[Leish-l] Simple method to promastigote culture from peripheral blood

[Mikko Aalto]

Dear All
O meus colegas 

Corresponder em ingles e em portugues.  

Can anyone advice a simple method how to culture promastigotes from human peripheral blood? 
I have used promastigote culture as diagnostic method for rK39 RDT negative Visceral Leishmaniasis patients. 
I have inoculated spleen and bone marrow aspirates to 10ml vials with autoclaved house made medium consisting of Tryptone, Yeast Extract, NaCl sealed with rubber stoppers and 5% human blood is added there after, kept at 80-100C for 15 minutes to inactivate inhibitory components. 
Just before inoculation I have added 1-2ml 10% filter sterilized human urine as an overlay, serving also to dilute complement and other inhibitory components in the maybe around 50µl aspirate. With strict sterility antibiotics have not been necessary. 
Incubated in a cooling incubator at 25C, the living motile promastigotes have been visible in wet preparation in 48 hours to 30days. 
But only around 70% of cultures have been positive. 

Now I would like to start doing promastigote cultures also from peripheral blood. 

The 2002 method by Shyam Sundar* and M. Rai (Laboratory Diagnosis of Visceral Leishmaniasis) seems feasible to my very basic conditions.  
"Aseptically collected blood (1 to 2 ml) is diluted with 10 ml of citrated saline, and the cellular deposit obtained after centrifugation is inoculated in culture media." 

But the recently published methods using buffy coats and micotiter plates etc seem too complicated to simple conditions. 

So I would like to hear advices on simple but good techniques to do promastigote cultures from patients' peripheral blood. 
And also if Streptokinase might help to eliminate complement and other inhibitory factors, without inhibiting amastigotes and promastigotes. 

Yours 
Mikko 


Dr Mikko Aalto 
Bosaso General Hospital 
Bosaso Somalia