|Basic Malaria Microscopy (part I and II) (WHO - OMS, 1991, 72 p.)|
By the end of this Unit you should:
· know that thick and thin blood films must be examined in a specific way for consistency
· know that the thin film is examined only exceptionally for malaria parasites and know what the exceptions are
· be able to demonstrate the skills needed for systematic examination of both thick and thin films by the standard methods
· know why parasite counts must be recorded
· be able to use accurately the two methods described in this Unit for establishing parasite density.
Examining the thin film
Since it takes almost 10 times as long to examine a thin film as to examine a thick film, routine examination of thin films is not recommended. Only a very few could be properly examined in a days work.
However, examination of thin films is recommended in the following circumstances:
· when it is not possible to examine a thick film because it is too small, has become autofixed, or is unexaminable for some other reason;
· when it is necessary to confirm the identification of a species.
When a thin film does have to be examined, this should be done in a systematic, standard way as follows:
Step 1 Place the slide on the mechanical stage.
Step 2 Position the x 100 oil immersion objective over the edge of the middle of the film (as shown by the x mark in the diagram on page 66).
Step 3 Place a drop of immersion oil on the edge of the middle of the film.
Step 4 Lower the oil immersion objective until it touches the immersion oil (as described on page 38).
Step 5 Examine the blood film, following the pattern of movement shown in the diagram, that is by moving along the edge of the thin film, then moving the slide inwards by one field, returning in a lateral movement and so on.
Step 6 Continue the examination for approximately 100 fields to determine whether the blood film is positive or negative for malaria. If doubtful diagnosis makes it necessary, more fields (up to 400) may be examined.
Examining the thick film
Routinely, it is thick blood films that are examined. Provided that they have been well made and stained before autofixation could take place, there should be no problems in identifying the species of malaria parasites. Occasionally, however, you may find that it is difficult to tell the difference between the later, mature trophozoites and the gametocytes of P. vivax and between P. malariae trophozoites and rounded P. falciparum gametocytes. Also, it is not possible to distinguish between late trophozoites and gametocytes of P. malariae in thick films, but the need to know whether gametocytes are present in blood is usually confined to P. falciparum, and this is a relatively easy diagnosis to make.
Routine examination of a thick film is based on examination of 100 good fields. That is, a slide can be pronounced negative only after no parasites have been found in 100 fields of the blood film. If parasites are found, a further 100 fields should be examined before a final identification of species is made. This ensures that there is little possibility of a mixed infection (more than one species present in the blood film) being overlooked.
The technique for thick film examination is as follows:
Step 1 Using the x 40 objective, scan the film for any microfilariae that may be present. At the same time, select a part of the film that is well stained, free of staining debris, and well populated with white blood cells. If the film is well made and of even thickness, this should present no problems; poorer quality films may need to be quite extensively searched.
Step 2 Place immersion oil on the thick film.
Step 3 Swivel the x 100 oil immersion objective over the selected portion of the blood film.
Step 4 Lower the objective so that it touches the immersion oil.
Step 5 Confirm that the portion of the film selected is acceptable and continue to examine the slide for 100 oil immersion fields. Move the blood film by one oil immersion field each time, following the pattern shown in the diagram on page 67. Remember to use the fine adjustment for focusing.
Step 6 To assist with your examination, you should use a hand tally counter to count the fields as they are examined. (You will also use this to help you carry out a parasite density count later, in another exercise.)
At the end of the examination, record your findings on the appropriate record form. Your result will probably include a parasite count.
Establishing a parasite count
It is necessary to establish a parasite count for the blood film for the following reasons:
· The physician may want to know how severe the malaria is.
· The physician may need to know whether the malaria parasites are responding to the antimalarial treatment being given. This can be monitored over time by plotting the parasite count on the day of treatment and comparing it with the count in a blood film made at some specified later time.
· Parasite counts are especially important in P. falciparum infections which are potentially fatal.
· The district health officer needs to know the severity of malaria infections being seen in the local health facilities.
· The data may be needed for special purposes, such as testing the sensitivity of parasites to antimalarial drugs.
Two methods are used to establish the parasite count. You would not start this procedure until you had completed your 100-field examination and identified the parasite species and stages present.
Method 1: parasites per microlitre of blood
This is a practical method of reasonable and acceptable accuracy. The number of parasites per microlitre of blood in a thick film is counted in relation to a standard number of leukocytes (8000). Although there are variations in the number of leukocytes between healthy individuals and even greater variations between individuals in ill health, this standard allows for reasonable comparisons.
You will need two tally counters, one to count parasites and the other to count leukocytes.
(a) If, after 200 leukocytes have been counted, 10 or more parasites have been identified and counted, record the results on the record form in terms of the number of parasites per 200 leukocytes.
(b) If, after 200 leukocytes have been counted, 9 or fewer parasites have been counted, continue counting until you reach 500 leukocytes on your tally counter; then record the number of parasites per 500 leukocytes.
In each case, the number of parasites relative to the leukocyte count can be converted to parasites per microlitre of blood by the simple mathematical formula:
In effect, this means that if 200 leukocytes are counted, the number of parasites is multiplied by 40 and if 500 leukocytes are counted the number of parasites is multiplied by 16.
Note: It is normal practice to count all the species present and to count and record separately the gametocytes of P. falciparum and the asexual parasites. This is particularly important when monitoring the response to schizontocidal drugs, which would not be expected to have any effects on the gametocytes.
Method 2: the plus system
A simpler method of counting parasites in thick blood films is to use the plus system. This system is less satisfactory, however, and should be used only when it is not possible to carry out the more acceptable count of parasites per microlitre of blood.
The system entails using a code of between one and four plus signs, as follows:
= 1-10 parasites per 100 thick film fields
= 11-100 parasites per 100 thick film fields
+ + +
= 1-10 parasites per single thick film field
+ + + +
= more than 10 parasites per single thick film field