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Chapter 4: Confirmatory laboratory diagnosis


Serological tests
Histopathology
Immunohistochemistry
Virus isolation
Electron microscopy
PCR

Procedures for the laboratory diagnosis of Nipah virus infections include serology, histopathology, immunohistochemistry, electron microscopy, polymerase chain reaction (PCR), and virus isolation. The recommended initial screening tests are ELISA serology and immunohistochemistry, neither of which amplify infectious virus, and so are safer tests in the laboratory.

Serological tests

In determining a sampling strategy it should be remembered that Nipah virus infection is highly contagious in pigs. By the time a farm is suspected to be infected, it is likely that a substantial proportion of pigs will have antibodies. As a guide, if it is expected that more than 20 percent of the pigs may have already seroconverted, 15 serum samples from each age group (adult, grower and weaner) will give a 95 percent probability of detecting seropositive animals (Daniels et al. 2001b).

ELISA

ELISA serology can be conducted safely and quickly without access to PC4 facilities, and can be a most useful diagnostic tool. Where laboratories are establishing an ELISA capability, it is recommended that as well as standardizing against positive and negative controls, the test should be validated against a reference panel of at least 500 sera representative of those to be routinely tested. Testing of a random sample of the 500 sera by serum neutralization test in a PC4 facility will give assurance that the sera are indeed negative for antibodies to Nipah virus. It also allows for an estimate of ELISA test specificity relative to the SNT to be calculated (Daniels et al. 2001b).

The current ELISA configuration developed by CSIRO Australian Animal Health Laboratory has a blocking step to minimize non-specific reactions. The negative control antigen is prepared in Vero cells in an identical manner to the virus-infected cell lysates, and used in a pre-absorption step and as a mock antigen in parallel with viral antigen on the test plates. In this way any high levels of non-specific binding are removed or identified. Recombinant Nipah virus G and M protein antigens, generated using baculovirus expression systems, have been used experimentally but have not yet been adopted routinely (Daniels et al. 2001b).

ELISA serology can also be a useful surveillance tool. It is emphasized that surveillance programmes need to be designed carefully, based on epidemiological principles, and in the knowledge that the ELISA screening test does not have 100 percent specificity. Thus, there will be false positives. The response to such ELISA reactors must be planned with the relevant veterinary and public health authorities in advance. To the pig producers false positives in the ELISA create much anxiety, while to the public health authorities the possibility of false negatives is a concern. The sensitivity of the testing procedure can be addressed through careful epidemiological design of the sampling strategy (Daniels et al. 2001a; Daniels et al. 2001b).

Serum neutralization tests

The serum neutralization test (SNT) is the accepted reference serological test, but because Nipah virus is a BSL4 level agent, biosafety considerations require that this work be carried out in a PC4 facility. In developing diagnostic and surveillance capabilities for Nipah virus, a partnership with an international reference laboratory with PC4 capabilities is strongly recommended (Daniels et al. 2001b).

Specimens for submission for serology:

Serum should be removed from the clotted blood samples within 24 hours to avoid haemolysis. For air transport to a laboratory (for example, an overseas reference laboratory), the serum samples should be packed by a trained person in accordance with International Air Transport Association (IATA) packing instruction 602 (see Appendix 6). The recipient country will require a valid import permit, so prior consultation with the reference laboratory is necessary.

Histopathology

The pathogenesis of Nipah virus infection involves primarily vascular endothelium in all species. In pigs, the respiratory epithelium is also involved. Although formation of syncytia is a feature of Nipah virus histopathology (Hooper et al. 2001), these structures are not identifiable in all cases, so histopathological changes are not pathognomonic. While histopathology is a useful diagnostic tool, it should be noted that specificity may be lacking where diseases causing lung and/or brain pathology (Aujeszky’s disease, Swine fever, Enzootic pneumonia) coexist with (or precede) Nipah virus infections.

In pigs, most of the principal histopathological lesions of Nipah virus infection were observed in the lung tissues. Generalized vasculitis with fibrinoid necrosis, haemorrhages, and infiltration of mononuclear cell sometimes associated with thrombosis were observed notably in the lungs, kidneys, and lymphoid tissues. There was moderate to severe interstitial pneumonia with widespread haemorrhages in the interlobular septa. Lesions seen in the bronchi and bronchioles were those of hyperplasia of the columnar epithelium, peribronchiolar and peribronchial infiltration of lymphocytes, exudation to the lumen of live and dead cells and other debris and single cell necrosis of columnar cells. Numerous neutrophils were seen within the alveoli and within bronchioles and bronchi. Syncytial cell formations were seen in the endothelial cells of the blood vessels of the lung and within the alveolar spaces. In the brain, some degree of meningitis, characterized by oedema and infiltration of lymphocytes, plasma cells and macrophages, as well as vasculitis characterized by swollen vessel walls containing some macrophages were observed.

Immunohistology has shown a high concentration of the viral antigens in the endothelium of the blood vessels, particularly in the lungs. Evidence of viral antigens has also been seen in cellular debris in the lumen of the upper respiratory tract (Hooper et al. 2001).

Immunohistochemistry

Immunohistochemistry (IHC) is highly recommended for initial Nipah virus diagnosis. It is one of the safest of tests as it is performed on formalin-fixed tissues. Since the primary pathology occurs in the vascular endothelium, viral antigen can be detected in a range of tissues (see Appendix 4). Thus it is important that laboratory submissions include a wide range of tissues, and not just lungs. Nipah virus antigen has been detected in porcine meninges (but not brain tissue), lungs, trachea and kidneys. In pregnant animals the uterus, placenta and foetal tissues should be submitted (Daniels et al. 2001a; Daniels et al. 2001b).

Immunohistochemistry, as with other laboratory tests, will not have perfect sensitivity and specificity. Imperfect sensitivity can be compensated by sampling an adequate number of animals at necropsy, perhaps over a period of a few days if disease is progressing on the farm. Importantly, an adequate range of tissues should be sampled from each animal. Laboratories using IHC should practise the test, keeping records of their observations. On some occasions, there will be apparent reactions that are difficult to interpret, and the specificity of the test in any laboratory will be greatly improved if the operators are familiar with the conditions and artefacts that are normally seen in their region. Consultation and sharing of specimens with colleagues in other laboratories and countries is recommended for mutual self-help. This is one of the key points in development of a laboratory quality assurance system for IHC (Daniels et al. 2001b).

Specimens for submission for histopathology and Immuno- histochemistry:

A wide range of (10 percent) formalin-fixed tissues packed (for air transport) in minimal formalin and in accordance with IATA packing instruction 650 (see Appendix 7). Multiple lung and airway samples are recommended.

Virus isolation

Ideally, to confirm any new Nipah virus outbreak, virus should be isolated. Because Nipah virus is a BSL4 level agent, biosafety considerations require that this work be carried out only in a PC4 facility. Nipah virus grows well in Vero cells, with development of characteristic syncytia with the nuclei arranged around the periphery of the multi-nucleated cell (Figure 4). This arrangement differs from most syncytia seen in cell cultures with the closely related Hendra virus (Hyatt et al. 2001). Brain, lungs, kidneys and spleen should be cultured (see Appendix 4). CPE usually develops within 3 days, but two 5-day passages are recommended before discontinuing the attempt (Daniels et al. 2001a). Identification of virus isolates may be attempted by immunostaining of fixed, infected cells, neutralization with specific antisera, PCR of culture supernatants, and electron microscopy. Suspected new isolates should be sent to an international reference laboratory for molecular characterization (Daniels et al. 2001b). Teamwork among the international scientific community is strongly recommended in the handling of emerging diseases such as Nipah virus.

Figure 4: Nipah virus syncytium with nuclei characteristically forming a ring around the periphery of the multinucleated cell (in this case with the cytoplasm stained to demonstrate Nipah virus antigens)

Specimens for submission for virus isolation:

A wide range of fresh tissues (lungs, spleen, kidneys, tonsil, central nervous system) packed (for air transport) by a trained person in accordance with IATA packing instruction 602 (see Appendix 6).

Electron microscopy

Negative contrast EM and immuno-electron microscopy are useful to rapidly obtain information on the structure and antigenic activity of viruses in cell culture. Details of both techniques, and their application to the detection and analysis of Nipah virus (and Hendra virus) infections are described by Hyatt et al. (Hyatt et al. 2001).

PCR

Diagnostic assays for Nipah (and Hendra) virus are in routine use by the CSIRO Australian Animal Health Laboratory (based on the M and N genes) and the US Centers for Disease Control (based on the N gene). While a valuable tool, the methodology warrants strict attention to internal quality assurance to avoid spurious results (Daniels et al. 2001a).


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