Leishmania donovani in Eastern Sudan an innovative experimental approach for parasite cultivation and in vitro drug sensitivity testing

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Elamin, Hayam
Hassan Sharief, Abdalla
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University of Khartoum
From the early 1900s, visceral leishmaniasis (VL) has been among the most important health problems in Sudan, particularly in the main endemic areas in the eastern and central regions. Several major epidemics have occurred, the most severe--in Western Upper Nile province in southern Sudan, detected in 1988-- claiming over 100,000 lives. The disease spread to other areas that were previously not known to be endemic for VL. Major upsurges in the number of cases were noted in the endemic area. These events triggered renewed interest in the disease. In this study we described a relatively simple medium formula using common, available and inexpensive ingredients that can be used in place of medium that requires fetal calf serum enhancement for promastigote forms. This medium supported the growth of parasites at rate comparable with those obtained with serum supplemented RPMI-1640. Parasites grown in this medium shown moderate to high infectivity rates when being inoculated to pass through macrophage cell line (about 27- 84% of macrophage where infected) and no difference was observed in their infectivity and replication potentials inside cells (for both responsive and unresponsive parasites) between these parasites and that ones grown in parallel in RPMI-1640. Six L donvani. Isolated and one L. major was in vitro tested to Pentostam and Amphotericin B in their amastigote form (using J774 murine macrophage like cell line and as axinecally grown amastigote). The number of parasite in the infected decreased steadily at drug concentration 7.8μg/ ml to 250μg/ ml (in Pentostam) and 0.22μg/ml to 0.75μg/ml (in Amphotericin B) and the capacity of parasites to replicate was also affected. L.major showed the same response as other tested strains. In conclusion: The use of new CML medium can easily take the place of NNN or any other medium, because of it easy to prepare and used instantly, economic, available when needed, also the shelf life is about 30- 45 days. The fact that this medium is similar to other culture media as far as durability and quantity of produced parasites are concerned might give it an advantage over the others currently used. We also showed that the in vitro drug sensitivities did not match clinical unresponsiveness in the six L.donvani isolates, and the in vivo unresponsiveness does not necessarily mean primary parasite resistance. Active and dividing populations of axenically cultured amastigotes were generally more susceptible to Pentostam and Amphotericin B than their corresponding promastigotes. All the tested isolates were found to be highly susceptible to amphotericin B, that mean Amphotericin B is suitable second line drug if patients can tolerate it is toxicity
A Thesis Submitted For the Fulfilment of the Requirements of Master Degree in Immunology