P.falciparum malaria proportion was found highest in Balochistan and Sindh, ... respectively (National HMIS report, 2000), which decreased in Punjab up to 19% ...
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"0" )*4/ +) * !5 !& 6!7%66898& % ) 2 : ! * & / - ;9 50 per minute in infants below 12 months of age, and > 40 per minute in children of 1259 months. The case record form was used to record the general information and the clinical observations of each patient under screening. Particular care was given to record the full patient’s address in order to be able to trace them at home, in case they did not come to the health facility at scheduled visits. 0nce the patient met all the inclusion criteria, was enrolled in the study. Doses were calculated according to the formula given and subjects were treated with chloroquine, basoquine or sulfadoxine-pyrimethamine. Thick blood smears were made & stained with Giemsa. The parasite density was calculated by counting the number of asexual parasites per 200 white blood cells, assuming a mean white blood cell count of 6000/μl. Each blood smear was independently examined. Gametocyte density was not examined. The parasitemia /μl were calculated by using the formula: parasitemia per μl = number of parasites x 8000 / number of leukocytes When 500 parasites were counted without having reached 200 leukocytes, the count was stopped after completing the reading of the last field, and the parasitemia was calculated. 100 fields of the second thick film were examined for the exclusion of mixed infections, which were confirmed on the thin film in case of any doubt. If the examination of the thin film was not conclusive, the patients were excluded from the study after complete treatment. Follow-up of test cases was done for monitoring the course of asexual
35 parasitemia (Bruce.Chwatt, 1981). Those did not return were followed-up at their homes for follow up blood sample collection. Table 2.1: Formulation of different antimalarial drugs Sr.No Drugs Used 1 2
3
Chloroquine (Roche) Basoquine (Roche) Sulfadoxinepyrimethamine (Roche)
Formulations Tablets 150 mg base as phosphate or sulphate. Tablets 100 mg base as phosphate or sulphate. Syrup 50 mg base as phosphate or sulphate. Tablets 150 mg base as chlorhydrate Syrup 50 mg base as chlorhydrate Tablets 150 mg base as sulfadoxine & 25 mg base as Pyrimethamine
2.1 Rapid Diagnostic Test (RDT) The whole blood was collected for detection of P.falciparum by the rapid test, which was compared in this study with the traditional microscopy. Total 200 patients with fever were identified and enrolled for study in district Sheikhupura. Thick and thin smears for traditional microscopy were made and for rapid test blood samples were collected in the capillary tube available in the Kit. Smears were considered negative if no parasite were seen in 500 oil-immersion fields on a thick blood film (Mills et al., 1999). Blood collected for rapid test was added to the well A and substrate to well B. Results were read after 15 minutes and recorded. One pink band appeared in the C (control) area of the test card, showed negative for P.falciparum. One pink band appeared in the C (control) area and second in the B (test) area of the test card, showed the positive for P.falciparum. It was assured by checking the strips from every batch against known positive blood samples for P.falciparum. Stability of all reagents of the kit was intact at 37°C.
2.2 Data Analysis All subjects studied by in vivo technique were kept under observation for 28 days or until treatment failure or loss to follow-up, if either occurred in the interim. Survival probability was determined by using the Kaplan-Meier method. Predictors of the treatment failure by analyzing the patient’s baseline characteristics (age, sex, weight, parasitemia at day 0) were also analyzed. For prediction of early and late failure time was divided into 0 to 28 days. Differences in proportions were
36 analyzed using chi-square tests (Fontanet and Walker, 1993). The results were interpreted as already described
2.3 Place of Work The major part of the proposed study was done in the field in selected districts of Punjab, Pakistan. Laboratory work was done in the Parasitalogy Laboratory of Zoology Department, University of the Punjab Lahore. Different anti malarial drugs, slides, prickers, chemicals and equipments etc required for study were managed by the department.
37
Annexure-I Questionnaire Case No.
Date _________
Name ______________________ Age _______ Sex Address ___________________________________ History 1. Have you suffered from fever?
Yes No a. In case of yes
i. When ii.
What type: (Intermittent or continuous) ________________
iii.
Vomiting ________________________________________
iv.
Shivering ________________________________________ Sweating ________________________________________
v. 2. Have you taken?
any treatment? Yes No
b. In case of Yes i.
From whom
ii. When 3. Have you traveled to any other area? Yes No c. In case of Yes i.
Where _________________________________________
ii. When _________________________________________ iii. Physical examination 1. Weight _____________________________________________________ 2. Temperature _________________________________________________ 3. Spleen size __________________________________________________
38
Annexure-II Consent The Ministry of health is interested in knowing how well the current treatment for malaria is working in our country. To do this, we are carrying out a study in which we are treating a group of patients with malaria and then following them for 28 days to see if their infection is cured. This is not a new treatment; the test drugs are chloroquine, basoquine and sulfadoxine-pyrimethamine. If you agree to participate in this study, we would like you to come to the clinic 7 times more over the next 4 weeks, so that we can monitor the progress of the treatment. It is very important that we see you on these days, 1, 2, 3, 7, 14, 21 and 28, so if you feel you will not be able to return on these days, please let us know now. At each visit you will receive a full medical examination and on 3 of these visits, we will take a small amount of blood by finger prick to make blood smears to see if you still have malaria parasites. Your participation is completely voluntary. If you do not want to participate in this study, you will receive treatment as usual at this clinic. Participation in this study will not cost you or your family anything. You may also withdraw from the study at any time and for any reason. You will be benefited for participating in this study because you will be closely followed over the next 28 days. If you continue to suffer from malaria, you will receive an alternative treatment, which will cure the illness.
39
3. RESULTS 3.1 IN VIVO 3.1.1 Mono therapy The results of 28-day follow-up of 404 subjects treated with chloroquine for uncomplicated falciparum malaria in five districts of Punjab, Pakistan are reported here. During the non-transmission season of the year 1999 to 2000 and 2008, among the rural populations 5952 persons were screened for malarial parasites. P.falciparum Positivity Rate (FPR), P.vivax Positivity Rate (VPR) and slide positivity rate (SPR) were found 24.02%, 3.32%, 27.35% respectively (Table 3.1.1.1). Table 3.1.1.1: Showing the slide positively rate, Plasmodium vivax positivity falciparum positivity rate in five districts of Punjab, Pakistan. Slides Total Study Area Pv Pf SPR VPR Examined Positive Sheikhupura 876 13 195 208 23.74 1.48 Muzaffargarh 1090 61 319 380 34.86 5.59 Multan 925 54 218 272 29.4 5.83 Jhang 1893 36 394 430 22.715 1.90 D.G.Khan 1168 21 283 304 26.02 1.79 5952 185 1409 1594 27.35 3.32 Total
rate and Plasmodium FPR 22.26 29.26 23.56 20.81 24.22 24.02
Pv= Plasmodium vivax, Pf= Plasmodium falciparum, Out of 409 subjects enrolled, 5(1.22%) were absconded and 404 (98.78%) completed the study and tested by in vivo technique for assessing the resistance status of P.falciparum. All subjects followed for seven days (on day 1, 2, 3, 7, 14, 21 and 28). 261(64.60%) declared sensitive and143 (35.39%) resistant subjects (Figure 3.1.1.1.A). A
B
1%
12%
35%
88%
64%
Absent
Sensitive
Resistant
Resistant I
Resistant II
Figure 3.1.1.1: A) Showing absent (1%), sensitive (64%) and resistant (35%) subjects by in vivo
40 technique against Plasmodium falciparum with chloroquine in five districts of Punjab, Pakistan. B) Showing resistance I and II Out of 143 resistant subjects 126 (88.11%) were Resistant 1 (RI) and 17(11.89%) RII (Figure 3.1.1.1 .B) and RIII were not found.. Maximum RI resistance was noted 30.95% (39/126) and RII 35.39% (6/17) in Multan. Minimum RI was 7.93% (10/126) and R II 5.88(1/17) in Sheikhupura. Trends of resistance in different districts have been shown in Figure 3.1.1.2.
12
R I%
R II%
Resistance (%)
10 8 6 4 2 0 SHR
MZ
MN
JG
DGK
SHR; Sheikhupura, MZ; Muzaffargarh, MN; Multan, JG; Jhang, DGK; D.G.Khan
Figure 3.1.1.2: Showing the area wise number of resistant subjects of chloroquine against Plasmodium falciparum by in vivo study in five districts of Punjab, Pakistan. The enrolment of male and females subjects was 63.6 1% (257/404) and 36.3 8% (147/404) respectively. Total resistant subjects were 35.39% (143/404).
41 Table 3.1.1.2: Showing the total study subjects, sensitive and resistant with gender, age and parasite density/μl by in vivo technique on chloroquine in five districts of Punjab, Pakistan. Age(years)
Gender
Parasite density/μl Total
Male
Female
5
5 to 15
15
3000
3000 to 6000
6000
Sensitive
166
95
22
135
104
115
72
74
261
Resistant
91
52
9
69
65
25
41
77
143
Total
257
147
31
204
169
140
113
151
404
Gender wise further analysis of resistance, described that there were 63.63% (91/143) male and 36.36% (52/143) females had resistant strains of P.falciparum. Males showed 27.27% more chances of treatment failure as compared to females (Odds ratio ‹OR›for male = 1.674‹95% CI=1.062.622›).
S u b je c ts (n )
450 400 350 300 250 200 150 100 50 0
404
Total Male Female 261
257 147
166
143 95
91 52
Total Sensitive Resistant Figure 3.1.1.3: Showing the total, sensitive and resistant subjects of Plasmodium falciparum in male and female subjects against chloroquine by the in vivo technique in five districts of Punjab, Pakistan.
Among total study subjects 22.52% (9 1/404) were found male resistant subjects and 12.87% (52/404) were females. On analysis of resistant subjects’ male among male study subjects and females among females, the result was found 35.40% (91/257) resistant males and 35.37% (52/147) females.
42
Total 16years 6 to 15 years 5 years
404
S u b je c ts(n )
450 400 350 300 250 200 150 100 50 0
261 204 169
143
135 104
65 69 31 Total
22
9
Sensitive
Resistant
Figure 3.1.1.4: Showing the sensitive and resistant subjects of chloroquine against Plasmodium falciparum in different age groups by in vivo technique in different areas of the Punjab, Pakistan. On analysis of age groups, it was noted that the 1st group 5 years of age had resistance 6.29% (9/143), 2nd group 6 to 15 years had 48.25% (69/143) and 3rd 15 years had 45.45% (65/143). Among the total study subjects the young one group (5) contributed 2.22% (9/404) resistance, 2nd age group (6 to 15) had 17.07% (69/404) and among 15 years age group had 16.08% (65/404) resistant subjects (Figure3.1.1.4).
S u b je c ts(n )
450 400 350 300 250 200 150 100 50 0
Total >6000 3000-6000 261 6000
S u b je c ts(n )
200 140
150 100
83 87 68
50
88 61 4336
3000-6000 6000 3000-6000
