4.3. Capítulo 3 2
4.3.2. Consecuencias del uso de las plantas medicinales 2
The first blood slide that was prepared was thick blood smear for initial screening. Thick blood smear was made by placing a large drop of blood on a clean glass slide. The corner of another slide was used to spread the blood, to make a circular patch of moderate thickness and allowed to dry in air. After drying the blood smear was stained for 10-15 minutes with 10% Giemsa stain.
The slide was examined under a microscopy at magnification of x100 with oil immersion lens, for the presence of parasitaemia. This was done by counting the number of asexual parasites and the number of white blood cells in a limited number of microscopic fields. Adequate parasitaemia for enrolment was 1000 asexual parasites per mm3 or more.
Patients with adequate or required level of parasitaemia were bled a second time to make a second slide. This second slide had a thick blood smear on one side and a thin blood smear on the other side. The thin blood smear was made by placing a small drop of blood on the glass slide. The smooth edge of another clean glass slide (spreader) was applied at an angle of 45O till the blood spread along its edge. The spreader was pushed forward maintaining the same angle to give a thin blood film. The smear was then kept to dry in air. The thin blood smear was fixed with methanol and then stained with 3% Giemsa for 30-35 minutes. Microscopic examination of the thin blood film, which was for species identification, was done at magnification of x 100 with oil immersion lens. The thick blood smear on this second blood slide was used to calculate parasite
density. Parasite density was determined by counting parasites in the same field as 200 leucocytes in the thick film. Density was calculated using: 133
(No of parasites counted against 200 leucocytes x 8000) μL-1 200
where 8000 is the average white blood cell count/μL.26 The parasite counting was done by myself and verified by an independent assessor who did not know the treatment group the patient belonged to. In general, there was an agreement between the counts by the author and those of the independent assessor with differences less than 5%.
The same technique was employed for establishing parasite counts on each of the follow-up visits.
Haematological assessment
Packed cell volume (PCV) was determined using microhaematocrit capillary tube blood sample. Blood was collected into one heparinized microhaematocrit capillary tube filled up to two-thirds. The dry end of the tube was sealed with heat from gas flame. The tube was then centrifuged for 5 minutes at 15,000 revolutions per minute. Using a microhaematocrit reader, the base of the blood in the capillary tube was aligned with the zero mark and the bottom of the meniscus of the plasma with the hundred marks. The red cell column extended from the base to the buffy coat. The value of the PCV was taken directly from the microhaematocrit reader. The reading was taken
immediately after centrifuging. Packed cell volume was estimated on DAYS O and 14 of the study. Subjects with PCV of less than 15% were not enrolled into the study (See Appendix VI).
Drug Treatment
The subjects were randomly allocated to a treatment using a table of random numbers. The random numbers were sealed in envelopes and were opened after a patient had been enrolled into the study. The randomization was to one of two treatment groups: Chloroquine (Chloroquine Sulphate 150mg base by May and Baker Nigeria PLC) or Sulphadoxine -pyrimethamine (Sulphadoxine 500mg + Pyrimethamine 25mg by Swispha Pharmaceutical Company Nigeria Plc.). The manufacturing, expiry date and batch number of the administered drug were recorded into the patient’s case record form. All drugs were in tablet forms and were administered by the investigator. The treatment with chloroquine was a three-day course with the following doses:
DAY –0 10mg/kg body weight Day – 1 10mg/kg body weight DAY – 2 5mg/kg body weight.
Sulphadoxine-pyrimethamine was given as a single dose equivalent to 25mg/1.25mg /Kg body weight on DAY 0. In practice it is difficult to often divide the tablets into fractions containing the stipulated dose. For this reason a list of dose regimens for use in various weight groups, adjusted to nearest manageable fractions of the tablets was used (Appendix VIIl). The older children were allowed to swallow the drugs under supervision while younger ones were given their drugs crushed and mixed with 2ml of water in a clean container before
swallowed under supervision. The mouth was examined to ensure the drugs were swallowed. Each child was observed for 30 minutes after each such supervised drug treatment to ensure that the drug was not vomited or spat out.
Where vomiting occurred within 30 minutes, the dose was repeated. If the child vomited more than two times he or she was excluded. Additional management given included the administration of an antipyretic-paracetamol at a dose of 10 – 15mg/kg body weight every 8 hours for 24 hours when the temperature exceeded 38.5oC. Parents were instructed to tepid –sponge or expose the child during the initial 24 to 48 hours of the illness.
During the follow-up visits, when a patient developed other infections, other than malaria; he or she was treated accordingly and then excluded from the study.
Follow-up Procedures
The subjects enrolled into the study were followed up on DAYS 1,2,3,7 and 14. Chloroquine was completed on DAYS 1 and 2. Parents were asked whether their child had been ill or had experienced fever, vomiting, diarrhoea, pruritis, blurred vision or headache. Body temperature was recorded at every follow-up visit. Parasitaemia was determined on the DAYS 2, 3, 7 and 14.
Packed cell volume was repeated on DAY 14.
Parents were asked to bring the patient/child back to the clinic on any other day within the 14 days if he or she was ill. The addresses of the patient at enrolment were taken carefully for vigorous tracing of patients who failed to show up on the scheduled days.