MINIMUM REQUIREMENTS TO ESTABLISH A SMALL LEPTOSPIROSIS LABORATORY
Good laboratory service is essential. As methods in leptospirosis diagnosis may be difficult, different, three or four levels (depending on the local health system) of laboratory service can be envisaged:
The most important equipment is a good microscope with dark field illumination Do not forget the illumination itself. Always order for extra lamps. A good microscope costs a lot of money. Research is often hampered by the lack of even such basic equipment.
Glassware like tubes, bottles, flasks, pipettes and microscopic slides. The glassware used in leptospirosis research should be cleaned to virology standards. Most laboratories can only afford to use glass tubes which are fairly old, usually with scratches with are very difficult to clean. Glassware such as Pasteur pipettes are often reused and when washed manually the pipettes are not cleaned to the required standards.
Method of sterilization.
For the sake of expenses, can you share equipment with other laboratories?
Storage of serum samples, chemicals and antigens:
Water used for preparing culture media has to be pre treated by distilling it several times before use. In most cases a distillator is shared by other laboratories or the hospital.
Special laboratory facilities and skills are required, and are best centralized in a
bacteriological laboratory where trained staff members are available.
Leptospires require a lot of care. It is not sufficient merely to leave cultures and read
them ar regular intervals. Tubes need to be subcultured and filtered into fresh medium.
One needs to be well trained or to employ a well trained technician who should be solely
working on leptospirosis.
Leptospires require a medium with bovine serum albumin fraction V which is very
expensive. Alternative media are: Fletcher's and/or Korthof's. These alternative media
require a certain amount of rabbit serum.
When a laboratory is carrying out diagnosis, there is a need to maintain leptospiral
strains. This is not only expensive but requires expertise.
Research methodology includes clear definition of objectives, setting of priorities,
selection of appropriate technology, epidemiology. The techniques that will be used must
however be thoroughly taught, theory as well as practice, and they must be adapted to meet
local needs and conditions. Even simple techniques must be well taught. Make protocols
about every detail.
The need to determine the effects of the for example long storage under sub optimal
conditions in customs sheds, should be emphasized. When sufficient information is
available, individuals will still have to make choices about the technology to be used in
their research or other work on infections diseases. The factors involved in this choice
are not only about the best technique that can provide the desired information, but also
about: costs, speed, sensitivity, specificity, simplicity, and thus: required space,
equipment, reagents, personnel, training and experimental animals.
When leptospires were first recognized, the live organisms have been used in some form of microscopic agglutination test (MAT). The basic difficulty of the MAT is keeping the antigens alive despite failures in the electricity supply, availability of experienced technical staff, or financial constraints e.g. for preparation of EMJH medium which is rather expensive. There is also the failure to get the better of the customs officer who insists on keeping your parcel for a week or so in a warehouse like a bake-oven. The frustrations and set-backs inherent in running a laboratory in a developing country are legion. However, it is not only laboratories in countries with deficient infrastructures that have these difficulties. The ability to keep antigens alive is the basic minimum requirement for technology transfer, and large, smart buildings and large budgets are less important than the will to carry on despite all setbacks. Laboratories do not really need an ELISA reader, they can wash their plates by hand. Their work can be done without laminar flow. They may use cylinders with propane in stead of piped gas supply, and non-electric bench-top autoclaves in stead of fancy sterilizers. There is a great deal to be gained from the transfer of technology, both for donor and recipient. In many cases metrolabs depend on outside sources for the biological samples that they need to test their new procedures on large numbers of sera.
Standard microbiological laboratory safety procedures are required when working with leptospires. Leptospires are susceptible to drying, to acid, to phenolic or detergent dezinfectants and antiseptics, and to heat. Spillages and splashes on laboratory floors and benches and animal house floors should be disinfected. The main danger to laboratory staff is in laboratory accidents, especially those caused by pricks and cuts and splashes into the eye from ill-fitting syringe needles used in animal inoculations. Mouth pipetting of cultures of leptospires is absolutely prohibited. All glassware must be safe (i.e. no sharp fringes) before washing up. Slides and pipettes should be discarded into disinfectant or detergent. In the event of an accident where staff are infected or believed to have run a risk of infection prophylactic treatment is advisable. Where fresh isolates and virulent strains are handled, all staff should be required to report febrile illness, and a serum sample has to be taken to establish a baseline MAT titer against the serovars they work with. Laboratory staff handling specimens of human blood or serum for culture or serology are also exposed to a risk of other infections (viral), which may be very serious. Precautions should be taken in order to prevent contact of hands, skin or clothing with serum or blood from spills or leaking containers. Heating serum samples (30 min., 56 C) will kill many - but not all - infectious agents.