be useful to try-out the above methods and adopt which ever gives you the better result.

CESTODES

Cestodes occupy the digestive tract, most frequently the small and large intestines. While searching through the digestive tract look-out for chains of tapeworm segments. Small tapes may appear as very thin whitish threads a few cm in length. When you locate a tapeworm, do not extract it, but carefully locate the scolex (at point of attachment). Observe this under the dissecting microscope, and with a dull scalped blade or probe attempt to dislodge the hooks or suckers. Tapeworm bodies are seldom strong enough to support even their own weight and will break very readily. The scolex is essential for identification and must be secured. Remember that larval tapeworms may be lodged in almost any organ of the body. They are common in the liver, spleen, lining of the body cavity, and in the fat layers under the skin, so look-out for them. They may appear sac-like, pigmented or white, fibrous or cyst-like.

PRSERVATION

Adult and larval tapeworms are generally preserved on slides. Transfer small specimens so that they will conveniently fit under a coverslip. Blot away excess water, add a coverslip to hold them into place, and fix with FAA and stain with borax carmine as descried above for tematodes.

Large species are cut into sections small enough to go under coverslips. The head (scolex), “neck”, and first few segments are kept intact. Some immature segments are preserved and some of the terminal segments which should be ripe are also secured. After cutting them into convenient lengths, place specimens on a glass plate, cover with coverslips or slides and fix with FAA as above.

More “muscular” and robust tapeworms have to be relaxed in their entirety in order to render them flat enough and thin enough for taxonomic uses. Place the entire animal in a dish of saline and put this in the refrigerator (not the freezer) until it is thoroughly chilled

Note, gently hold the anterior end and gently pull the opposite end and on the glass plate, in order to stretch the specimen. Remove excess water from the distal end so that it will not contract again, cover the desired sections of the tapeworm with glass slides, and fix by adding FAA for 30 mm specimens may be stained in borax carmine as described above Dehydrate in ascending concentration of alcohol, clear in xylene and mount in DPX or Canada balsam.

NEMATODES

Nematodes may be found in various tissues and organs o the host. There should be little difficulty in identifying these.

PRESERVATION:

Very small to small nematodes (those which will conveniently fit under a coverslip) may be fixed in one of 2 ways

– (1) Pipette the specimens into a sheet of glass with a minimal amount of water and fix by squirting them with hot FAA (60oC) which has 10% glacial acetic acid.

-(2) Pipette specimens into a concave slide depression and warm this above a burner flame until the worms stop wriggling. Do not boil or overheat Remove excess water and fix by flooding with FAA. Mount on gelatin DPX or Canada Balsam.

Specimens too large for slide preparation should be reserved in 5% formal in or 75% alcohol. Straighten the specimens and place them on a piece of cloth. In the meantime, bring a beaker of water to a temperature near boiling (80oC). Rom the cloth and grasp both ends with forceps; remove them after 90 seconds. Then place the specimens in a dish of 5% formalin to fix for 24 hours. Store in 5% formalin or 75% alcohol.

ACANTHOCEPHALA

Acanthocephalans are routinely obtained during collection of other parasites from the digestive tracts of vertebrates. Adults will have their proboscis firmly embedded in the gut wall. Usually they can be extracted by grasping them between the fingers and pulling gently. Other worms may dislodge themselves when left alone in the saline dish. Remove the worms to dishes of clean saline where they will alternately contract and extend the proboscis. Leave them in the solution long enough to free the body of debris.

PRESERVATION

Attempt to kill the Acanthocephalans with their proboscis extended. After they are killed, preserve large specimens in 50% formalin, while the smaller specimens should be mounted on microscope slides. In addition to ensuring that the proboscis is extended, fix these worms in a position such that they will fit under a standard coverslip . For small to medium size worm suitable for slide-making, the worms may be killed with their proboscis averted as follows

Transfer each specimen with a moderate quantity of saline to a sheet of glass. Position the worm so that it will fit under a coverslip. When the proboscis is averted, quickly drop microscope slide on top of the worm. Usually, the suction created between the glass plate and the slide by the saline will maintain enough pressure to keep the proboscis averted Otherwise, slight additional pressure could be applied to the slide during the killing. Prepare FAA and put the solution on one side of the slide and put it around the specimen by absorbing water on the opposite side of the slide with a piece of filter paper as before.

An alternative and simple method is to place the worm in the refrigerator until they are thoroughly chilled and expanded, Next siphon off the water and pour hot (50 to 60oC) FAA solution over the worms

A technique that works fairly well for large acanthocephalans is to chill them as directed above, then inject, with a fine hypodermic needle placed in the posterior part of the body cavity, sufficient FAA solution to expand the worm and the proboscis to be averted. Stain with Ehrlich’s acid hematoxyline for 2 hours.

PERSPECTIVES ON THE DIAGNOSIS OF PARASITIC DISEASES IN

DEVELOPING TROPICAL AFRICAN COUNTRIES

INTRODUCTION The major cause of morbidity and mortality in tropical developing countries is the high

prevalence of infectious diseases. Besides the plethora of microbes causing

gastrointestinal and respiratory infections, there are major diseases of parasitic origin

caused by pathogenic protozoa, helminthes and nematodes. Table I summarizes the major

infections of developing tropical African countries.

Parasitic Infections;

Malaria

Amoebiasis

Ascariasis

Hookworms

Trichuriasis

Filariasis

Schistosomiasis.

Prevalence

800,000,000

400,000,000

900,000,000

800,000,000

500,000,000

250,000,000

200,000,000

Mortality/y

1,200,000

30,000

20,000

55,000

low

low

750,000

Two major programmes of the world Health organization (WHO) are focused on the

control of acute respiratory infections and diarrhoeal diseases with the central objective

of reducing severity and mortality. The main objective of these programmes is the

improvement of acute case management; - diagnosis does not have high priority.

In the developing world, specific diagnostic exploration is often impossible. A febrile

patient will be automatically treated for malaria before other pathogens are taken into

consideration. In many health centers and small hospitals in rural areas, laboratory

facilities are rather limited owing to lack of trained personnel, adequate equipment or

sufficient operating funds. In contrast, in major cities, the diagnostic laboratory potential

is very often no different from that of industrialized countries.

The field of diagnostics has made enormous progress in the last decade, primarily

as a result of advance in biotechnology. Recombinant DNA and hybridoma technologies

as well as peptide chemistry have allowed the production of highly specific reagents for

the diagnosis of infectious disease. The Polymerase-Chain-Reaction (PCR) with its

phenomenal power of amplifying very small amounts of specific DNAs, has boosted

sensitivity to levels beyond imagination. New “generations” of test kits for viral

hepatitis, HIV infections e.t.c. testify to the rapid development of diagnostics. If they can

be produced in conjunction with an appropriate robust and simple test design, and at a

low cost, such techniques could easily make their way to the rural health centres and

hospitals. An example of this, is the new “dipstick” test for antibodies to HIV developed

by the Programme for Appropriate Technology in Health (PATH) in Seattle, USA. which

is currently being used in most hospitals around the country.

For most acute viral or bacterial diseases, the major diagnostic aims are either to detect

present infection or to analyse the immune status. For many parasitic disease diagnostic

issues are more complex. This stems from two factors. Firstly, the patient often has a

lifelong history of contact with the disease secondly, each disease has a wide clinical

spectrum ranging from states of latent infection or symptomatic carriers to acute or

chronic pathology, including life threatening condition Relevant diagnostic questions

associated with individual cases might be related to identifying the stage of infection,

assessing morbidity, identifying subjects who are at risk of developing severe morbidity,

assessing the parasite load (as in helminthes infections) and so on. Diagnostic issues are

different depending on the purpose for which they are required, for example, for public

health purposes, diagnostic tools are used to facilitate effective treatment, whereas an

epidemiologist uses them to quantitate transmission or assess the impact of control

measures on incidence, prevalence, overall morbidity or transmission.

It is obvious that one single diagnostic test cannot answer all those questions.

HIGHLIGHTS ON ADVANCES AND PROBLEMS IN THE DIAGNOSIS OF

SOME TROPICAL PARASITIC DISEASE

Malaria Specific diagnosis of malaria is usually achieved by microscopic examination of a blood .

However, the detection of a malaria case might be a time-consuming and costly exercise,

especially in areas with a low level of transmission. For an experienced microscopist, 10

to 20 parasites per µl blood are considered to be the detection limit when analyzing a

thick blood film. For public health purposes (– e.g to assess the impact of control

measures), a more efficient method would be of great advantage. During the last decade,

alternative diagnostic approaches have been developed using three techniques, namely

DNA probes, Antigen detection and Quantitative Buffy Coat (QBC) analysis.

Most DNA probes for Plasmodium falciparum (either genetic probes or

systematic oligonucleotides) detect a family of 21 base-pair tandem repeats, which

comprise about 10 per cent of the genome (for review see 1) with the most. Sensitive

procedures, using isotope-labelling and long exposure time the sensitivity, claimed for

parasites obtained from in vitro culture, was 20 to 50 parasites per 1 However, using

blood samples from infected individuals sensitivity was considerably lower (approx, 200

to 1,00 parasites 1. A comparative study of four hybridization assays with different

probes and procedures (part of a vaccination trail) confirmed that all of them were less

sensitive than microscopic examination of thick blood films or in vitro cultivation (2) In

fact, the DNA probes had a disappointing sensitivity of only 5 to 28 percent compared to

culture and 13 to 40 percent competed to thick films. The application of DNA

hybridization as an alternative diagnostic method for malaria is thus still in its initial,

phase further improvements, especially of sample preparation and of the detection system

may increase sensitivity.

A second alternative approach to the diagnosis of malaria is the immunological

detection of red blood cell-associated antigens or soluble antigens in the serum. Two test

principles have been used so far. In the first, a competitive radio-immunoassay, malaria

antigens (in blood lysates) were used to bind polyclonal (3) or monoclonal antibodies (4)

and thus inhibit the subsequent binding of these antibodies to antigens bound to a solid

phase. The results of preliminary attempts to use this method were far from satisfying.

The sensitivity was much too low (several thousand parasites 1) A much better sensitivity

was reported by a different techniques an immunoradiometeric assay (IRMA). (5) An

enrollment correlation was reported between IRMA binding activity and parasitaemia for

in vitro cultured parasites. However, this correlation was less satisfactory when blood

samples from patients were analysed. A major problem is the interpretation of antigen-

positive results from microscopically negative individuals. One explanation is the

persistence of antigen from an infection that has been treated. Antigens could be detected

by IRMA as long as two weeks after the disappearance of parasitamia. This could cause

confusion and lead to false diagnosis of other febrile illnesses. In addition, considering

the common practice of self-medication for malaria in endemic areas, parasite antigen

detection in human blood might not be a satisfactory alternative to conventional

microscopy in prevalence surveys.

Antigen detection assays have proven their merits for detecting infective

mosquitoes. (6) Two – site immunoenzymatic methods using monoclonal antibodies

against the epitivitive epitope of the . P falicparum circumsporozoite antigen are now

reliable and applicable tools in the field measuring impact of control on transmission or

giving valid answers to entomological questions. (7)

An effective method for the rapid detection of acute infections – where the lenghy

procedure of DNA hybridization and present antigen detection assays are inappropriate –

was recently developed by flections – Dickinson (Franklin Lakes, N. J. ). The clever

principle of this QBC techniques, staining parasitic DNA With acridine – orange and

concentrating infected red blood cells by centrifugation, is based on a modified

microhamatocrit tube. Infected red blood cells can be easily detected in a 1 – 2 mm

broad band using fluorescence microscopy. High sentitivity is to be expected since a

larger blood sample can be readily examined (55 1 v s about 0.4 1 equivalent to 200

fields of a thick – film preparation). In a first field evaluation, the QBC Method appeared

to be a least eight times as sensitives as conventional microscopy, detecting an additional

10 per cent of infections not diagnosed by conventional microscopy (8) In our experience

with imported malaria cases QBC has proved to be as sensitive as classical microscopy,

(9) and in holoendemic area we confirmed a slightly higher sensitivity of QBC. The

great advantage is the remarkable time gain for microscopic reading the test need s about

one compared with 10 to 15 minutes for thick films. This is especially important if there

are many negative samples to be screened. For dependable species diagnosis, however,

blood has to be re-examined by conventionally stained blood films

AMOEBIASIS

It is estimated that approximately 10 per cent of the worlds population are infected by

Entamoeba histolytica and that in some developing countries invasive amoebiasis is

among the 10 leading causes of death. (10) Imperfect diagnostic tests limit our

perception of the magnitude and severity of this disease. The two major problems

concerning diagnosis are to find more efficient methods to detect an intestinal infection,

and simpler ways to distinguish pathogenic and non-pathogenic forms (isolates). Even

using concentration methods for microscopically detection, a reliable diagnosis needs

repeated stool examination. From our own data using the “SAF” – method, (11) which

involves fixation and concentration of the parasites, we calculated. From analyses on

multiple specimens – that at least four stool specimens are needed to guarantee a

predictive value for a negative result of 0.99. (9) If only one false – negative out of 1,000

stool specimens is acceptable, one would have to analyse 10 stool samples from each

patient

Since it is evident that dependable stool examination are strenuous, time

consuming, expensive and rely on high level of microscopic skills, alternative diagnostic

approaches were attempted. The first development of an ELISA test to detect stool

antigens was described in 1978. (12) using a commercial immunozyme test kit

(Millipore Corp. Bedford, Mass.) conflicting results were reported with regard to

sensitivity and specificity. This test depended on polyclonal antibodies, but more recent

attempt utilized monoclonal antibodies for antigen capture. (13) Unfortunately the

excretion of antigens, like that of trophozoites or cysts, is irregular and it is therefore still

necessary to analyse multiple stool specimens to reach an acceptable level of sensitivity

(own results).

Recently, first results using a DNA hybridization technique as an alternative to

microscopy were reported. (14) The diagnostic clones, detecting highly repeated

parasite DNA sequences, reacted specifically with a few as 800 amoebae, but did not

distinguish between pathogenic and non-pathogenic zymogene of E. histolytic a. Further

investigations with multiple sampling of individuals are needed to determine reliable

predictive values.

When it comes to the analysis of (potential) virulence of an isolate, the current

“gold standard” is the zymogene analysis introduced by sergeant and co-workers (for

review see 15). The isoenzyme profiles obtained after electrophoresis allow the

distinction of virulent and avirulent E. histolytica isolates. However, this technique is too

time – consuming for most diagnostic laboratories. New ways of distinguishing E.

histolytica possessing pathogenic and nonpathogenic zymogene were opened by using

either genomic DNA (16) or DNA probes. (17) preliminary results indicate that

pathogenic isolates of E. histolytica are genetically distinct from nonpathogenic isolates.

In addition to their diagnostic use, these probes could serve as tools to investigate the

molecular basis of pathogenicity.

Serology is an important tool to aid in the diagnosis of suspected extra intestinal

involvement or cases of bloody diarrhoea or chronic colitis. A wide range of methods,

ranging from the very simple (e.g. Latex agglutination) to the rather sophisticate (time

resolved fluoro –immunoassay), were evaluated for review see 18) for all of them the

major dilemma is to interpret a positive serological finding in an endemic area. This is

related to the fact that antibodies due to past infections persists and that antibody titers

can be low in the early stages of liver abscess. Formation. A solution to this might be

the choice of (an) appropriate diagnostic antigen(s) produced by recombinant DNA

technique or as synthetic peptide(s) – in combination with class – or subclass-specific

antibody detection of an early immune response after invasion. In view of the high

mortality due to invasive amoebiasis, the development of a robust and reliable immune

assay (e.g. dipstick or dot blot test) seems to be an urgent priority.

INTESTINAL NEMATODIASIS

The detection of intestinal infections due to nematodes is still in the field of classical

microscopical techniques. Since morbidity is related to worm burden, the quantitative

assessment of egg counts is relevant, Recent studies have clearly demonstrated the impact

of nematode infections on health, growth and physical fitness. Programmes for the

control of nematodiasis are now being actively reassessed by WHO the World Bank.

UNESCO and UNICEF. However, there is no consensus on whether diagnostic screening

before treatment is a required component of such programmes. The issue of mass

treatment versus diagnostic screening has recently been discussed. (19) The central

question still remains unanswered: Is it acceptable to treat individuals without knowing

their infection status? Apart from the economic issue – a screening component increases

programme cost by a factor of 2 to 6 – diagnostic screening is laboratory equipment.

Diagnostic screening would therefore only be possible if simpler diagnostic tools were

available. For severe hookworm, infections, haematocrit values have been shown to be a

possible indicator.

A negative consequence of any diagnostic screening, which often seems to be

ignored, is that a significant proportion of infected individual remain untreated since the

level of compliance with stool sampling is reported to be of the order of 50 to 70 per cent.

(19) if one opts for diagnostic screening (to be in concordance with good medical

practical) to add an educational component to the control programme in order to enhance

compliance. This problem illustrates the fact that the diagnosis of infections disease

includes more than technical laboratory aspects. A diagnostic procedure has to find its

place in a given health system, and has to be accepted by the health personnel and the

population concerned.

FILARIASIS AND ONCHOCERIASIS

There are major limitations to the parasitological diagnosis of tissue-dwelling nematodes

infections remain parasitologically unidentified during the long prepatent period (until

adult worms produce microfilariae) and in light and occult infections, as well as in

individuals with an acquired immunity to circulating microfilariae. In addition, the

periodicity of certain blood microfilariae necessitates the sampling of blood at night,

which presents a significant obstacle for epidemiological studies. The replacement of

night- blood by an alternative method is one of the defined diagnostic goals of the

tropical disease programme (TDR) of who. for one possible approach, the

detection of circulating antigens, a variety of immunological methods have been utilized,

with polyclonal or monoclonal antibodies as reagents. To take the example of a

monoclonal antibody-based immunoradiaometric assay (IRMA), evaluation have clearly

shown an association of paten infection with detection of the target epitope, and a good

correlation between levels of serum antigen and blood microfilarial counts. (20)

However, circulating antigens could also be detected in amicrofilariaemic subjects with

acute symptoms of lymphatic filariasis, as well as in about half of the symptomatic

amicrofilaraemic individuals. Similar finding were also reported using other test system.

The interpretation of a positive antigen test for an amicrofilaemic individual is

rather difficult. Does it mean that we are detecting a latent infection, or antigen detection

in “endemic controls” a sign for active immunity from what has been said and in view of

the possible interference with host antibodies detection of circulating antigens seems not

to be the approperiate approach to replace microfilarial blood counts, but it could be used

to follow the effect of filaricidal drug administration was shown in a study in papua New

Guinea. (21)

Specific DNA probes were produced to diagnose Brugia malayi. A Method to

detect mirofilae in blood speciment has been developed (22) Theree are ongoing efforts

to adapt this technique for field applcation.

For onchocerciasis, major advances can be reported on two diagnostic issues,

which are especially relevant for the onchocerciasis control programme (OCP) in West

Africa. The first is related to the differentiation of the savannah and forest forms of

Onchocerca volulus using specific. DNA probes. (23). These forms differ in the

symptoms produced. The savannah form more often leads to blindness. This

differentiation is therefore relevant with regard to identifying the potential pathogenicity

of parasites carried by vectors invading the control area. The second issue is investigating

the recrudescence of transmission in the control area, where an early indicator for

reification is an urgent need. It has been shown that in early infections specific antibody

detection is more sensitive than parasitological examination of skin snips. (24). The old

problem of the poor specificity of serological tests ca be overcome by using recombinant

antigens selected for high specificity. (25,26,28) owing to individual variations in the

immune response, a mixture of several recombinant antigens will increase sensitivity.

For that purpose a collaborative study, including candidate antigens from several

laboratories, has been organized within the TDR Programme with the aim of developing

a reliable field test.

SCHISTOSOMIASIS

Morbidity in chronic schistosomal infections is mainly related to the magnitudes of egg

production, which is a function of the number of that aspect taking the case of urinary

schistosomiasis. The identification of heavily infected individuals is therefore an

important diagnostic issues, in order to prevent morbidity by timely drug treatment. The

standard diagnostic procedure is the urine filtration technique, which droduces

quantitative egg counts. However; multiple daily sampling of infected individuals

revealed extreme fluctuation of the egg output over time (29) and even in heavy

infections. Egg-nagative results were not infrequently observed. Analysis on a single day

detected only 44 per cent of heavily infected children. A single urine examination is

therefore not a reliable indicator for measuring the actual worm load. Repeated urine

examinations require an excessive amount of work even for a single patient, and are

certainly not realistic for public health purposes.

When community diagnosis (identifying villages for which urinary

shcistosomiasis is a major health problem) is the diagnostic aim; have to be available.

Reagent strips to test uring for blood or protein have been evaluated (30) Both indicators

have a high sensitivity for detecting egg-positive uring specimens but specificity was not

optimal, especially for proteinuria. Results from two different endemic areas showe

significant variation, which shows that locally defined criteria for the interpretation of

test results are necessary. In a recent study in Tanzania, testing for microbhaematuria

was found to be a reliable indicator (93 per cent sensitivity and 92 per cent specificity)

for heavy infections (29).

An interesting approach is the immunological detection of parasite antigens in the

urine. Especially the groups of Drs. Deelder and de Jonge (University of Leiden, The

Netherlands) have put much effort in the development of antigen detecting assays over

the last ten years. The detection of the circulating anodic antigen” (CHA) in the urine by

a monoclonal antibody-based two-site immunoenzyme assay had a sensitivity of 97 per

cent (31) if further validation attests adequate specificity and a simpler test format can be

designed, such a test could become an interesting candidate for public health purposes.

Possible use of this antigen-detection assay to monitor the efficacy of chemotherapy. (32)

A completely different approach to measure morbidity in schistosomiasis is the

use of ultrasound. (33) various studies have shown that this is an efficient technique that

is applicable and acceptable in the filed at the community level

A completely different approach, which may prove valuable for public health

proposes, is the use of questionnaires based on the disease- perception of members of the

community. In a study of urinary schistosomiasis a questionnaire administered to

teachers and school children was tested as a diagnostic tool. (34). In comparison to urine

filtration, this cost – effective way of screening revealed a sensitivity of 100 per cent and

a astonishingly high specificity of 87 per cent for schools with a high infection rate using

this approach of rapid assessment with key informants combined with selective reagent

stick testing (performed by instructed teachers in school identified by the questionnaire),

the distribution of S. haematobium in a rural district with over 300, 000 people could be

mapped within a period of four months at a total cost of less than.

1. US cent per inhabitant (34) current this diagnostic approach is validated for

schistosomiasis in different African countries.

PERSPECTIVES ON FUTURE DEVELOPMENTS

The enormous potential of new technologies is of diseases that are of concern for the

industrialized world. Although recent advances in the diagnosis of tropical diseases are

discernable, only a few real breakthroughs have resulted in diagnostic tools that are

appropriate for widespread routine use. The development of a diagnostic test is a rather

complex and costly process (major stages are listed in table 2). For each step, the

definition of precise objectives is needed, this requires the multidisciplinary collaboration

of various specialists. Too of ten researchers are not fully aware of the problems of

developing countries with respect to urgent diagnostic needs and appropriate techniques.

On the other hand, field workers have insufficient knowledge about new diagnostic

possibilities. Field research networks were recently incorporated into the TDR

programme to recruit more scientists for field-related research. Operational. Programme.

Known as FIELDLINCES, have been initiated to improve interactions between

researchers, managers and health workers form health ministries and national disease

control programmes.

Training of scientists from developing countries, and the development of research

capacities there, are important components of programmes of the World Health

Organization (WHO), the United Nations Industrial Development organization (UNIDQ)

AND OTHERS. In the field of biotechnology, the international centre for Genetic

Engineering and Biotechnology, a major UNIDO project fulfils this role. Gbal-oriented

research in diagnostics has to include test developers and test users from the beginning in

order to ban costly mistakes and further due to ignorance o diagnostic needs, imprecise

diagnostic objectives, inappropriate techniques or irrelevant validation. A dependable

validation is a prerequisite for the production of a test kit such a validation has to include

evaluating the test with defined specimens (e.g. from serum banks) to validate technical

specifications. However, this is not enough; it is vital also to prove the appropriateness of

the method under real –life condition outside the developers laboratory, for its inclusion

in a health system or control programme the applicability of the method and its

acceptability has to be tested.

The final step is the production of a diagnostic test kit. For this, a partner from the

industry has to be found. However, the decline in the interest of industry in the

industrialized world in parasitic diseases is a major obstacle making profits from

diagnostic tools is apparently the driving force for test development. The aim of the

newly established product. Development Unit within the TDR programme is to accelerate

the development of high priority products, and to stimulate collaboration with partners

form the industry. In the future, partners may be more easility found in the developing

world, as a result of the programes mentioned above.

I am confident that a goal-oriented multidisciplinary approach, initiated and

steered by international collaboration, will produce more and more appropriate diagnostic

methods for large-scale use. When such methods are available, they will be able to make

a major contribution to achieving the ultimate goal of improving health in the tropics.

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