Laboratory

Laboratory notification

VIDRL maintains the National High Security Quarantine Laboratory (NHSQL), a Physical Containment 4 (PC4) suit laboratory and Victoria’s designated centre for VHF testing. VIDRL is available 24/7 for testing of specific VHF pathogens.

Testing for VHF must be discussed with and approved by the department. Testing should be coordinated in consultation with the department, treating clinician, infectious disease specialist/microbiologist, pathology/laboratory team of the health service and VIDRL.

On approval, a nominated departmental representative should be the primary person to liaise with VIDRL, the health service pathology and courier service, unless an alternative arrangement has been made and agreed on.

Testing and specimen transfer

Testing for VHF is conducted at the NHSQL in the PC4 laboratory. Definitive testing includes:

• reverse transcription polymerase chain reaction (RT-PCR) – for example, for Ebola virus, Marburg virus, Lassa virus, CCHF virus, and emerging agents like Lujo virus, Sabiá virus and Rift Valley fever virus
• confirmation by sequencing
• virus isolation in cell culture
• whole genome sequencing for outbreak surveillance.

Adjunct testing in the form of serology and transmission electron microscopy may also be considered.

Specimen collection

General principles of specimen collection include that:

• specimen collection should preferably occur at a designated hospital (RMH or RCH) whenever possible
• if urgent transfer is not required and delaying collection may compromise diagnosis, venepuncture should be performed at the initial admitting hospital by an experienced clinician
• only essential specimens should be collected, and safety must take priority, in accordance with the National High Security Quarantine Laboratory Guideline for Management of Quarantinable Viral haemorrhagic Fevers.

See Appendix 4: Specimen collection and transport for suspected VHF for collection and packaging processes.

Other clinical sampling, including pathology

Testing for other infections common in returned travellers (for example, malaria, typhoid and dengue), or blood cultures (if clinically indicated) could be considered by clinicians in cases with appropriate epidemiological risk factors.

In general, differential diagnostic assays and routine pathology should be avoided while the possibility of VHF is being evaluated, and only performed at a Physical Containment 2 (PC2) laboratory when or if VHF has been excluded.

When VHF is suspected, DO NOT send any specimens to the laboratory unless VHF is excluded. DO NOT run blood gas testing on emergency department/intensive care unit analysers used for other patients. If necessary, consider using a handheld blood analyser (iSTAT machine) for point-of-care testing. However, these must be decontaminated after use and must not be used on other patients.

If other clinical specimens have been sent to the onsite/affiliated laboratory inadvertently (prior to suspicion of VHF being raised), alert the onsite/affiliated laboratory immediately.

Molecular assays

Nucleic acid testing, performed primarily on serum or plasma, has become the preferred diagnostic method for VHF pathogens. VIDRL uses laboratory-developed conventional and real-time RT-PCR assays. In silico analysis of primers and probes is conducted regularly to ensure fitness for detection of currently circulating strains. The commercial FilmArray BioThreat Panel (BioFire) is also used on suspected cases of VHF as an adjunct test.

Viraemia can be reliably detected from the time of presentation for at least 14 days; occasionally for as long as 21 days. To prevent false negatives, results obtained in the first 48 hours of illness should be confirmed by a second specimen obtained after this time when a high degree of clinical suspicion exists.

Genomic sequencing

Sanger sequencing of amplicons generated by conventional RT-PCR can be used to confirm the specific detection of a VHF pathogen. Whole genome sequencing using next-generation sequencing technologies can also be used to confirm the molecular detection and support contact tracing efforts, through detailed phylogenetics and genomic epidemiological analysis.

Virus isolation

Virus isolation from serum or other clinical material remains a reference method for confirmatory testing and can also be used for reagent production, test validation or research. Postmortem liver tissue is the most suitable source of virus after serum, while throat washings or urine samples have less commonly yielded virus.

PC4 containment is required for virus amplification in cell culture. Viral replication can be monitored by microscopy, immunofluorescence or real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR).

Note: The filoviruses, Ebola virus and Marburg virus, are Tier 1 security sensitive biological agents in Australia. They may only be stored or manipulated in an appropriately accredited facility by authorised staff like the NHSQL VIDRL.

Serological tests

Antigen testing

Detection of viral antigens, most commonly using monoclonal antibodies in an antigen-capture enzyme immunoassay (EIA), has been widely used in the laboratory and in the field in endemic settings. Antigenaemia has been shown to clear with the appearance of immunoglobulin M, (IgM) on average, one to two days before RT-PCR became negative. Rapid antigen tests are generally not recommended in settings where RT-PCR is available due to poor sensitivity.¹

Antibody testing

Serology can be useful for sero-epidemiological purposes in endemic settings or outbreaks and as an adjunct to direct virus detection, especially in survivors during the convalescent period. Its use in acute diagnosis is limited, however, by the frequent failure for an antibody response to be mounted in fatal cases.

In addition, antibody responses may not be detectable in the early stages of infection, and cross-reactivity with other viruses in the same genus has been seen.² Virus neutralisation assays require live virus and containment facilities, limiting their use in resource-constrained settings.

The VIDRL does not currently use serological assays for diagnosis or confirmation of VHF.

Reporting

On validation of a positive or negative VHF result, the VIDRL microbiologist will notify the clinical microbiologist of the referring diagnostic laboratory, the LPHU and the department by phone.

A report will be issued to the referring laboratory and the department will be automatically notified by the VIDRL Laboratory Information Management System.

Quality assurance

VIDRL’s assays are National Association of Testing Authorities (NATA) accredited to the ISO 15189 standard. Assay primers and probes are regularly assessed for applicability to currently circulating strains by in silico analysis. The VIDRL also participates in The Royal College of Pathologists of Australasia Quality Assurance Program covering proficiency testing for VHF pathogens.

Notes

¹ Bettini, A., Lapa, D., & Garbuglia, A. R. (2023). Diagnostics of Ebola virus. Frontiers in Public Health, 11, Article 1234567.

² World Health Organization. (2025, February). Marburg virus disease – Tanzania and Rwanda outbreaks 2025.