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 countries1.In 2016 there were 216 million cases of malaria reported worldwide and there have been 445000 deaths due to malaria in the same year1.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 nature2.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 standard stain used3.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 components4.It has been used previously for the assessment of malarial parasite before5.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:This is a prospective study.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.To prepare a peripheral blood smear, 1-2 drops of blood are placed close to one end of a slide.Then a spreader slide(with smooth edges) is taken and placed in front of the drop of blood.It is held at a 30-45° angle approximately and it is drawn back against the drop of blood.The blood is allowed to spread along the edges of the slide.It is then pushed forward for the blood to uniformly spread out.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°C overnight with occasional shaking6.It is left to cool.After cooling it is filtered and then the stain is finally applied on the slide.The air dried smear is flooded with Leishman stain and it is set aside for 2 min.Then double the quantity of distilled water is added and is set aside for 8 min.It is then carefully washed in a stream of tap water. The slide is wiped dry with cotton and set aside for drying for 5 min.It is then examined in a microscope.Toluidine 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 overnight6t.It is filtered before it is used on the slides. The dried smear is flooded with Toluidine blue stain and it is kept aside for 1 min.It is then carefully washed in a stream of tap water. The slide is wiped dry and set aside for 5 min.It is then examined in a microscope. 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:Positive results from this study will prove the efficacy of Toluidine blue stain in identification of the malarial parasite along with its various life stages when compared to the routine Leishman’s stain used for peripheral smear staining. It can be a better alternative because it is cheaper,less time consuming,easier and requires less expertise when compared to the standard7.References:1.Fact sheet about Malaria. (n.d.).from http://www.who.int/mediacentre/factsheets/fs094/en/2.Annam, V., Mohan, C. N., & Mrinalini, V. R. (2013). Rapid Detection of Malaria Parasite by Toluidine Blue Method: A New Staining Method. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3958985/#!po=50.00003.4.Sridharan, G; Shankar, AA (2012). “Toluidine blue: A review of its chemistry and clinical utility”. J Oral Maxillofac Pathol. 16: 251–5. doi:10.4103/0973-029X.99081. PMC 3424943? . PMID 22923899.5.Shravan N, Yelikar BR. A novel use of toluidine blue in diagnosing malaria. Australasian Med J. 2010;3(8):554.6.Awale, R., Maji, R., Patil, P., Lingiah, R., Mukhopadhyay, A. and Sharma, S. (2018). Toluidine blue: rapid and simple malaria parasite screening and species identification. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5681009/#!po=62.00007.