疟原虫

Morphology

P.v morphology

P.f morphology

Ring forms (early trophozoites)

P. vivax thin smear, showing early trophozoites.
The infected red cells are enlarged and show some stippling. Giemsa. ×1000. Enlarged by 5.4.

P. vivax thick smear, early trophozoites.
The red cells are lysed. Giemsa. ×1000. Enlarged by 5.4.

Late trophozoites

P.vivax thin smear.
Showing late trophozoites (amoeboid stage). The infected red cells are enlarged and show marked stippling. Giemsa. ×1000. Enlarged by 5.4.

Schizonts

P. vivax thin smear.
A mature schizont about to rupture. A clump of malarial pigment can be seen in the center. Giemsa. ×1000. Enlarged by 5.4.

P. vivax thin smear.
Merozoites lying free. Malarial pigment is seen as a clump on one side. Giemsa. ×1000. Enlarged by 5.4.

Male gametocytes

Female gametocytes

Exflagellation

Zygote(No pic available now)

Ookinete

Ookinete, from the midgut of an infected mosquito. Giemsa. ×800. Enlarged by 5.4.

Oocyst

Sporozoite

Exo-erythrocytic forms(EEF)

红外期裂殖体

Pre-erythrocytic schizont in liver.
These mature in 6-14 days’ time liberation merozoites into the blood stream. Giemsa-colophonium. ×400. Enlarged by 5.4.

Pathogenesis

1. Incubation period: P.v. 14-17 days P.f. 8-12 days

2. An attack occurs because of the sudden liberation of merozoites, malarial pigment and RBC debris into the blood stream.

Three stages of each paroxysm

(1) The cold stage (chill) lasting for 30min-1hr

(2) The hot stage(fever) lasting for 1-4hr (P.v. P.f. and P.o. once every other day; P.m. once every 2 days. )

(3) Sweating stage 1-2hr

4. Recrudescence and Relapse

P.f and P.m. have only recrudescence, but, P.v. and P.o. both have relaps and recrudescence.

remnant of parasites in RBC result in recrudescence

hypnozoites cause relapse

5. Anemia; splenomegalia and fatal malaria-cerebral malaria caused by P.f. (small vessels are plugged)

Diagnosis

A. Clinical symptoms and history

B. Microscopic examination of blood.

1 Thin film and Thick(Giemsa's stain)

To master the morphology of parasites and changes of infected red cells

2 P.f.: Only Ring forms and gametocytes can be found in blood film.

C. Other methods: Immunologic/Biochemical/Molecular diagnosis.

Laboratory Diagnosis

There are four species of malaria which commonly infect man, P.falciparum, P.vivax, P.malariae and P.ovale. The most important of these is P.falciparum because it can be rapidly fatal. P.vivax is the most commonly seen species. P.malariae is present in Africa, South America and in one area of New Guinea. P. ovale is relatively unusual outside Africa although some cases are now being identified in other regions(eg. Southern States of India).

A number of new techniques based on the "dipstick" format, have recently become available for the diagnosis of malaria. These include the ICT-Malaria Pf, OptiMALr and the Determine kits. The methods are based on the principle of the detection of plasmodial histidine rich protein-2 (HRP-2) or parasite-specific lactate dehydrogenase (pLDH) which is present in P. falciparum infections, with a number of reports claiming sensitivities and specificities approaching 100%. Some of these "dipstick" methods have been extended to include screening for other forms of malaria but to date results have not been quite so impressive.

In this laboratory we have found these kits to be very useful screening or confirmatory tests, especially when there is difficulty in identifying scanty ring forms in blood films. They are particularly useful out of hours when more junior, less experienced staff are likely to be on duty. However we would like to emphasise, that we regard these methods as additional to the long established method of examining thick and thin blood films (outlined below), which is still regarded as the "gold standard", NOT as replacement methods.

Examination of a thick blood film should be the first step, if parasites are seen then the species should be confirmed by the examination of a thin film. Blood is best collected when the patient's temperature is rising.

Preparation of thick and thin blood films :-

Thick films:- place a drop of blood in the middle of a clean microscope slide and with the corner of a second slide spread the drop until it is about the size of a five cent coin. The thickness should be such that it is just possible to see news print through it. Thin films are made in the standard manner. Allow the films to dry, do not leave on the bench in a laboratory which is not fly proofed otherwise the film will be eaten.

When the films are dry, fix and stain the thin films in the conventional manner but be careful about the pH of the stain, a slightly alkaline stain is recommended (pH 7.2) as an acid stain may fail to show the parasites. When only a few thick films are to be stained it is best to use dilute Giemsa stain (1/20), using a staining jar so that the film is in an upright position, this will allow any debris to fall to the bottom of the jar. Do not fix the sample prior to staining. Stain for about 30 minutes, wash gently with clean water and allow to dry. If available use a positive control. When a large number of thick films require staininq, Field's stain is preferred because it is very quick. Field's stain comprises two solutions; a polychrome methylene blue (A) and eosin (B). The solutions are kept in covered staining jars.

1. Dip the dry but unfixed film into solution A for 1 or 2 seconds.
2. Remove from solution A and immediately rinse in clean water ( a 250ml beaker with water gently flowing into it is suitable)
3. Dip the film into solution B for 1 or 2 seconds.
4. Rinse in clean water for a few seconds.
5. Place in a vertical position to dry.

If films are old or too thick the red cells may not lyse completely in the brief staining time. If this is likely dip the film in clean water for a few seconds or until the haemoglobin has dispersed before staining. Instructions for preparing Field's stain can be found in many laboratory text books.

Under the microscope examine the thick film first, using an oil immersion or high dry lens to determine if parasites are present. Be aware of the patient's platelet and leucocyte counts. Malaria is usually associated with a normal or reduced leucocyte numbers. A leucocytosis is only found in terminal cases. Platelet numbers are moderately or markedly reduced in some 80% of patients with malaria. Parasites may appear distorted if the patient has been treated or has had inadequate prophylaxis.

Mixed infections are not uncommon.

The illustrations show the characteristics of the four species.

Diagnostic points:- Red Cells are not enlarged. Rings appear fine and delicate and there may be several in one cell. Some rings may have two chromatin dots. Presence of marginal or applique forms. It is unusual to see developing forms in peripheral blood films. Gametocytes have a characteristic crescent shape appearance. However, they do not usually appear in the blood for the first four weeks of infection. Maurer's dots may be present.

Diagnostic points:- Red cells containing parasites are usually enlarged. Schuffner's dots are frequently present in the red cells as shown above. The mature ring forms tend to be large and coarse. Developing forms are frequently present.

Diagnostic points :- Ring forms may have a squarish appearance. Band forms are a characteristic of this species. Mature schizonts may have a typical daisy head appearance with up to ten merozoites. Red cells are not enlarged. Chromatin dot may be on the inner surface of the ring.

Diagnostic points :- Only found in Africa. Red cells enlarged. Comet forms common (top right) Rings large and coarse. Schuffner's dots, when present, may be prominent. Mature schizonts similar to those of P. malariae but larger and more coarse.

Treatment

1. Three principles:

• Control of clinical symptoms
• Eradication of gametocytes
• Prevent relapse

Malaria Treatment

P. falciparum.
This species was originally sensitive to chloroquine, however, strains resistant to this and other antimalarial drugs are now commonplace. Because the parasite is able to multiply very rapidly and sequester within the microvasculature, a life threatening illness may develop in a very short space of time.

Uncomplicated malaria (where patients can take oral therapy) can be treated with one of three regimens:

1. Quinine sulphate 10 mg salt/kg 8 hourly for seven days plus doxycycline 100 mg daily for 7 days. Patients will usually develop 'cinchonism' (tinnitus, high-tone hearing loss, nausea, dysphoria) after 2-3 days but should be encouraged to complete the full course to avoid recrudescence.
2. Malarone^TM (atovaquone 250 mg plus proguanil 100 mg) 4 tablets daily for 3 consecutive days. This combination therapy has only recently come on the market and is relatively expensive. Data on efficacy are promising but limited.
3. Mefloquine (Larium^TM) given as 15 mg/kg in a divided dose followed by 10 mg/kg the following day. Antipyretic and antiemetic agents may need to be given prior to mefloquine administration to reduce the risk of vomiting.

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