Methylene blue vital staining for Trypanosoma cruzi trypomastigotes and epimastigotes

Abstract

SUMMARYThe morphological identification of Trypanosoma cruzi is currently considered to have a high specificity, but its sensitivity, which depends on the volume of the sample examined, is rather low. Trypanosome developmental stages suspended in blood, reduviid feces, and culture media are routinely searched for by means of fresh film examination (about 2 µL). High speed centrifugation of blood samples separates the buffy coat, where most trypomastigotes concentrate. As the parasites are transparent and colorless, their detection is mostly dependent on their motility. The fluorescent vital stain acridine orange has been used to enhance image contrast, as exemplified by the QBC (Quantitative Buffy Coat) technique. Staining blood, buffy coat, reduviid feces, and culture media samples with methylene blue (also a vital dye) is a means of producing sharp, well contrasted images of motile or non-motile T. cruzi developmental stages, only standard laboratory microscopes being required. Slides previously coated with a thin layer of methylene blue are used to stain fresh blood films. Photomicrographs exemplify the results of methylene blue staining applied to living and fixed parasites. KEYWORDS: Chagas disease; Morphological identification of Trypanosoma cruzi;Fresh blood films; Buffy coat; Methylene blue vital staining.The morphological identification of the etiological agent of Chagas disease, Trypanosoma cruzi, in fresh preparations of peripheral blood (about 2 µL) collected with anticoagulant has been for a long time standard technique. Such films are examined under a microscope fitted with ×10 and ×40 dry objectives. As the objects of our investigation are transparent, colorless motile protozoa, their detection is to a great extent dependent on their motility, which causes the red blood corpuscles to move 6 , most frequently the first indication of their presence. A time lag between collection and examination of blood samples is one of the factors that can, by affecting the motility of trypomastigotes, be the cause of false-negative diagnoses. Imageenhancing means have been recommended for routine use, viz: reduction of the numerical aperture of the condenser diaphragm; dark field; phase-contrast microscopy 6 , and staining. The use of dark field and phase-contrast techniques requires microscopes with expensive components. The reduction of the numerical aperture of the condenser diaphragm is not effective enough when the parasites are not motile. Romanowsky-stained films usually produce sharp and easily recognized images of blood parasites. This applies especially to thin films. However, they require much time and expertise; their use is restricted to selected cases.On account of such characteristics as cost effectiveness and high specificity, wet blood films are still used, without major modifications, in routine clinical and epidemiological investigation. Other factors remaining constant, the sensitivity of techniques involving the direct examination of blood films depends on the volume of the sample. As the vol...

Evidence of meaningful levels of Trypanosoma cruzi in platelet concentrates from seropositive blood donors

“…Filtration takes advantage of differences in particle size to separate and, as has already been stated, centrifugation is based on the specific gravity of blood cells. The trypomastigote form of T. cruzi has a size of 16 to 20 µm and a specific gravity between PLTs and WBCs . According to these data, it is reasonable to think that, since PLTs are concentrated along with WBCs throughout the apheresis procedure, trypomastigotes are concentrated along with WBCs in the PLT fraction rather than in the plasma or RBC fraction.…”

“…The trypomastigote form of T. cruzi has a size of 16 to 20 mm 18 and a specific gravity between PLTs and WBCs. 27,28 According to these data, it is reasonable to think that, since PLTs are concentrated along with WBCs throughout the apheresis procedure, trypomastigotes are concentrated along with WBCs in the PLT fraction rather than in the plasma or RBC fraction. Therefore, the PLT fraction initially collected by apheresis contains WBCs, although it is leukoreduced during the procedure by elutriation or after the procedure by filtration.…”

Haematological and immunological characteristics of eastern hellbenders (Cryptobranchus alleganiensis alleganiensis) infected and co-infected with endo- and ectoparasites

“…In other vertebrates, vital stains such as new methylene blue can be useful for evaluating regenerative responses to anaemia by highlighting immature erythrocytes, termed reticulocytes, and for identifying oxidative injury to RBCs ( Campbell and Ellis, 2007 ; Fallon et al ., 2013 ). New methylene blue has also been used to highlight trypanosome organisms in humans ( Ferreira et al ., 2006 ).…”

Photodynamic antimicrobial chemotherapy (PACT) using phenothiazine derivatives as photosensitizers against Leishmania braziliensis

“…Phenothiazines possess great affinity by trypanosomatides 35–37. Trypanothione and trypanothione reductase (TryR)‐based redox metabolism found in Leishmania sp.…”