Laboratory preparedness: Ebola and other emerging infectious diseases

The current Ebola virus disease (EVD) outbreak has been in progress for more than a year in Western Africa, and unfortunately has spread across international borders. It has affected thousands within the countries of Liberia, Sierra Leone, and Guinea, as well as small numbers of patients in several other countries around the world.

Ebola virus belongs to the Filoviridae family of linear, negative-sense, single-stranded RNA viruses. Within this family, the current outbreak species, Zaire ebolavirus (commonly named Ebola virus) is one of the five distinct species within the Ebola virus genus.¹ Most Ebola viruses can cause severe and often fatal hemorrhagic disease in humans. 

Ebola virus was first described in Zaire in 1976, and over the past forty years there have been numerous small outbreaks with fewer than five hundred cases per outbreak. In contrast, the present Zaire ebolavirus epidemic devastating Western Africa has resulted in more than 9,000 confirmed deaths and nearly 23,000 suspected cases.² The case-fatality rate in this epidemic is currently around 70%.

People infected with Ebola virus disease commonly present with non-specific symptoms such as fever, severe headache, muscle pain, weakness, fatigue, and, importantly, vomiting and other gastrointestinal symptoms such as severe diarrhea. Less than half of cases demonstrate hemorrhage.³ The reservoir for the virus is probably in fruit bats and is transmitted either directly to humans or indirectly via interactions with apes.

Human-to-human spread occurs by direct contact with bodily fluids. The most probable sources of infectious virus responsible for person-to-person transmission are blood, urine, vomitus, and diarrheal fluids, but the virus has been detected in most body fluids. While respiratory spread via aerosolization of infective viral particles has been postulated in the current outbreak, both the basic biology of the virus and the epidemiology of it suggest this is extremely unlikely. Secondary cases among individuals who did not have direct contact with virus-laden material are rare and poorly documented, and the virus does not survive in droplet nuclei. Nevertheless, many of the victims of past outbreaks and the current outbreak have been healthcare workers.

Assessing the risks

Hospitals and laboratories planning the management of any emerging infectious diseases should begin by characterizing the risk of infection by the offending agent. Factors that affect the risk from an emerging pathogen include:

• The route of transmission
• The location of the virus in the body during infection
• How much infectious virus is present
• How common the infection is
• How pathogenic the agent is
• What clinical activities are performed on infected or potentially infected patients.

Performing a risk assessment allows the facility both to gauge the degree of risks to employees and to mitigate known and expected risks. 

As the Ebola epidemic evolved in 2013 and the threat to the United States increased, numerous governmental and nongovernmental bodies, including the Centers for Disease and Control and Prevention (CDC) and the American Society of Microbiology (ASM), issued guidance on preventing transmission of the Ebola virus in the clinical laboratory setting.^4,5 The guidelines from each organization outlined actions that healthcare facilities and their laboratories may take in categories including test ordering, sample collection, processing, and the use of personal protective equipment (PPE) to minimize the risk of transmission of Ebola virus to healthcare workers. They highlight the need to follow standard precautions for specimen procession and handling, and they suggest additional steps to follow with regard to contact, droplet, and airborne precautions as they pertain to healthcare workers. 

However, guidelines from different sources contain significantly different recommendations for important tasks including sample collection and labeling, transport, processing, and performance of routine testing. Individual laboratories are faced with the unenviable task of performing risk assessments and sorting through contradictory guidelines to create local policies. 

It is critical to assess risks in the context of clinical settings and patient populations and to recognize that zero risk is unattainable. Also, as the outbreak progresses, the epidemiology and quantitative risks evolve and vital lessons may be learned along the way. Thus it is necessary for laboratories not only to create policies and procedures, but also to review and revise them as the outbreak continues.

The laboratory context

Laboratory issues exist that all institutions performing testing on or taking care of patients with the virus must address.

• What tests should be performed?
• Who collects the specimens?
• How is the specimen transported?
• How are patients at-risk or infected with Ebola virus identified?
• How is the laboratory notified of patients and of specimens from these patients?
• How are lab personnel who handle specimens tracked and assessed for potential exposure?
• What PPE will be used for each exposure?
• How will PPE competency be established and maintained?
• How will the sample be handled once in the laboratory?
• How will spills be managed?

Assessing and planning the workflow of patient samples is exceedingly important.

For example, when a patient infected with Ebola virus arrives at a hospital for treatment, the institution must determine how patient specimens will be handled to most effectively reduce risk to those handling them. They must assess the types of testing that must be done to ensure that appropriate levels of care are maintained as well as the way in which the laboratory does the specified testing. Is the risk of testing a patient sample in the lab low enough to utilize enhanced PPE with standard sample processing, or would it be better to keep patient testing at the bedside? If a spill occurs in the laboratory, will the response affect the availability of testing for other patients?

The American Association of Blood Banks (AABB) has assessed the transmission risk of Ebola virus via blood transfusion. The organization’s risk characterization of Ebola virus transmission through the blood supply and public health risk estimates concludes that there is insufficient data to make recommendations regarding donor deferral periods of patients previously infected with Ebola virus or those individuals who had high risk contact.⁶ However, when an Ebola-infected patient requires transfusion therapy, the AABB recommends exposing laboratory personnel to patient blood as little as possible during pre-transfusion testing, by transfusing type O Kell-negative blood.⁷ 

The POC perspective

The use of point-of-care (POC) testing with regard to the Ebola virus has been discussed and implemented in hospitals in the United States that have taken care of Ebola virus-positive patients. POC testing potentially avoids specimen transport, centrifugation, and aliquoting, reducing the risks of droplet formation or aerosolization. Patient samples are contained within the patient’s isolation room or suite, reducing the number of people exposed. However, a facility faced with more than one potentially infected patient will need to maintain multiple devices. Additionally, the test menus of CLIA-waived POC devices are limited and may correlate poorly with laboratory-based test equipment, while more complex equipment may require dedicated space near the patient that few facilities possess. Particularly in patients for whom Ebola virus disease is only one item on a complicated differential diagnosis, the exclusive use of POC testing may compromise quality of care. Whatever procedures are used to test samples from patients at-risk for Ebola, they should not obstruct the capability to deliver appropriate medical care to patients.⁸ 

Policies and procedures

While current policy in the United States is to manage patients with diagnosed Ebola virus infection in specialized facilities, most acute-care facilities will need to plan to assess, rule out, and refer patients with appropriate travel and clinical histories. The two cases of healthcare-transmitted Ebola disease in Dallas last year have made institutions extremely cautious about the handling of patients and samples associated with EVD.

In addition to the promulgation of policies and procedures for handling samples associated with potential EVD, practice and drills of those procedures, particularly the donning and doffing of PPE, are essential. Ultimately, all staff caring for patients and processing specimens need to be well-versed in the plan, with drills being performed so that staff is aware of how to communicate with management. 

Further, specimen collection, labeling, and transport must be a priority in overall planning. While stringent precautions for handling samples from patients with potential or proven EVD are essential, and hospital policies and procedures should include early identification of patients at risk and notification of laboratories, laboratories should recognize that patients with EVD may present with confusing or inadequate histories, and specimens may be sent to the laboratory before the possibility of EVD infection is raised. Reinforcing careful adherence to current standard precautions against blood-borne pathogens is an essential precaution against EVD, other emerging pathogens, and the known hazardous pathogens found in routine laboratory practice. 

References

1. Kuhn J, Becker S, Ebihara H, et al. Proposal for a revised taxonomy of the family Filoviridae: Classification, names of taxa and viruses, and virus abbrevia. Archives of Virology .2010;155(12): 2083–2103. 
2. CDC: Ebola: 2014 Ebola Outbreak in West Africa – Case Counts. http://www.cdc.gov/vhf/ebola/outbreaks/2014-west-africa/case-counts.html. Accessed February 11, 2015.
3. Feldmann H, Geisbert TW. Ebola haemorrhagic fever. Lancet. 2011;377(9768):849-862. doi:10.1016/S0140-6736(10)60667-8.
4. Interim Guidance for Specimen Collection, Transport, testing, and Submission for Persons Under Investigation (PUIs) for Ebola Virus Disease (EVD) in the United States. October 2014 Center for Disease Control and Prevention.http://www.cdc.gov/vhf/ebola/hcp/interim-guidance-specimen-collection-submission-patients-suspected-infection-ebola.html. Accessed February 2, 2015.
5. Interim Laboratory Guidelines for Handling/Testing Specimens from Cases or Suspected Cases of Hemorrhagic Fever Virus (HFV). September 2014. American Society for Microbiology. https://www.asm.org/images/PSAB/Ebola9-10-14.pdf Accessed February 2, 2015.
6. Supplemental. Ebola virus. Transfusion. 2009: volume 49, Appendix 2.
7. Koepsell S. AABB, Annual meeting education program. Ebola and Transfusion Medicine. Ebola Hot Topic. October 2014.
8. New York State Department of Health. Revised NYS/NYC Laboratory Guidelines for Handling Specimens from Patients with Suspected or Confirmed Ebola Virus Disease.

Emerging viruses of North America: are labs ready?

By Linda L. Williford Pifer, PhD, SM(ASCP), GS(ABB) and Wyenona Hicks, MS, MT(ASCP)SBB

During the past 30 years, newly emergent viruses have swept out of Africa and the East with stunning regularity. HIV-1 has invaded every continent since its official recognition in the early 1980s. In 2003, severe acute respiratory syndrome (SARS) took lives from Guangdong province in China to Taipei and beyond. This zoonotic coronavirus ultimately caught flights aboard human hosts to Toronto, where 5,000 Canadians were quarantined, and to Bangalore, India, before subsiding.^1,2 Recent memory and fear of Ebola virus are painfully fresh, as deaths continue in 2015, although the epidemic appears to be fading, with a mortality rate exceeding 9,000.³ Influenza, though far less exotic, has presented cases worldwide. During the World War I era it is estimated to have killed more than 50 million people worldwide, which is far more than those claimed by bombs, bullets, and poison gas.⁴

What about North America? Is our environment pristine with regard to de novo emergent viruses? Although the viruses described above were imports, the United States has its own “home grown” viruses. The Centers for Disease Control and Prevention (CDC) announced several months ago the development of a blood test to confirm the new tick-borne Bourbon virus (an orthomyxovirus), named after Bourbon County, Kansas. A man died with symptoms including fever, fatigue, muscle aches, and severe appetite suppression. His condition worsened and ultimately advanced to renal and respiratory shut-down and shock. Other tick-borne illnesses were ruled out. The genetic material of Bourbon virus is quite similar to that of viruses seen in Africa, Asia, and Eastern Europe, but no virus like this has ever been identified in the U.S., according to Dana Hawkinson, an infectious disease specialist at the University of Kansas Hospital. He indicated that a commercial laboratory test would likely be forthcoming soon for routine screening for the virus.⁵

This comes only two years after the discovery of the Heartland virus. This novel agent was reported in two Missouri men who fell acutely ill after having been bitten by Lone Star ticks (A. americanum).⁶ Symptoms include fever, headache, muscle aches, anorexia, nausea, fatigue, leukopenia, thrombocytopenia, disseminated intravascular coagulation, and cerebral hemorrhage. Intensive study revealed that the agent involved was a phlebovirus of the Bunyaviridae family similar to one recently identified in China called severe fever with thrombocytopenia syndrome virus, or SFTSV. Heartland virus was confirmed by ELISA antibody testing and by next generation full genome viral RNA sequencing by the CDC.⁷ 

In 2014, U.S. children experienced an unusually high prevalence of cases of enterovirus D-68 (EV-D68) respiratory infections. Although enteroviruses are not prominent causes of this pathology, mild cases were characterized by fever, coryza, and muscle aches; patients with severe cases reported wheezing and difficulty breathing. Last October, the CDC announced that it would accept specimens from physicians for its more rapid test for the virus.⁸ Parents became alarmed by 90 cases of what appeared to be EV-D68-associated limb weakness, or what pediatricians termed “acute flaccid myelitis,” with MRIs showing spinal cord damage. Dr. James Sejvar, a CDC neuro-epidemiologist, stated, “We are one or two steps shy of definitively stating that [the virus] is causing these cases.” The investigation is continuing.⁹

In the Makonde dialect of the West African Swahili language, “chikungunya” means, “he who walks all bent up,” referring to the severe joint pain suffered by those who suffer from the virus of the same name. In 2014, chikungunya virus (CHIKV) was not a notifiable viral disease in the U.S., although it can be reported to ArboNET, the national surveillance system for arthropod-borne diseases. This is an Aedes mosquito-borne viral infection causing joint swelling, aches, pain, fever, and often rash that is more severe in infants and the elderly. According to the CDC, as of January 13, 2015, 2,344 chikungunya cases were reported in the U.S. in 2014, with 11 having been locally transmitted in Florida. All others were imported from the Caribbean, Asia, or Pacific islands. Viral RNA can be detected by RT-PCR in acute serum specimens during the first seven days with results available within one or two days. The concern is that the virus will become established endemically in native U.S. mosquito species.¹⁰

Of the viruses mentioned in the first paragraph, only HIV-1 and influenza have thus far proven to require a full-court press by the laboratory from a diagnostic and testing standpoint. However, arthropod-borne viruses present a serious legitimate growing threat not only to North America, but to the world. Many species of ticks in the U.S carry at least 14 different infectious diseases, including those of viral, parasitic, rickettsial and bacterial etiologies.¹¹ These should legitimately create unease among lab managers (and all of us), especially if predicted increases in global temperatures become reality. Such warming trends will favor tick population increases and the northerly spread of chikungunya-virus infested mosquitoes which already breed within the continental U.S. 

Lab managers’ concerns about new viruses arise somewhat in this order: 

1. Is the new virus really a threat? 
2. Will it be a problem in my geographic area and in my lab’s patient population? 
3. Will a test become available, and when? 
4. Where will it be available, and will it be cost-effective in-house?
5. Will the new test require new technology and accompanying special technical or safety training (think Ebola virus)? 
6. Will the virus become a “regular” in the population like HIV, undergo antigenic shift over time like influenza, infect populations sporadically like hantavirus or SARS, or never achieve a toehold at all in the U.S., like Ebolavirus? 
7. How will lab staffing, work flow, and economics be affected?

The lab manager is in the position of needing to weigh all odds and make an educated conservative determination as to how to respond, with the emphasis on “educated.” These are situations in which even professional virologists, clinicians, and epidemiologists cannot be certain which viruses will approach epidemic or even significant status. We are at a point where models can be projected with a small degree of confidence, but there are still too many variables. Models have not been in play long enough to have established trust, especially where both health and economics are concerned.

Vigilant medical laboratory managers can stay on top of breaking news about emerging viruses and other infectious diseases by free online subscription to the Morbidity Mortality Weekly Report published by the CDC at www.mmwr.cdc.gov and daily bulletins published by the International Society for Infectious Diseases’ Program for Monitoring Emerging Infectious Diseases at www.promedmail.org.

In closing, Nobel Laureate Joshua Lederberg once stated that, “The biggest single threat to man’s continued dominance on this planet is the virus.”¹² In these days of shifting viral winds and tides, it would be wise to take his warning to heart.

CDC updates Guidance for managing and testing possible Ebola clinical specimens

The U.S. Centers for Disease Control and Prevention (CDC) has released an updated “Guidance for U.S. Laboratories for Managing and Testing Routine Clinical Specimens When There is a Concern About Ebola Virus Disease.”

CDC recommends that Ebola testing be conducted only for persons who meet the criteria for persons under investigation (PUIs) for EVD. A PUI is a person who has consistent signs and/or symptoms, including:

• Elevated body temperature or subjective fever or symptoms, including severe headache, fatigue, muscle pain, vomiting, diarrhea, abdominal pain, or unexplained hemorrhage, AND
• An epidemiological risk factor within the 21 days preceding the onset of symptoms.

U.S. hospitals or clinical laboratories concerned about a patient with potential Ebola virus exposure should contact their local and/or state public health authorities. These agencies will work with CDC to determine whether a patient is or is not a PUI, and whether testing is indicated. Patient status should be determined as quickly as possible in order to ensure that patient care is not compromised.

Presumptive testing for Ebola virus is available at more than 50 LRN laboratories located throughout the United States. Any presumptive positive Ebola test result must be confirmed at the CDC to inform public health decisions.

If it is determined that testing for Ebola virus is indicated, at least 4 mL of whole blood collected in a plastic tube preserved with EDTA is the preferred sample for testing. Specimens should be shipped with refrigerant to maintain 2° to 8°C to the designated LRN laboratory. If the PUI symptoms have been present for less than three days, a second sample collected 72 hours after onset of symptoms may be required to definitively rule out Ebola.

To minimize risk to personnel, a site-specific risk assessment must be performed by the laboratory director, safety officer, and other responsible persons prior to receiving specimens in order to determine the potential for exposure from sprays, splashes, or aerosols generated during all laboratory processes, procedures, and activities. Risks should be mitigated by implementing engineering controls, administrative and work practice controls, and use of appropriate personal protective equipment (PPE).

Ebola virus is classified as a Category A infectious substance by the U.S. DOT. Transport of samples from PUIs or patients confirmed or suspected of having EVD is regulated by DOT’s Hazardous Materials Regulations 49 CFR 171-180. Specimens for shipment should be packaged following the basic triple packaging system consisting of 1) a primary container (a sealable specimen container) wrapped with absorbent material; 2) a secondary container (watertight, leak-proof); and 3) an outer shipping package.