Laboratory for leptospirosis
Author: Miladin Kostovic

MINIMUM REQUIREMENTS TO ESTABLISH A SMALL LEPTOSPIROSIS LABORATORY

#### LABORATORY SERVICE

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:

- Level 1. Inoculating cultures and serological screening.
- Level 2. Culturing and confirmation of screening method by standard serological methods.
- Level 3. Reference laboratory to provide reagents (sera, antigens, perhaps culture medium), to maintain a culture collection, to perform typing, to perfom quality control, to train, to publish results, to reprot to and to communicate with health autorities.
- Separate level: Field work to trace infection sources and to collect (human and/or animal) samples from the field.
- Staff required: Clinicians, microbiologists, technicians, veterinarians, epidemiologists, public health specialists.

#### WHAT EQUIPMENT OR OTHER ITEMS ARE ALREADY AVAILABLE?

#### Microscope:

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:

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.

#### Sterilization:

Method of sterilization.

#### Equipment:

- fluorescence microscope
- gel electrophoresis apparatus
- centrifuge
- automatic dispensers and micropipettes
- accurate balance

For the sake of expenses, can you share equipment with other laboratories?

#### Storage:

Storage of serum samples, chemicals and antigens:

- refrigerator
- freezer

#### Culture medium preparation:

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.

#### LABORATORY FACILITIES AND SKILLS

#### Staff and skills required:

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.

#### Theoretical skills:

- Understanding leptospirosis as a zoonosis
- Understanding diagnostic procedures
- Interpretation of laboratory tests
- Understanding occupational and public health aspects.

#### Technical skills in the laboratory:

- For separating serum
- Bacteriological examination of cultures, inoculating culture media with blood specimens from the patient, perhaps even at the bedside, testing if necessary in a screening test.
- To examine blood culture media
- Examination and identification of positive cultures to serogroup level.
- Subculturing and maintenance of isolates.
- To carry out serological tests i.e. screening, MAT, ELISA and/or others.

#### Equipment:

- Containers for serum
- Pasteur pipettes, dilution pipettes
- Centrifuge, dark field microscope, incubator if necessary
- Culture media, filters, sterile syringes, needles
- Refrigerator (deep freezer), autoclave (pressure cooker)

#### TECHNICAL SKILLS IN THE FIELD

#### Training:

- In trapping and identifying animals
- In killing and autopsying them
- In identifying and taking tissues
- In the handling of live and dead animals
- In trapping techniques

#### Equipment:

- Cages and other trapping facilities
- Instruments for autopsy and/or for field autopsy

#### CONSUMABLES

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.

#### METHODOLOGY AND TECHNOLOGY

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.

#### TRANSFER OF KNOWLEDGE AND TECHNOLOGY

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.

#### SAFETY N THE LABORATORY

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.

#### REFERENCES

1. Leptospirosis on the African continent. Proceedings of a CES/STD3 Research Meeting, Harare, Zimbabwe, 17-20 February 1992.
2. Faine, S. Guidelines for the control of leptospirosis, WHO Offset Publication No.67, 1982.