Reference ID: 2018-02036 Title:Rapid and simple screening of malarial parasite by toluidine blue and evaluation of efficacy of the method compared to Leishman’s stain.Introduction:Malaria is an infectious communicable disease that is often life threatening which spreads through the bite of the female anopheles mosquito.It is more prevalent in the asian and african countries.In 2016 there were 216 million cases of malaria reported worldwide and there have been 445000 deaths due to malaria in the same year.In India there are a lot of reported cases of malaria due to various factors such as high population of vectors,favourable weather etc.Early detection and management of the disease can prevent complications.

There are various laboratory diagnostic tests to detect malaria but microscopy remains the gold standard due to its effectiveness,cheap and rapid nature.In microscopy,pap smears will be prepared and stained to visualize RBC’s in a microscope.Various dyes are used for this purpose of staining.Leishman’s stain is the dye recommended by WHO.This paper aims at demonstrating the use of toluidine blue as a more rapid,cheaper alternative and an easier technique.

Toluidine blue is a basic dye with affinity for acidic components.It has been used previously for the assessment of malarial parasite before.However it has not been utilised in a larger scale for the diagnosis of malaria.

Objectives:The objective of the study is to compare the efficiency,cost-effectiveness and ease of staining of Leishman’s and Toluidine blue in hopes of establishing that Toluidine blue is a better alternative for the diagnosis of malaria.Methodology:Blood samples will be collected from 50 people with clinical suspicions of malaria.Following collection thin blood smears are to be prepared by slide method.The slide will then be left to dry.After air drying the slides will be alternately subjected to staining with Leishman’s and Toluidine blue stains.

Leishman stain will be prepared in a flask by mixing 1.5 grams of Leishman powder with 500 ml methanol followed by warming the mixture to 37 degree Celsius for overnight with occasional shaking. After cooling the stain will be filtered and will be and used. Leishman stain will then be applied on the slides. The air dried smear is flooded with Leishman stain, after 2 minutes double quantity of distilled water will be added and thoroughly mixed with the help of a pipette and kept for 8 minutes followed by washing in a stream of tap water. The back side of the slide will be wiped clean with dry cotton and set upright for drying for 5 minutes. The Leishman stained smear will then be subjected to microscopic examinationToluidine blue stain will be prepared in a flask by mixing 5 grams of Toluidine blue stain with 100 ml of 95% alcohol and 400 ml of distilled water followed by keeping the mixture at room temperature overnight.

The stain is then filtered and used on the slides. The dried smear is flooded with Toluidine blue stain for 1 minute followed by washing in a stream of water. The backside of the slide will be wiped clean with dry cotton and set upright for drying for 5 minutes. The toluidine blue stained smear will then be subjected to microscopic examination. The slides will be reported by two different qualified lab professionals in a blindfolded manner. The findings will be noted and data will be analyzed.Implications:Reference ID: 2018-02036 Title:Rapid and simple screening of malarial parasite by toluidine blue and evaluation of efficacy of the method compared to Leishman’s stain.

Introduction:Malaria is an infectious communicable disease that is often life threatening which spreads through the bite of the female anopheles mosquito.It is more prevalent in the asian and african countries.In 2016 there were 216 million cases of malaria reported worldwide and there have been 445000 deaths due to malaria in the same year.

In India there are a lot of reported cases of malaria due to various factors such as high population of vectors,favourable weather etc.Early detection and management of the disease can prevent complications.There are various laboratory diagnostic tests to detect malaria but microscopy remains the gold standard due to its effectiveness,cheap and rapid nature.In microscopy,pap smears will be prepared and stained to visualize RBC’s in a microscope.Various dyes are used for this purpose of staining.Leishman’s stain is the dye recommended by WHO.This paper aims at demonstrating the use of toluidine blue as a more rapid,cheaper alternative and an easier technique.

Toluidine blue is a basic dye with affinity for acidic components.It has been used previously for the assessment of malarial parasite before.However it has not been utilised in a larger scale for the diagnosis of malaria.Objectives:The objective of the study is to compare the efficiency,cost-effectiveness and ease of staining of Leishman’s and Toluidine blue in hopes of establishing that Toluidine blue is a better alternative for the diagnosis of malaria.Methodology:Blood samples will be collected from 50 people with clinical suspicions of malaria.Following collection thin blood smears are to be prepared by slide method.

The slide will then be left to dry.After air drying the slides will be alternately subjected to staining with Leishman’s and Toluidine blue stains.Leishman stain will be prepared in a flask by mixing 1.5 grams of Leishman powder with 500 ml methanol followed by warming the mixture to 37 degree Celsius for overnight with occasional shaking. After cooling the stain will be filtered and will be and used. Leishman stain will then be applied on the slides. The air dried smear is flooded with Leishman stain, after 2 minutes double quantity of distilled water will be added and thoroughly mixed with the help of a pipette and kept for 8 minutes followed by washing in a stream of tap water.

The back side of the slide will be wiped clean with dry cotton and set upright for drying for 5 minutes. The Leishman stained smear will then be subjected to microscopic examinationToluidine blue stain will be prepared in a flask by mixing 5 grams of Toluidine blue stain with 100 ml of 95% alcohol and 400 ml of distilled water followed by keeping the mixture at room temperature overnight.The stain is then filtered and used on one of the slides. The air dried smear is flooded with Toluidine blue stain for 1 minute followed by washing in a stream of deionized water. The backside of the slide will be wiped clean with dry cotton and set upright for drying for 5 minutes.

The toluidine blue stained smear will then be subjected to microscopic examination. The slides will be reported by two different qualified lab professionals in a blindfolded manner. The findings will be noted and data will be analyzed.Implications:Expected results from this study will prove that Toluidine blue staining is a better method compared to the standard Leishman’s in identification of the malarial parasite along with its various life stages.It will be a better alternative because it is cheaper,less time consuming,easier and requires less expertise when compared to the standard.