RECENT ADVANCES IN DIAGNOSIS OF MALARIA

• LAB DIAGNOSIS• 1 .Microscopically: A microscopically examination of a blood film

forms one of the most imp. Diagnostic procedures in malaria. It is a good clinical practices to take both thin and thick films at the same time so that the parasite may be quickly detected in the thick films and than thin film is examined for identifying the species.

• Both thick and thin film are stained with Leishman’s Stain. The smear are then examined under oil immersion lens of microscope.

• The parasite are most abundant in peripheral blood late in the febrile paroxysm, Because blood for smear should be collected at this period.

• Occurence of multiple rings with accole forms is daignostic of P. falciparum infection.

• Malaria pigments may be demonstrated inside monocyte and PMN leucocytes

• Presence of malaria pigment only in presence of malaria parasite, suggests P falciparum infection.

• Schuffner’s dot -------------P . vivax• Maurer’s dot----------------P. falciparum• Ziemann’s dot---------------P. malaria• James’s dot----------------P. ovale, respectively• RBC’s are enlarged in P. vivax infection.• Parasites are more along the upper and lower margin of “tail” of

the film.• At least 250-300 oil fields should be examined before the smears

are considered negative.

• The staining of films with acridine orange can be read on flourescence Microscope or microscope equipped with an interferance filter system, allows quicker screening of films, because Parasites are more readily recognized and a lower power lens may be used.

• Conventional light Microscopy• Advantage ----1.It is sensitive and detects densities as low as

10-40 Parasities /ul of blood. 2. It is informative – When parasites are found , they can be

charactrised by terms of their species and of circulating stage. 3. Relatively inexpensive 4. Can provide permanent record.

• Disadvantge • 1. Time consuming • 2. It depends absolutely on good techniques, reagents,

microscoes and trained technicians.

2. RDTS( Rapid diagnostics Test)---- Are based on detection of antigens and derived from malaria pts. In lysed blood using immunochromatographic methods. Mostly they employ a dipstic or test strip bearing monoclonal antibodies directed against the target parasite antigen’s. The tests can be performed in about 15 mins. Several commercial tests kits are currently available.

Antigen targeted by currently available RDTS1. Histidine rich protein-11 (HPR-11)-: Water soluble protein produced by

trophozoites and young gametocyte of P.falcioparum. Commercial kits currently available detect HRP-11 from P.falciparum only.

2. Parasite lactate dehydrogenase(PLDH) is produced by asexual and sexual stages(gmetocytes) of malaria parasites. The tests currently available can distngiush P. falciparum from non falciparum species, but can not distinguish between P.vivax , P ovale and P. malariae.

General kit procedure :- • Test strip( most often nitrocellular) consists of a sample pad, 3 Detction lines

containing capture antibodies specipic for P. falciparum, all Plasmodium spec. and control antibody responsible and on adsorbent pad depending on kit, 2-50ul of finger prick blood specimen is lablled on a sample pad with a buffer soln that contain a haemolysing compd. and specific antibodies that is labelled with a visually detectable marker such as colloidal gold. If malaria antigen antibody complexis formed.

• The labelled Ag-Ab complex migrates up the test strip by capillary action toward the detection lines containing capture Abs.

• A washing buffer is than added to remove Hb and permit visulization of any coloured line on the strip.

• If blood contains malaria Ag the labelled Ag-Ab complex will be immobilized at the corresponding pre deposited line of capture Ab and will be visully detectable in 5-15 mins.

• RDTs for P. falciparum have sensitivity and specificity of. > 90% at parasite densities above 100 parasites per ul of blood.

Advantages of RDTs over microscopy1. Simple to perform and interpret2. Does not require electricity, special equipment or training in

microscopy.3. Health worker with minimal skill can be trained.4. Since RDTs detect circulating Ags, they may detect P.

falciparum infection even when the parasites are sequestered in deep vascular compartment and thus undetectedable by microscopy examination of peripherl blood smears.

• Disadvantages1. More expensive2. Can not diff. bet P.vivax, P.ovale and P.malariae, nor they can

distinguish pure P. falciparum infection from mixed infection that include P.falciparum.

Ouantitative Buffy Coat (QBC) TestDeveloped by Becton and Dickenson.It involves staining of centrifuged blood and compressed red cell

layer with acridine orange and its examination under uv light source.

• The QBC tube is a high precision glass hematocrit tube, pre-coated internally with acridine orange stain and pot. Oxalate. It ‘s filled with 60ul of blood from a finger, ear or heel puncture. A clear plastic closure is attached . A precisely made cylindrical float is inserted . The tube is centrifused at 12000 rpm for 5 min. The components of the buffy coat seprate according to their denesities, forming discrete bands. Because the float occupies 90% of internal lumen of the tube, the leucocyte and the thrombocyte cell band widths and the top most area of red cells are enlarged to 10 times the normal. The QBC tube is placed on the holder and examined using a std. white light microscope equipped with uv microscope adapter. Fluroescing parasites are then observed at RBC/WBC interface.

• Red cells containing plasmodia are less dense than normal ones. and conc. Just below WBC’s at the top of RBCcolum.

• The float forces all the surrounding red cells into 40 um space b/w its outside circumference and the inside of the tube.

• Since the parasites contain DNA which takes up acridine orange stain, they appear as bright specks of light among the non fluorescing red cells.

Culture examination Is not required for diagnosis except, only in special circumstances

when there is diffculty in differentiating the trophozoite.Trager and Jensen in 1976 could successfully cultivate human malarial

parasite(P. falciparum) to observe erythrocyte schizogony using

• RPMI 1640 medium in vitro continuous culture technique in which human erythrocytes (AB positive) were in a shallow stationary layer covered with shallow layer of medium. The medium was made to flow slowly and continuously over the layer of settled red cells, under an atmosphere with 7% CO2 and 1.5% oxygen.

Value of culture methods 1. To obtain erythrocyte stages for study of antigenic structure.2. As a source of Ag for sero-epidemiological investigation.3. To study sensitivity/resistance of the parasite to various

drugs.

• To observe immune prophylactic activity. BLOOD COUNT----AnemiaModerate leucopenia with monocytosis in chronic malaria. SEROLOGICAL TESTS – are not necessary for diagnosis of acute

infection, but are used for studying immunological aspects of population living in endemic areas and leading to latent infection in subjects used as blood donors.

1. Henry Mclanin flocculation test-: Positive test indicate high serum globulin. Also positive in African Trypanosomaiosis, visceral Leishmaniasis and Hepatitis.

2. CFT

4 . Passive Heam agglutination test Positive result indicates recent infection. Infected RBC’s are

agglutinated by homologus antiserum. It is particularly applicable to P.falciparum infection where enough infected erythrocyte are present.

5 . Immunofluorescence test(direct and indirect) The flourescence dye used in the test congugate with

gamma gloublin of serum to be tested. If such congugated gamma gloublin contains material Ab, it will adhere to the relevant malarial parasite and will be recognized as a glistening particle under fluorescent microscope.

5 . Gel ppt. Test-: Ag from P.falciparum is used . Incidence of positive result is very high in hyperendemic areas.