Leishmaniasis is caused by Leishmania, a family of parasitic protozoa that includes 40 different taxa. Identifying the particular strain of Leishmania is critical to treatment. The Biological Resources Centre of Leishmania (CRB-Leish) in Montpellier, France, is a 39-year-old biobank that includes 6,353 strains belonging to 36 Leishmania taxa. Pratlong et al. (2016) discuss technical and organizational changes the collection has undergone between 1975 and 2014, describe its current state and outline its future.1
Over the life of the biobank, Pratlong et al. have modified their culture and cryopreservation techniques, identification methods, collection management and quality control procedures. In the CRB-Leish’s initial stages, the laboratory used biphasic Novy-MacNeal-Nicolle (NNN) medium for parasite isolation and culture. The researchers prepared it in-house, adding rabbit blood collected from animals kept on the laboratory premises. Today, NNN medium remains the reference medium for primary strain isolation, and protocol is unchanged. For mass culture, they used brain-heart infusion agar supplemented with rabbit blood until 2009. They then replaced this with an axenic liquid medium, supplemented with 10% fatal calf serum. Pratlong et al. also simplified their cryopreservation procedure.
Between 1980 and 1997, the laboratory typed strains using multilocus enzyme electrophoresis. However, in 1998, they added a molecular typing approach, initially based on sequencing part of the large subunit of the RNA polymerase II gene. Since 2006, they have routinely performed multilocus sequence typing of seven coding regions. The biobank has also used several databases over its lifetime. Beginning with dBase II, including only basic information, they moved to a new database using Microsoft Access in 1995. They now use the more advanced laboratory information management system BRC-LIMS. BRC-LIMS allows them to monitor quality and activity indicators defined in their laboratory and easily manage all stocks, including strains, culture media, reagents and laboratory material. Consequently, they now meet all requirements of the NF S 96-900 standard, including those of the ISO 9001:2008 standard, the worldwide reference. Pratlong et al. note that the BRC-LIMS program and database fully meet the NF S 96-900 quality requirements, and in 2012 they fully implemented the NF S 96-900 standard, as well as a quality manual with defined quality indicators. As of 2004, the biobank has had an online strain catalogue, defined by a CRB-Leish strain code (LEM) and a WHO strain code, with safety procedures in line with current biological sample standards.
Their collection includes strains from the Old World (Africa, Asia and Europe; n = 5,032 strains [82%]) and the New World (the Americas; n = 1,108 [18%]). France is the most sampled country (n = 1,283), followed by Spain (n = 363), Portugal (n = 293) and Greece (n = 245), while Africa has the most strains (n = 1,906), many from the Maghreb area. Of the 36 Leishmania taxa in the collection, the most abundantly represented are Leishmania infantum (n = 2,559), followed by Leishmania major (n = 945), Leishmania tropical (n = 497), Leishmania guyanensis (n = 345), Leishmania braziliensis (n = 257) and Leishmania donovani (n = 191).
The CRB-Leish facility is working toward obtaining NF S 96-900 certification in the coming years. It is also changing its typing methods and developing mass spectrometry–based approaches for faster identification compared to DNA sequencing. Pratlong et al. hope that their long-term experience with parasite storage will encourage field epidemiologists and clinical practitioners in endemic countries to secure their own strain collections, with the help of the French CRB-Leish.
1. Pratlong, F., et al. (2016) “The Montpellier Leishmania collection, from a laboratory collection to a biological resource center: A 39-year-long story,” Biopreservation and Biobanking [Epub ahead of print].