Use of glycerol as an alternative to freeze‐drying for long‐term preservation of antigen for the direct agglutination test

Abstract

The potential of glycerol for long-term preservation of the direct agglutination test (DAT) antigen was evaluated at a fluctuating laboratory temperature of 25-37 degrees C and at constant temperatures of 37 and 45 degrees C for a period of 222 days. DAT titres recorded for the three antigen aliquots preserved in 50% (v/v) glycerol and stored at 25-37, 37 or 45 degrees C at 11 time intervals were within the same range of the control antigen kept at 4 degrees C. Performance of the glycerol-preserved antigen stored at 45 degrees C was compared with that of a freeze-dried version on 24 visceral leishmaniasis (VL) and 54 non-VL patients. For all non-VL patients, a maximum DAT titre of 1/800 was recorded for either of the two antigens. For all VL patients, in comparison with the freeze-dried, the glycerol-preserved antigen always had equal or higher titre; in 16 of the 24 VL sera tested, the latter antigen scored three- to sixfold higher titres. As this glycerol preservation method is economical and easy to perform, it has better potential for wider-scale application than freeze-drying.

Local Production of a Liquid Direct Agglutination Test as a Sustainable Measure for Control of Visceral Leishmaniasis in Sudan

“…The introduction of an additional step of preservation in 50% glycerol has further improved stability of the LQ-DAT under the harsh field conditions encountered in eastern Sudan. 4,5 Because of a lack of facilities for cryopreservation, and thus, an inability to continuously use the standard 1-S strain, our efforts were directed toward recovering the parasite as part of the diagnostic procedures for patients presented at our laboratory or any of the collaborating laboratories. We observed relatively higher sensitivity for VL detection with the LQ-DAT as compared with the FD-DAT, probably because of the use of the endemic autochthonous L. donovani (eastern Sudan), and not the standard 1-S strain (Upper Nile, southern Sudan), for antigen processing.…”

“…Glycerol preservation was introduced to further ensure safe long-term antigen storage under the harsh rural conditions prevailing in eastern Sudan. 4,5 On the basis of the achievements thus far, we think that the LQ-DAT can significantly contribute to the control of VL in Sudan provided the finance is available to import the badly needed raw materials. Locally produced, ready-for-use LQ-DATs could then be dispatched to peripheral hospitals on a regular basis.…”

rKLO8, a Novel Leishmania donovani – Derived Recombinant Immunodominant Protein for Sensitive Detection of Visceral Leishmaniasis in Sudan

“…The stability of DAT was improved by using freeze-dried and glycerol preserved antigens which does not require storage at 4°C thus making the test suitable for field application [28], [29]. However, the test procedure and the need for overnight incubation give limitations for the field use.…”

Diagnosis of visceral leishmaniasis: developments over the last decade

“…DAT is an easy to perform, widely applicable technique with high sensitivity (90-100%) and specificity (95-100%) and is routinely used in some regions for the past two decades (Sreenivas et al 2002;el Harith et al 2003;el Mutasim et al 2006;Jacquet et al 2006). The test can be carried out using plasma, serum, or even urine samples, making it suitable for both field and laboratory application (Chappuis et al 2006;Sundar et al 2007;Mandal et al 2008;Bhattarai et al 2009;Oliveira et al 2009).…”