Conceptos Básicos de la Teoría de Piaget Los estadios

Whole blood was used for the diagnosis of P. falciparum malaria using thick and thin blood smears for microscope detection and malaria plasmodium falciparum rapid test device (CARESTART^TM Malaria HRP2 (Pf) by ACCESS BIO,INC. USA) which is a chromatographic immunoassay for the qualitative detection of circulating p. falciparum antigen in whole blood. It contains a membrane strip, which is pre-coated with a monoclonal antibody as a single line across a test strip. The monoclonal antibody is specific to the HRP2 (histidine-rich protein 2) of the P. falciparium. The conjugate pad is dispensed with antibody absorbed on gold particles, which are specific to HRP2 of P. falciparium.

The thick blood film concentrated red blood cells (RBCs) on a small surface and was more sensitive than the thin film method in detecting low levels of parasitaemia. Giemsa stain an alcohol based Romanowsky stain was used. It is a mixture of eosin which stains the parasite chromatin red or pink and methylene blue which stains the parasite cytoplasm blue. The RBCs were lysed during the staining process leaving white blood cells, platelets and any parasites.A drop of blood was placed on the centre of a clean grease free slide and spread in a circular

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manner on the slide with the corner of another slide till it covered an area four times its original area. The film was allowed to dry properly in the incubator at 37⁰ C for 30 minutes before immersing in a staining jar containing Giemsa stain (freshly diluted with buffered water (10%) for 30 minutes before washing off. The slides were stood upright to air dry. Slides were viewed using X100 objective (oil immersion) for detection of malaria parasite. In the thin blood films, RBCs were fixed and a parasites inclusion in RBCs was demonstrated. The morphological identification of the parasite to the species level was much easier and provided greater specificity than the thick film examination. For the procedure, a small drop of blood was placed at the bottom of the slide and using a spreader, a thin film was made. This was air dried and stained using Leishman stain first for 2 minutes before double diluting in buffered water and staining further for 8 minutes. Subsequently, the slides were washed and air dried before viewing using X100 objective. For the rapid malaria parasites test, whole blood was used for the diagnosis using malaria plasmodium falciparum/pan rapid test device (CARESTART^TM Accessio USA).

Principle

The principle is based on a rapid chromatographic immunoassay for the qualitative detection of circulating P. falciparum antigen in whole blood. Immunochromatography relies on the migration of liquid across the surface of a nitrocellulose membrane. The immunochromatographic tests are based on the capture of parasite antigens from peripheral blood using monoclonal antibodies prepared against a malaria target and conjugated to gold particles in a mobile phase. A second capture monoclonal antibody applied to a strip of nitrocellulose acts as the immobile phase. The migration of the antigen – antibody complex in the mobile phase along the strip enables the labeled antigen to be captured by the monoclonal antibody of the immobile phase, thus producing a visible coloured line. The test cassette (CARESTART^TM) contains a membrane strip, which is pre-coated with a monoclonal antibody as a single line across a test strip. The monoclonal antibody is specific to the HRP2 (histidine-rich protein 2) of the P. falciparium. The conjugate pad is dispensed with antibody absorbed on gold particles, which are specific to HRP2 of P. falciparium. The assay buffer borax buffered SDS and saponin solution. The mouse monoclonal antibodies react with the malaria antigen if present in the sample they move along the membrane chromatographically to the test region and form a visible line as the antibody – antigen – antibody gold particle complex with high degree

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of sensitivity and specificity. Both the test line and control line in the result window are not used for procedural control and should always appear if the test has been performed correctly.

Procedure (as described by the manufacturer)

Briefly, 5µl of the whole blood specimen from the subjects was delivered unto labeled round specimen wells in the cassettes, using the specimen loops provided. 2 drops of assay buffer (supplied with the test device) was added into square assay diluents wells. After 20 minutes, the results were read.

For positive result:

P. falciparum positive: the presence of two colour bands (“P.F” Test line and “C” Control line) or three colour bands (“P.F”, “Pan” Test lines and “C”Control) within the result window, no matter which bands appears first, P. falciparum positive result would be indicated.

Procedure for parasite count (as described by Cheesbrough, 2005)

The parasite count was done using thick blood films. The films were stained using Giemsa stain as previously described. The density of malaria parasites were estimated by counting the number of parasites against 200 white blood cells (WBCs) in the thick film. The parasite density (per µl) was obtained using the formula:

No of parasite counted x WBC count WBC counted (200) 1 3.19. Estimation of serum lipids.

(a)Total Cholesterol

(a)Total Cholesterol was estimated by Cholesterol enzymatic End-point Method (Roeschlau et.al., 1974). The kit was purchased from Randox Diagnostic LTD,Cat. No. CH 200.

Principle

Cholesterol is determined after enzymatic hydrolysis and oxidation. The indicator quinoneimine is formed from hydrogen peroxide and 4- amino antipyrine in the presence of phenol and peroxidase (Trinder, 1969).

Cholesterol ester + H2O cholesterolesterase Cholesterol + fatty acids Cholesterol + O2Cholesterol oxidase Cholestene-3-one + H2O2

2H2O + phenol + 4-Aminoantipyrine peroxidase quinoneimine + 4H2O

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10(ul) of serum, distilled water, control and standard cholesterol was collected and transferred into four test tubes labelled sample ,reagent blank,control and standard respectively.1000(ul) of reagent(R1) was added into all the test tubes, mixed and incubated for 5 minutes at 37^OC. The absorbance of the sample (A sample) against the reagent blank was read spectrophotometrically within 60 minutes at 500nm.

(b) Triglycerides

Triglycerides was estimated by enzymatic method (Tietz, 1990 and Trinder, 1969).

The kit was purchased from Randox Diagnostic LTD,Cat. No. TR 210.

Principle

The triglycerides are determined after enzymatic hydrolysis with lipases. The indicator is a quinoneimine formed from hydrogen peroxide,4-aminophenazone and 4-chlorophenol under the catalytic influence of peroxidase.(Tietz,1990 and Trinder,1969).

Triglycerides + H20 ^lipases glycerol + fatty acids Glycerol +ATP ^GK glycerol-3-phosphate +ADP

Glycerol-3-phosphate + O2GPO dihydroxyacetone + phosphate + H2O2

2H2O2 +4-aminophenazone +4 chlorophenol ^POD quinoneimine +HCl +4H2O

Procedure

10(ul) of serum, distilled water, control and standard cholesterol was collected and transferred into four test tubes labelled sample ,reagent blank, control and standard respectively.1000(ul) of reagent(R1=reagent mixture R1a +R1b) was added into all the test tubes, mixed and incubated for 5 min at 37^OC. The absorbance of the sample (A sample) against the reagent blank was read spectophotometrically within 60 minutes at 500nm.

(c) High Density Lipoproteins-cholesterol (HDL-C).

The kit was purchased from Randox Diagnostic LTD,Cat. No. CH 203.

Principle

Low Density Lipoprotein (LDL AND VLDL) and chylomicron fractions are precipitated quantitatively from serum by the addition of phosphotungstic acid in the presence of

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magnessium ion. After centrifugation, the cholesterol concentration in the HDL (High Density Lipoprotein) fraction which remains in the supernantant is determined.

Procedure

(i) Precipitation reaction, 500(ul) of sample and standard was transferred into two test tubes, each containing 1000(ul) of precipitant (R1),and labelled, sample and standard respectively. The tubes were mixed and allowed sitting for 10minutes at room temperature. Then centrifuged for 10 minutes at 4000 rpm, or 2 minutes at 12000rpm. The cleared supernatant was separated off within two hours and was used for determining the cholesterol content as follows.

(ii) 100(ul) of supernatant, distilled water and standard supernatant was collected and transferred into three test tubes labelled sample ,reagent blank and standard respectively.1000(ul) of reagent (R1) was added into all the test tubes, mixed and incubated for 5 min at 37^OC. The absorbance of the sample (A sample) against the reagent blank was read spectophotometrically within 60 minutes at 500nm.

(d) Low Density Lipoprotein Cholesterol (LDL-C) in mmol/l was estimated by a clearance method for the direct measurement of LDL cholesterol using Randox Diagnostic LTD kit.

Principle

The assay consists of two distinct reaction steps

Elimination of chylomicrons, VLDL-Cholesterol and HDL-Cholesterol by cholesterol esterase, cholesterol oxidase and subsequently catalase.

Specific measurement of LDL-Cholesterol after release of LDL-Cholesterol by Cholesterol enzymatic End-point Method (Roeschlau et.al., 1974).

(e) Atherogenic index (AI)

Atherogenic index was calculated by dividing the values of low density lipoprotein cholesterol with that of high density lipoprotein cholesterol.

(f) Coronary risk index (CRI)

Coronary risk index was calculated by dividing the values of total cholesterol with that of high density lipoprotein cholesterol.