JKIMSU, Vol. 6, No. 1, January-March 2017 ISSN 2231-4261 ORIGINAL ARTICLE New Methylene Blue Stain for Malaria Detection on Thin Smears Himanshu D. Mulay1, Teena D. Murthy1*, Savitri M. Nerune1, Amrutha M. R1 1Department of Pathology, B.L.D.E.U'S Shri. B. M. Patil Medical College, Hospital & Research Centre, Vijayapura-586103 (Karnataka) India Abstract: Introduction: Background: Malaria is the most important parasitic Malaria is the most important mosquito borne infection of man. Microscopy remains gold standard in parasitic infection of man caused by parasites of malaria diagnosis. Management of malaria requires the genus Plasmodium [1]. Four species of malaria rapid detection of parasite in human blood. Hence parasite infect human beings; they are Plasmodium there is a need to develop another diagnostic method falciparum, Plasmodium vivax, Plasmodium ovale with less limitation, which will address this issue. Aim and Plasmodium malariae [2]. It is a leading cause and Objectives: To find a low cost reliable and accurate method for malaria detection on peripheral smear. of morbidity and mortality worldwide. Malaria is Material and Methods: A prospective study of 40 cases prevalent in tropical and subtropical regions was done. Two thin smears were prepared for each because of significant amount of rainfall, warm case; one was stained with Leishman stain and other temperature, high humidity and stagnant water in with new methylene blue stain and examined under oil which the larvae mature providing mosquitoes immersion. The smears were examined individually ideal environment needed for continuous breeding by two pathologists and results were prepared. [1]. The symptoms of malaria such as fever, Different parasitic morphologic forms were looked headache, malaise, fatigue and abdominal for. Parasitemia percentage was calculated. We also discomfort are very non specific which may lead to compared number of fields required to diagnose with over treatment of malaria in endemic areas without both stains in positive cases. Results: In this study we laboratory diagnosis [3]. Early diagnosis of malaria found that 25 (83.3%) cases were detected in less than 50 fields using New Methylene blue stain against 18 is essential for malaria treatment and control. (60.0%) cases with Leishman stain. We also found Microscopy remains gold standard in malaria 100% sensitivity and specificity for New Methylene diagnosis even though rapid diagnostic tests are blue stain, whereas Leishman stain showed 90% increasingly used [4]. The advantage of sensitivity and specificity of 85%. Conclusion: The microscopy is that it provides a quantitative detection of malaria parasite was considerably easy assessment of peripheral blood parasitemia and with New Methylene Blue stain and required less time parasite stages as well as information on other in comparison with Leishman stain. blood elements [5]. Diagnosis of malaria based on Keywords: Malaria, New Methylene Blue Stain, microscopy has central importance for species Leishman stain, Parasitemia differentiation, parasitic quantification and Ó Journal of Krishna Institute of Medical Sciences University 76 JKIMSU, Vol. 6, No. 1, January-March 2017 Himanshu D. Mulay et al. management of severe disease [2]. Microscopy Leishman stain and other with new methylene requires technical expertise of the microscopist and blue stain and examined under oil immersion. is time consuming. To overcome the limitations of The samples were divided into 3 groups based on conventional microscopy many alternative rapid the percentage of parasitemia. The smears by both diagnostic non microscopic methods such as the stains were examined individually by two demonstration of acridine orange stained parasites pathologists and results were prepared. Smears in capillary centrifuged blood, rapid immuno- were examined for different parasitic morphologic chromatographic assays and the sensitive forms like trophozoites, schizonts and gametocytes. molecular techniques like DNA hybridization and Also hemozoin pigment character was observed in polymerase chain reaction have been introduced. the smears. Parasitemia percentage was calculated. These methods also have their own limitations and The smears were scanned carefully and minimum should be used as complementary method to of 200 oil immersion fields were examined before conventional microscopy [6]. Management of labeling a slide as 'Negative for Malaria'. If only malaria requires rapid detection of presence of occasional infected red cells are seen then 'less parasite in human blood and early institutionali- than 1%' value was given. One negative smear zation of antimalarial drugs. According to WHO, makes diagnosis of malaria unlikely but in all cases of suspected malaria should be confirmed clinically suspected cases; smears were repeated by microscopy or rapid diagnostic tests before every 6-12 hours for 48 hours to rule out malaria. administering treatment [2]. Hence there is a need Also presence of malarial pigment in circulating to develop another diagnostic method with less neutrophils and monocytes was looked for. limitation, which will address this issue. Results: Material and Methods: Malaria parasite can be detected by peripheral A prospective study of 40 cases including 30 cases smear examination using Leishman stain and new positive for malaria and 10 cases negative for methylene blue stain as in Table 1. malaria diagnosed by Leishman stained smears, The percentage of parasitemia was calculated by was done in haematopathology section of calculating number of infected red blood cells per Department of Pathology, BLDE University's, 1000 red cells and expressed as percentage (Table Shri B. M. Patil Medical College, Vijayapura, 2). Karnataka. All universal precautions were taken Parasitemia % = Number of infected red cells while preparing the smears. Two thin smears were 1000 RBCs prepared for each case; one was stained with Ó Journal of Krishna Institute of Medical Sciences University 77 JKIMSU, Vol. 6, No. 1, January-March 2017 Himanshu D. Mulay et al. Table 1: Detection of Malaria Parasite by Peripheral Smear Examination using Leishman Stain and New Methylene Blue Stain Method Ring Stage of Malaria Parasite under Microscope Nucleus Cytoplasm Pigment New Methylene Blue Stain Dark blue - Refractile brown-black Leishman Stain Red Blue Brown-black Table 2: Distribution of Cases According to Parasitemia Percentage Parasitemia Number of Cases Percentage (Positive Cases = 30) <2% 16 2-5% 10 5-10% 4 Table 3: Comparison between Positive Cases using Leishman Stain and New Methylene Blue Stain Identified Positive Cases in Leishman Stain New Methylene Blue Stain Number of Fields (positive cases = 30) (positive cases = 30) <50 Oil Immersion Fields 18 25 50-100 Oil Immersion Fields 8 5 100-200 Oil Immersion Fields 4 - Table 4: Shows Sensitivity and Specificity of Leishman and New Methylene Blue stain Stain Sensitivity Specificity Leishman Stain 90% 85% New Methylene 100% 100% Blue Stain Ó Journal of Krishna Institute of Medical Sciences University 78 JKIMSU, Vol. 6, No. 1, January-March 2017 Himanshu D. Mulay et al. Fig. 1: a) Plasmodium Falciparum showing Two Rings on Leishman Stain b) Plasmodium Falciparum showing Two Chromatin Dots on New Methylene Blue Stain c) Schizonts of Plasmodium Vivax on Leishman Stain d) Schizonts of Plasmodium Vivax Showing Pigment and Nuclear Chromatin Dots on New Methylene Blue Stain Fig. 2: a) Plasmodium Vivax showing One Ring on Leishman Stain b) Plasmodium Vivax showing One Chromatin Dot on New Methylene Blue Stain c) Engulfed Malaria Parasite within Neutrophil on Leishman Stain d) Basophilic Stippling within RBC which can be Confused with Malaria Parasite in Leishman Stain Ó Journal of Krishna Institute of Medical Sciences University 79 JKIMSU, Vol. 6, No. 1, January-March 2017 Himanshu D. Mulay et al. Discussion: This is a novel and one of its kind studies as here blue stain than Leishman stain. It was observed we have compared the number of oil immersion that hemozoin pigment was better appreciated fields required for making the diagnosis. It is with new methylene blue stain than Leishman recommended that at least 200 oil immersion stain (Fig. 1 and 2). fields should be examined before labeling a In this study we found that 25 (83.3%) cases were peripheral smear negative for malaria. In a detected in less than 50 fields using new laboratory in the resource poor endemic zone, this methylene blue stain against 18 (60.0%) cases has become cumbersome for pathologists to with Leishman stain. (Table 3) We found 100% screen multiple cases stained with Leishman stain. sensitivity and specificity for new methylene blue Thick smears have a higher sensitivity of stain, whereas Leishman stain showed 90% detection of 5-10 parasites/µl but due to red cell sensitivity and specificity of 85% because of false lysis, morphology and species identification is positive and false negative results due to artifacts difficult, for which a second thin smear stained (Table 4). with Leishman stain needs to be done. Thick Lema et al. (1999) [6] found 98% sensitivity and smear method is tedious with multiple steps. Thin 100% specificity for field stain which has new smears stained with Leishman stain are used for methylene blue as a component and 92% species identification and have a sensitivity of 200 sensitivity and 85% specificity for Giemsa stain parasites/ µl. But if the single thin smear prepared, which is also a Romanowsky stain like Leishman is stained with new methylene blue stain