A simple and rapid staining method for detection of hemozoin pigment by methylene blue stain.

Sir,

Light microscopy using Giemsa stain is considered as the gold standard for malaria diagnosis, but its sensitivity varies depending upon the expertise of the observer.[1] In addition, low parasitemia conditions (e.g., chronic infection, early stage of disease and partially treated condition exhibiting only hemozoin pigments) remains challengeable.[2] Detection of hemozoin pigment within parasite and mononuclear cells is observed to be an important tool for the diagnosis of malaria and considered as one of the signs of severity.[34] These pigments can be detected by flow cytometry, spectrophotometer, polarizing microscope, etc., which found to be expensive and not very sensitive and specific.[5] Methylene blue dye remains a major component of Giemsa stain, but its use for the detection of hemozoin pigment and malaria parasite has not been studied much.

A pilot study was carried out for detection of malaria parasite and hemozoin pigment using methylene blue stain and Giemsa stain. A total of 30 blood samples positive for malaria by peripheral blood smear and rapid diagnostic tests were included in this study. The samples were divided into three groups based on parasitic count, i.e., >5000 parasites/μl (Group A, n = 8), ≥3000 to ≤5000 parasites/μl (Group B, n = 8), <3000 parasites/μl (Group C, n = 14). Simultaneously, another set of peripheral smears were made from the samples and stained with methylene blue stain for 45 min. Smears by both stains were examined for different parasitic forms and hemozoin pigment character. Parasitemia and various morphologic forms (early trophozoite, late trophozoite, schizont, gametocyte and pigment containing leukocytes) were calculated for each individual slide by both stains. All the smears were independently observed and scored by three experienced microbiologists. A questionnaire based evaluation was carried out by 11 other resident doctors of the microbiology department for the assessment of both stains [Appendix 1].

All smears (n = 30) found positive for asexual forms of malaria parasite by Giemsa stain in comparison to 29 by methylene blue stain [Table 1]. Gametocytes of Plasmodium vivax was seen in 83.3% (25/30) and 90% (27/30) of the blood smears stained with Giemsa and methylene blue stain, respectively. Asexual forms were found more in Group B and C with Giemsa stain, whereas the gametocyte count was observed 2-3 times higher in all groups with methylene blue stain [Table 1]. The total number of slides positive for pigment containing leucocytes was also higher with methylene blue stain [Table 1]. However, the average parasitemia remained nearly equal by both stains. As per the result of questionnaire evaluation, 6/11 and 4/11 observers found gametocyte detection to be good and very good with methylene blue stain [Appendix 1]. For the detection of asexual stages, six observers scored methylene blue stain to be a poor stain and four found it to be good in comparison to Giemsa stain. Majority of the observer graded methylene blue as very good for the detection and characterization of hemozoin pigment. Out of 11, 8 observers were found methylene blue as a very good stain for the detection of pigment containing leucocytes.

Table 1

Comparative evaluation of peripheral blood smears by Giemsa stain and methylene blue stain

Methylene blue is a polychromed basic dye, which is further methylated to Azure B, Azure A, Azure C and Threonine.[67] Azure B remains one of the major components of Giemsa stain, which maintains the normal appearance of neutrophils including its granules and nucleoli.[8] The average parasitemia observed to be nearly equal by both stains suggesting the total number of asexual forms observed by both stains was found nearly the same. However, as per the result of the questionnaire, the morphology of the asexual forms is better appreciated in Giemsa. The gametocyte count was found invariably higher in all groups with methylene blue stain in comparison to Giemsa. The result of the questionnaire showed complete concordance with the above finding, in which the gametocyte morphology and the pigment character was better appreciated by methylene blue stain in comparison to Giemsa stain [Figure 1]. Background stain deposit always remains a major limitation with Giemsa stain and creates confusion between parasitic forms and artifacts. In contrast, smears stained with methylene blue have a very pale background without giving any color contrast to stain deposits or artifacts and only exhibiting blue color for parasitic forms and leucocytes. Therefore, the number of false positivity and false negativity was found lesser than Giemsa stain and screening rate of slides with methylene blue was found to be more rapid. During the evaluation period, we also observed that the gametocyte forms with methylene blue stain were easier to identify. However, the morphology of the asexual form was less appreciated in comparison to Giemsa. Hence, methylene blue can be added with Giemsa stain in the endemic areas as a screening test to screen the carrier stage. It can be useful to give accurate result in chronic conditions, relapsed cases and partially treated patients showing only leucocyte containing pigment. It is cost-effective, easy to prepare and unlike Giemsa, do not need time for maturation. As per the result of our evaluation, gametocyte, pigment containing leukocytes and the hemozoin pigments were easily identified with this stain and need less expertise. However, the number of slides with pigment containing leucocytes was less, which is a limitation to the study. Further, study can be planned with more number of samples positive for pigment containing leucocytes to evaluate the efficacy of both stains for the detection of hemozoin pigments.

Figure 1

Peripheral blood smear showing (a) gametocyte of Plasmodium vivax stained with Giemsa, (b) trophozoites stained with Giemsa, (c) gametocyte of P. vivax stained with methylene blue and (d) trophozoites stained with methylene blue

In the present study, peripheral blood smear with methylene blue stain found to be an easier and better method for the identification of gametocyte of Plasmodum spp. and hemozoin pigments. It is accurate, rapid and cost-effective in comparison to other methods available for pigment detection. Hence, it can be useful as an adjunct to Giemsa stain in mass survey for detection of malaria in low parasitemic conditions and identification of hemozoin pigments.