Chapter 2: Working safely with Nipah virus

Risk assessment in field investigations - general principles
Safety procedures on Nipah-infected or suspected farms
Safety procedures in the laboratory with Nipah-infected or suspected samples

Nipah virus is classified internationally at the highest biosecurity level - BSL4 - and as such warrants the highest level of care in the field and laboratory. What precautions are necessary during investigations on farms where Nipah virus infection may be suspected? How should diagnostic specimens be handled in the laboratory where Nipah virus infections are suspected but not confirmed? How should sera from suspected outbreaks be handled? In addition to the discussion herein, it is recommended that a recent review of the principles of working safely during investigations of dangerous zoonotic agents (Abraham et al. 2001) be read.

Risk assessment in field investigations - general principles

A necessary prelude to any investigation of possible zoonotic disease is an assessment of possible risk to those involved in the investigation. The following approaches (from the CSIRO Australian Animal Health Laboratory’s Standard Operating Procedures for the Field Investigation of Animal Disease) are suggested:

• Review the situation prior to commencement of any examination of live or dead animals. Consider differential diagnoses based on the species involved, clinical syndromes, previous diagnostic tests and epidemiological features of the disease including whether people are already known to be affected.
• Inquire whether the area has a history of particular zoonoses.
• Note the presence of any assistants, farm workers or other people at the investigation site, and their likely proximity to potential sources of infection.
• Note the location of the investigation site in relation to any environmental features which may increase the spread of the infection as a result of the investigation (such as proximity to watercourses, dams, public thoroughfares and other farming establishments).
• Review what appropriate precautions may have already been taken by yourself and others. For example, restricting public access, or vaccination of personnel where a vaccine exists.
• Special precautions should be taken for personnel who are pregnant, immunocompromised or inexperienced.
• Communicate clearly any concerns or advised precautions to assistants and other people at the investigation site. Manage the investigation site in accordance with a duty of care.
• Avoid contact with secretions, excretions and body fluids of potentially infected animals while conducting clinical examinations or collecting specimens. Wear suitable protective clothing, including examination gloves.
• Keep the use of sharps to a minimum and be sure to dispose of scalpel blades and needles in an appropriately designed “sharps” container.
• Apply insect repellent (such as DEET) in areas/situations where insect vectors are seen as a potential hazard. Apply to any exposed parts of the body and protective clothing.
• During examination and sampling of live animals, ensure adequate restraint to reduce the risk of accidental infection of personnel.
• Wash hands and equipment after examinations or specimen collection. Disinfect protective clothing, refuse and biological waste, or otherwise dispose of safely.

Safety procedures on Nipah-infected or suspected farms

It is emphasized that precautions needed for working safely on farms extend beyond issues of personal protective equipment. Thought must also be given to appropriate work procedures to ensure that the activities of the investigation do not spread the infection, or increase the risk of exposure, to other locations. The following approaches are suggested (Daniels et al. 2000):

• On arrival at the farm, designate a “clean” area (which commonly includes the farmhouse, offices and the departmental vehicles), and operate from that area using procedures to ensure that any potential infection is not introduced from the animal pens back to that area. Place buckets of disinfectant at the boundary of the clean area and the potentially infected farm area. Use viricidal disinfectants such as sodium-hyperchlorite, Betadine, Dettol, Lysol, Virkon or Savlon. Ensure the boundary is easily identified by all staff on site.
• Within the clean area, put on appropriate protective clothing: long sleeve overalls, rubber boots, gloves (preferably two sets, taped to the overall sleeve cuffs), eye protection (goggles, safety glasses or safety mask), and nose and mouth protection (a face mask that will filter virus particles). People conducting necropsies on affected animals should preferably wear positive air pressure respirators (e.g. 3M Racal ^TM) and double glove with puncture resistant gloves (Figure 2).

Figure 2: Protective equipment worn by those performing necropsies (note long sleeve overalls, double puncture-resistant gloves taped to overalls, and positive air pressure respirators)

• Before moving into the infected area, organize all equipment to minimize the number of times staff will have to return to the clean area from the infected area. If it is necessary to return to the vehicles during the course of operations, ensure disinfection of boots, gloves, etc. at the boundary before moving from the infected to the clean areas.
• Enter the infected area (animal pens) and conduct a visual examination of the disease situation. Note the health status of all animals, the distribution of any sick or dead animals, the location of any classes of animals to be sampled, and suitable locations to either establish a sampling coordination area or to conduct post mortem examinations. If pigs are to be sampled for serum, establish a work area where tubes can be labelled and recorded. The use of collection tubes which facilitate clotting avoids the need for centrifugation (and the associated possibility of aerosols being created). If necropsies are to be conducted, select a site where contamination of other animals can be minimized and which can be cleaned and sterilized after the job is done.
• Within the infected area, carry a spray bottle of disinfectant so that hands and equipment can be progressively washed and sterilized throughout the course of operations, to prevent the build up of contamination on people and equipment.
• When operations have been completed collect all rubbish into appropriate containers. Place all needles or disposable scalpel blades into a “sharps” container. Assist the farm owner to dispose of necropsied carcasses, by placing in a body bag ready for burial or burning. Spray the outside of the bag with disinfectant.
• Wash all visible contamination (blood, faeces) from equipment, boots, hands and clothing. Proceed to the boundary of the clean and infected areas and sterilise all clothing, aprons, equipment and samples. Spray all clothing, and wash boots in the buckets of disinfectant. Wash all equipment in the disinfectant before taking it to the vehicles. If waterproof overalls are to be reused, ensure that these have been completely sprayed with disinfectant.
• The outside of sampling containers (blood tubes, tissue jars) should be cleaned and sprayed with disinfectant. The containers should be tied in a plastic bag then placed in a transport container (ideally a plastic or metal container, but at least a plastic bag) and the outside of this extra container also sprayed with disinfectant.
• When everything has been disinfected return to the vehicles, store samples and equipment and remove protective clothing. If cloth overalls have been used, wet these in disinfectant and store in leak-proof plastic bags. If disinfected waterproof overalls are to be reused, store these in clean plastic bags.
• Spray face masks and safety glasses again, and store for reuse.
• Discard items such as gloves and any other rubbish into biohazard or other strong plastic bags and tie the bag. At the laboratory, burn or autoclave the bag.
• Change into clean clothes before leaving the premises. (It is good practice to leave removal of the inner pair of gloves to the last step in the undressing procedure.) Wash all clothing at least daily and do not use the same clothing between farms.
• Wash vehicles, including tires and wheels, with disinfectant before leaving the farm.

Care of equipment

• The Racal positive air pressure respirators (PAPRs) supplied by the 3M company comprise a battery operated motor and air filter in a plastic case worn on the back, a head mask with perspex face shield and a flexible air hose linking the two. All exterior surfaces should be sprayed with disinfectant after operations. All components will be reused and should be kept clean and decontaminated.
• The batteries are re-charged between use, ideally with complete discharging first (3M supply a unit for this purpose). When not in use, batteries should be discharged and recharged at regular intervals to prolong battery life and to ensure equipment is always ready for immediate use.
• Filter cartridges in units need not be changed too frequently, say at intervals of a month if in heavy use. Protect the filter cartridge by placing a dust filter on top of the cartridge inside the lid of the unit.

Safety procedures in the laboratory with Nipah-infected or suspected samples

Where Nipah virus infection is suspected as a possible differential diagnosis, appropriate protective clothing and safe work procedures should be adopted (Daniels et al. 2000).

Receipt of Blood Samples

• Blood samples should arrive ideally in an inner plastic or metal container, or at least bagged, and in a leak-proof outer container (see Appendix 7), and the tubes already disinfected at the time of collection. Even so, staff opening containers or receiving blood tubes should be appropriately dressed with a long sleeve laboratory gown that does not open at the front, shoes that offer protection to the feet, gloves that pull over the sleeves of the gown, eye protection (goggles or safety glasses), and nose and mouth protection (face mask that will filter virus particles).
• Conduct all operations in a Class II Biohazard cabinet where possible, and:
  • open the outer container wearing full protective clothing described above;
  • spray the inner container containing the tubes with disinfectant;
  • place the inner container of tubes in the biohazard cabinet and open it carefully, checking for broken or leaking tubes;
  • spray tubes thoroughly with disinfectant, wipe dry, and place in rack for transport to the centrifuge; and
  • record tube numbers and prepare labelled serum tubes for receipt of separated serum.

• Should centrifugation of the blood collection tubes be necessary to clear the serum, use a closed laboratory centrifuge. (Centrifugation can create aerosols - using collection tubes which facilitate clotting and thereby avoiding the need for centrifuging should be considered.) After spinning, allow the centrifuge to sit 5 minutes before opening. When opening the centrifuge, be sure to wear full protective clothing including mask and eye wear.
• In the biohazard cabinet and wearing full protective clothing, open each tube and use a disposable pipette to transfer serum to a labelled tube. Dispose of pipettes and blood tubes in a biohazard plastic bag contained within the cabinet.
• Whenever withdrawing tubes, waste disposal bags or the hands from the cabinet, disinfect with a disinfectant spray first.
• Allocate, label and record a testing (accession) number for the sera.
• Store sera at 4°C to await processing.

Serum processing

• Where sera from herds or animals suspected of Nipah virus infection are processed, sera are aliquoted into inactivation buffer in masterplates as outlined in the ELISA protocol supplied with the reagents. This should be done in a separate room from the blood separation procedure, and this room is not used for any other purpose. The only people to work in this room should be trained operators, wearing full protective gear and working in a certified biohazard cabinet. After the heat inactivation step, the samples are considered non-infectious. (Sera may be treated by heat inactivation at 56°C for 30 minutes following a 1:5 dilution in PBS buffer containing 0.5 percent Tween20 and 0.5 percent Triton-X100 prior to testing. Alternatively, irradiation is an option (Daniels et al. 2001b).)
• The remaining serum is stored in a -20°C freezer that is not used for any other purpose and that is kept secure. These sera are considered still infectious until tested negative, and this is clearly indicated on the freezer.

Use standard operating procedures (SOPs) that utilize a step-wise diagnostic approach, with more dangerous procedures being undertaken only if the results of less dangerous screening tests indicate a need. Built into the SOP should be sampling strategies (for suspect outbreak and surveillance) to ensure that an adequate number of sera are collected, an adequate number of animals necropsied, and an appropriate range of tissues collected.

Laboratories should consider carefully what can be done safely with their facilities, and develop standard operating procedures that are written down, approved by senior management, and in which staff are regularly trained and retrained. Relevant recommendations for SOPs have been developed by several authors (Daniels et al. 2000; Nor & Ong 2000). A comprehensive discussion of diagnostic tests is presented in Chapter 4.

It is timely to suggest that veterinarians adopt a basic universal precaution approach to handling all animals and samples submitted to laboratories to minimize the risk of zoonotic disease. A basic requirement of such an approach is the prevention of exposure of the skin and mucous membranes to the body fluids of sick or potentially infected animals. Hence internal examinations and necropsies should not be conducted without gloves and other protective measures such as appropriate clothing and footwear. Depending on the circumstances, personal judgement should be exercised regarding the need for protection of the mucous membranes of the face and the need for respiratory protection.