1. West Nile Virus Screening for Viremic Blood Donors
Karen A. Reiner, Ph.D. Student
Walden University
PUBH
Dr. Raymond W. Thron
Fall Quarter, 2010
Hello, my name is Karen Reiner. I am a Medical Laboratory Scientist specializing in the areas of Clinical Microbiology and Immunology. Today, I will be presenting on the increasing need for screening blood donors for the West Nile Virus. I will be presenting compelling facts that indicate the need for a national standard in screening for West Nile virology in blood.

2. Intended Audience Immunohematologists Blood banks specialists
Transfusion medicine personnel
Physicians
This in-service is intended for health professionals like you: Immunohematologists, blood blank specialists, transfusion medicine personnel, and physicians.

3. Learning Outcomes
After completing this presentation, participants should be able to:
Describe the etiology and epidemiology of West Nile Virus.
Describe the signs and symptoms of West Nile Virus.
Discuss potential interferences with current serological testing of West Nile Virus.
Discuss the importance of West Nile Virus screening of blood.
The objectives (learning outcomes) of this presentation are to:
Describe the etiology and epidemiology of West Nile Virus.
Describe the signs and symptoms of West Nile Virus.
Discuss potential interferences with current serological testing of West Nile Virus.
Discuss the importance of West Nile Virus screening of blood

4. Introduction West Nile Virus can cause serious illness
Seasonal epidemic in the US
Can be fatal
Humans and animals affected
Cases reported to the CDC (so far in 2010)
789 cases
33 deaths
The West Nile Virus is a potentially serious disease. Here in the United States, it is considered a seasonal epidemic beginning in the early summer and lasting into the fall months. So far, as of October 19 of this year, there have been 789 reported cases of West Nile Virus associated disease, 33 of which resulted in deaths.
The effects of West Nile virus have been particularly dramatic in North America, where widespread epidemics have occurred not only in humans, but in horses and certain species of birds. Although vaccination can control the disease in equids, no human vaccine is available, and thousands of people become ill each year in the United States and Canada.
References:
CDC. (2010). West Nile Virus: Statistics, Surveillance, and Control . Available from
CDC. (2010). West Nile Virus: Statistics, surveillance, and control . Available from

5. Background First isolated in 1937
Recognized as etiologic agent of meningoencephalitis
First identified in the US in 1999
The West Nile virus (WNV) was first isolated in 1937 from a febrile woman in the West Nile District
of Uganda. Thus the name. WNV was later recognized as a cause of severe human meningoencephalitis in elderly
patients during an outbreak in Israel in In the US, it was first identified in New York in 1999.

6. Etiology Caused by a single-stranded RNA virus Family Flaviviridae
Genus: Flavivirus
Red
Blood
Cells
Viron
The West Nile virus is a single‐stranded RNA virus of the family Flaviviridae, genus Flavivirus. It is a member of the Japanese encephalitis virus serocomplex, which contains several medically important viruses associated with human encephalitis: Japanese encephalitis, St. Louis encephalitis, Murray Valley encephalitis, and Kunjin virus (an Australian subtype of West Nile virus). The close antigenic relationship of the flaviviruses, particularly those belonging to the Japanese encephalitis complex, accounts for the serologic cross‐reactions observed in the
diagnostic laboratory.
Before 1994, disease occurred only sporadically in humans and horses, or as relatively small epidemics in rural areas, and severe neurological signs were uncommon in most outbreaks. Until 1999, West Nile virus was also confined to the Eastern Hemisphere. However, severe outbreaks were reported in Algeria, Romania, Morocco, Tunisia, Italy, Russia and Israel between 1994 and 1999, and West Nile virus spread to North America in 1999.
An increased incidence of neurological disease and a higher case fatality rate have been associated with these viruses. Some recent viral isolates also cause clinical signs in birds and can be quite virulent in reptiles. Consequently, West Nile fever has emerged as a significant human and veterinary health concern in the Americas, Europe, the Mediterranean basin and other areas. In the US, it is considered a reportable disease.

7. Epidemiology Transmitted primarily by: Other transmission routes are:
Infected mosquitoes
Other transmission routes are:
Blood transfusions
Organ transplant
Intra utero
Breast feeding
The West Nile Virus is transmitted to humans most commonly by an infected mosquito bite. Mosquitoes become infected when they feed on infected birds. Mosquitoes then spread the disease to other animals and humans.
West Nile Virus can also be transmitted through organ transplants, breast feeding, intra utero, and via blood transfusions. While these has been the least likely transmission modes, cases has been reported.
West Nile Virus is not transferred by casual touch or kissing.
Animated graphic available from

8. West Nile Virus Transmission Cycle
The transmission cycle for the West Nile Virus is depicted on this diagram.
Humans are considered accidental hosts.
Human to human transmission is also possible and of increasing concern.
This diagram depicts the West Nile Virus transmission cycle.
WNV transmission cycle. Available from

9. Distribution of West Nile Virus in the US
So far this year Arizona has the highest number of reported West Nile Virus cases.
The distribution of West Nile Virus in the US is shown here. So far this year (as of October, 2010), Arizona has the higher number of reported West Nile Virus cases.
References:
CDC. (2010). West Nile Virus: Statistics, Surveillance, and Control . Available from
CDC. (2010). West Nile Virus: Statistics, surveillance, and control . Available from

10. Arizona: Cases per Week
The graph below shows the increase in human disease cases during the summer months; consistent with increased mosquito activity .
Arizona cases per week are shown here. Notice the trend is consistent with mosquito activity; which is higher during the summer months.
References:
CDC. (2010). West Nile Virus: Statistics, Surveillance, and Control . Available from
CDC. (2010). West Nile Virus: Statistics, surveillance, and control . Available from

11. Epidemiology
39%
61%
Of the 789 West Nile Virus cases reported to the CDC so far this year, 479 (61%) were neuroinvasive disease cases, and 310 (39%) nonneuroinvasive disease cases.
Neuroinvasive disease cases, refers to severe cases of disease that affect a person’s nervous system. These include encephalitis which is an inflammation of the brain, meningitis which is an inflammation of the membrane around the brain and the spinal cord and acute flaccid paralysis which is an inflammation of the spinal cord that can cause a sudden onset of weakness in the limbs and/or breathing muscles.
Nonneuroinvasive disease cases refers to less severe cases that show no evidence of neuroinvasion, but demonstrate West Nile fever. West Nile fever is a notifiable disease; however, the number of cases reported (as with all diseases) may be limited by whether persons affected seek care, whether laboratory diagnosis is ordered and the extent to which cases are reported to health authorities by the diagnosing physician.
References:
CDC. (2010). West Nile Virus: Statistics, surveillance, and control . Available from

12. Cases by State as of October 2010
Epidemiology
Cases by State as of October 2010
This chart shows some of the states with higher reported cases of West Nile Virus as of October 19, It is important to understand that an individual’s blood can test positive for West Nile Virus, but not be considered a “case” by the CDC. The reason: A West Nile Virus "case" is defined as a person who has become ill and has been confirmed to have a West Nile Virus infection. This infection might be either West Nile Fever, a mild illness with fever, or West Nile encephalitis or meningitis, or other more severe illnesses.
Blood donors, who do not become ill and do not develop symptoms, are counted in a separate category because they are not considered "cases."
Therefore, estimates are only regarding “cases”.
So far this year, 98 presumptive viremic donors have been reported; with the highest incidence in Arizona, California, and Texas.
CDC. (2010). West Nile Virus: Statistics, surveillance, and control . Available from

13. Disease Manifestation
West Nile Virus infections may be:
Asymptomatic
80% of infections
Mild
10-20% of infections
Severe
1 in 150 infections
Fatal
3-15%; mostly elderly individuals
Approximately 4 out of 5 or 80% of WNV infections are asymptomatic. Only 10–20% of individuals present with febrile illness of sudden onset, often accompanied by malaise, anorexia, nausea, vomiting, eye pain, headache, myalgia, rash, and lymphadenopathy. These
symptoms generally last 3 to 6 days, and most cases are never diagnosed.
Approximately half of hospitalized U.S. patients have had severe muscle weakness and encephalopathy. Approximately 10% of patients with encephalitis develop a poliomyelitis‐like syndrome with flaccid paralysis. Neurologic presentations other than encephalitis or meningitis, which occur more rarely, include ataxia and extrapyramidal signs, cranial nerve abnormalities, myelitis, optic neuritis, polyradiculitis, and seizures. Myocarditis, pancreatitis, and fulminant hepatitis have been described in outbreaks occurring before Although recent outbreaks of West Nile virus seem to be associated with increased morbidity and mortality (approximately 3-15%, primarily among the elderly), severe neurologic disease remains uncommon. Two serosurveys conducted in New York City in 1999 and 2000 showed that approximately 1 in 150 infections resulted in meningitis or encephalitis.
References:
CDC. (2010a). Cases of West Nile Virus human disease. Available from
CDC. (2010a). Cases of West Nile Virus human disease. Available from

14. Signs and Symptoms Asymptomatic Mild Fever Headache Body aches Nausea
Vomiting
Stomach & back pain
Swollen lymph glands
Skin rash
Symptoms include:
Asymptomatic
Mild: fever, headache, body aches, nausea, vomiting, swollen lymph glands, skin rash, stomach & back pain
Severe symptoms involve neurological complications: High fever, headache, stiff neck, stupor, disorientation, coma, tremors, convulsions, muscle weakness, loss of vision, numbness, and paralysis

15. Signs and Symptoms Severe High fever Headache Stiff neck
Disorientation
Coma
Tremors
Convulsions
Muscle weakness
Loss of vision
Numbness
Paralysis
Severe symptoms involve neurological complications: High fever, headache, stiff neck, stupor, disorientation, coma, tremors, convulsions, muscle weakness, loss of vision, numbness, and paralysis

16. Diagnosis Detection of increased IgM in serum or CFS Serology ELISA
Nucleic Acid
Plaque reduction neutralization (PRN) test
Indirect Immunofluorescence (IFA)
Hemagglutination inhibition
In humans, West Nile virus infections are often diagnosed by serology. Diagnostic criteria include a rising titer or the presence of IgM in serum or cerebrospinal fluid (CSF). IgM in CSF indicates a recent infection; however, anti-WNV IgM can persist in the serum of some individuals for more than a year. For this reason, IgM in serum is suggestive but not definitive. Enzyme-linked immunosorbent assays (ELISAs) are the most commonly used serological tests. Other tests include the plaque reduction neutralization (PRN) test, indirect immunofluorescence (IFA) and hemagglutination inhibition. Two rapid tests, an optical fiber immunoassay and a microsphere-based fluorescence immunoassay, have recently been developed. In some serological tests, cross-reactions can occur with closely related flaviviruses including yellow fever, Japanese encephalitis, St. Louis encephalitis or dengue viruses. For this reason, positive reactions in ELISAs or other tests may be confirmed with the PRN test.
West Nile virus, viral antigens or nucleic acids can sometimes be detected in tissues, CSF, blood and other body fluids. WNV can usually be found in the blood of patients with West Nile fever, during the first few days after the onset of illness. Reverse-transcription polymerase chain reaction (RT-PCR) assays are often used to screen blood supplies for transfusion. However, viremia usually disappears before the onset of neurological signs, and viral RNA is generally absent from the serum of patients with neuroinvasive disease. CSF can be tested with RT-PCR, although this is rarely done in clinical practice. Immunohistochemistry to detect viral antigens is mainly used postmortem in cases of fatal neurological disease. Virus isolation requires level 3 biosafety containment, and is rarely performed.

17. Treatment No specific treatment Supportive care Experimental
Interferon
Antisense nucleotides
Experimental
Intravenous immunoglobulins
There is no specific treatment for West Nile Virus. Supportive care is provided for mild to sever cases. Some severe cases may require medical intensive care and mechanical ventilation.
Various therapies include:
Interferon
Antisense nucleotides
Intravenous immunoglobulin are currently being tested in clinical trials
Some antiviral drugs were promising in vitro, but most have been ineffective when tested in animal models or given to humans with severe disease. Screening for new drugs that may inhibit West Nile Virus reproduction is currently underway.

18. Prevention Infection prevention Vector elimination
Limit outdoor activities during peak biting times
Use mosquito repellent
Wear appropriate protective clothing
Long pants
Long-sleeved shirts
Vector elimination
Pesticides
Eliminate water holding containers
In most cases, West Nile Virus infections can be prevented by preventing mosquito bites. Limit outdoor activities when mosquitoes are active. Peak biting times are during dusk and dawn. Use mosquito repellents when you must be out during peak biting hours. Wear appropriate protective clothing such as long pants and long-sleeved shirts. Specialized fine mesh clothing, for example, mesh head coverings and jackets, is also available.
Measures to reduce mosquito populations include (vector elimination) include:
Application of adulticides and larvicides
Environmental modifications such as emptying containers that may hold standing water.

19. Prevention (continued)
Environmental surveillance
Sentinel birds
Death birds
Mosquito populations
Vaccine
No human vaccine is available
Education
CLICK on picture to view video on separate window.
Environmental surveillance can also contribute to detection and reduction of West Nile Virus exposures. These include:
Surveillance in sentinel birds, dead birds and mosquitoes.
Dead or sick birds should be reported to health, agriculture or mosquito-control agencies. Dead animals should never be handled without gloves and sanitary precautions, as feces and body fluids may be infectious in some species.
Human vaccines are not yet available, but some vaccines have entered clinical trials.
West Nile Virus Surveillance. Microbe World Video available from

20. Timeline of West Nile Virus Screening
West Nile Virus screening begins after the first transfusion-related infection reported
2003 – Another case of transfusion-related West Nile Virus infection
2003 – National blood donation screening for West Nile virus started
2005 – DFA approves NAT for screening blood donors
The first West Nile Virus transfusion-related infection was reported in 2002 and then another one in 2003.
As a result, national blood donation screening for West Nile virus result of the documented West Nile Virus transfusion-associated transmission (TAT) in 2002 started (June 2003).
References:
MMWR. (2004b). Update: West Nile Virus Screening of Blood Donations and Transfusion-Associated Transmission --- United States, Available from
MMWR. (2004b). Update: West Nile Virus screening of blood donations and transfusion-associated transmission --- United States, Available from

21. Current Criteria for Blood Screening
Mandatory blood screens include:
ABO Rh
Other antigenic screening
HIV-1 & HIV -2
Hep B
Hep C
HTLV-I and HTLV-II
Syphillis
After blood has been drawn, it is tested for ABO group (blood type) and Rh type (positive or negative), as well as for any unexpected red blood cell antibodies that may cause problems in a recipient. Screening tests also are performed for evidence of donor infection with hepatitis B and C viruses, human immunodeficiency viruses HIV-1 and HIV-2, human T-lymphotropic viruses HTLV-I and HTLV-II, and syphilis.
Type of specific tests are:
Hepatitis B surface antigen (HBsAg)
Hepatitis B core antibody (anti-HBc)
Hepatitis C virus antibody (anti-HCV)
HIV-1 and HIV-2 antibody (anti-HIV-1 and anti-HIV-2)
Nucleic acid amplification testing (NAT) for HIV-1 and HCV
HTLV-I and HTLV-II antibody (anti-HTLV-I and anti-HTLV-II)
Serologic test for syphilis
AABB. (2010). Blood FAQ. Available from

22. Current Criteria for Blood Screening
Optional Screening include:
West Nile Virus
Chaga’s Disease
CLICK on picture to view video on separate window.
While not required, blood donors are also being tested for West Nile virus (using nucleic acid tests). The FDA (the organization responsible for federally regulating the blood supply) also has licensed one test for the screening of blood for Chagas' disease (Treponema parasites), but has not required that all blood products be screened.
Neither of these tests are required by the FDA.
References:
AABB. (2010). Blood FAQ. Available from
FDA. (2010). Testing HCT/P Donors for Relevant Communicable Disease Agents and Diseases. Available from
First Donor Screening Test for West Nile Virus. Available from
AABB. (2010). Blood FAQ. Available from
FDA. (2010). Testing donors for relevant communicable disease agents and diseases. Available from

23. Challenges of Current Protocols
Current blood screening protocols:
Do not reflect current needs
Example:
Human T-Lymphotropic Virus (HTLV)
Last documented transfusion-related exposure was in 1985
Reportedly, less likely transmission for HTLV than for Hepatitis B
No other transfusion-related HTLV infections have been reported since
West Nile Virus
Steady increase in cases since 1999
The challenge is that we are no moving at the same speed as the demands at hand. New diseases appear and develop relatively quickly, but screening protocols lag behind. They do not reflect current needs.
Lets take a look, for example, at Human T-Lymphotropic Virus (HTLV):
The most current recommendations for HTLV date back from the 1986 (from a reported transfusion-related HTLV in November of 1985).
At that time the CDC indicated that the risk of bloodborne transmission from inadvertent exposure is considerably less for HTLV-III/LAV than for hepatitis B virus infection.
No other transfusion-related HTLV infections have been reported since
References:
MMWR. (1986a). Current trends in human T-Lymphotropic virus type III/Lymphadenopathy-associated virus: Agent summary statement. Available from
MMWR. (1986b). Epidemiologic Notes and Reports Transfusion-Associated Human T-Lymphotropic Virus Type III/ Lymphadenopathy-Associated Virus Infection From a Seronegative Donor – Colorado. Available from
MMWR. (1986a). Current trends in human T-Lymphotropic virus type III/Lymphadenopathy-associated virus: Agent summary statement. Available from
MMWR. (1986b). Epidemiologic notes and reports transfusion-associated Human T-Lymphotropic Virus Type III/ Lymphadenopathy-associated virus infection from a seronegative donor – Colorado. Available from

24. Challenges of Current Protocols
West Nile Virus transfusion-related infection reported in 2002
23 cases reported
500 viremic donations
Other transfusion-related West Nile Virus infections reported since
In contrast:
The first West Nile Virus transfusion-related infection was reported in 2002 and another one in Since then, the CDC Division of Vector-Borne Infectious Diseases has tracked presumptively viremic blood donors (PVD).
During the 2002 epidemic of West Nile virus (WNV) in the United States, a total of 23 persons were reported to have acquired WNV infection after receipt of blood components from 16 West Nile Virus-viremic blood donors and an estimated 500 viremic donations might have been collected. Because of the possibility of recurrent WNV epidemics in the United States, blood collection agencies have implemented West Nile Virus nucleic acid--amplification tests (NATs) to screen all donations and quarantine and retrieve potentially infectious blood components.
Reference:
MMWR. (2004a). Update: Detection of West Nile Virus in Blood Donations --- United States, Available from
MMWR. (2004b). Update: West Nile Virus Screening of Blood Donations and Transfusion-Associated Transmission --- United States, Available from
MMWR. (2004a). Update: Detection of West Nile Virus in blood donations --- United States, Available from
MMWR. (2004b). Update: West Nile Virus screening of blood donations and transfusion-associated transmission --- United States, Available from

25. West Nile Virus Viremic Blood Donor Activity in the United States
This chart shows the West Nile Virus Viremic Blood Donor Activity in the United States between 2003 and The number for 2010 are as of October 19, 2010.
Between 2003 to 2005, a total of 1,425 presumptive viremic donors were reported to CDC from 41 states. Of 36 investigations of suspected West Nile Virus transfusion associated transmission, 6 cases were documented.
As shown here, there is compelling evidence supporting routine, mandatory screening of blood for West Nile Virus.
References
CDC. (2010b). West Nile Virus: Statistics, Surveillance, and Control Archive
CDC. (2010b). West Nile Virus: Statistics, surveillance, and control archive. Available from

26. Proposed West Nile Virus Screening Methods
Approved methods for West Nile Virus testing:
COBAS TaqScreen West Nile Virus Test  Roche Molecular System, Inc.
Procleix West Nile Virus (WNV) Assay  Gen-Probe, Inc. (NAT)
CLICK on picture to view video on separate window.
The FDA has approved 2 methods for screening for West Nile Virus:
COBAS TaqScreen West Nile Virus Test  Roche Molecular System, Inc. – approved on 2007
Procleix West Nile Virus (WNV) Assay  Gen-Probe, Inc. – approved on 2005
The PROCLEIX® WNV Assay is the first NAT approved by the FDA for the direct detection of the West Nile virus in plasma specimens from individual donors.
How to test for West Nile Virus in blood. Available from
FDA. (2009). West Nile Virus nucleic acid testing. Available from

27. Summary West Nile Virus infections can be serious
West Nile Virus can be transmitted via blood transfusion
Many lives have been saved by screening
West Nile Virus screening of blood should be mandatory
West Nile Virus infections are serious. While cases of West Nile Virus infections have declined in the last 10 years, the fact that West Nile Virus can be transmitted via blood transfusion is sobering. I propose that based on the facts presented, West Nile Virus screening of blood should become part of mandatory blood screening.
Consider that since the introduction of FDA NAT screening in 2003, US blood centers have screened more than 29 million blood donations with the assay and intercepted more than 1,500 positive WNV donations. Implementation of West Nile Virus NAY has protected many patients who receive blood and other such products against West Nile infection. To date, there have been 30 documented cases of people who most likely acquired WNV from a blood transfusion, including nine who died.

28. Please e-mail all questions to: karen.reiner@waldenu.edu

29. References
AABB. (2010). Blood FAQ. Available from
CDC. (2010). West Nile Virus: Statistics, surveillance, and control . Available from
FDA. (2009). West Nile Virus Nucleic Acid Testing. Available from
Macedo de Oliveira, A., Beecham, B. D., Montgomery, S. P., Lanciotti, R. S., Linnen, J. M., Giachtti, C., Stramer S.L., Safranek T. J. (2004). West Nile Virus blood transfusion-related infection despite nucleic acid testing. Transfusion, 44(12),
MMWR. (1986a) Current trends Human T-Lymphotropic Virus Type III/ Lymphadenopathy-associated virus: Agent summary statement. Available from
MMWR. (1986b). Epidemiologic notes and reports transfusion-associated Human T-Lymphotropic Virus Type III/ Lymphadenopathy-associated virus infection from a seronegative donor – Colorado. Available from
MMWR. (2004a). Update: Detection of West Nile Virus in blood donations --- United States, Available from
MMWR. (2004b). Update: West Nile Virus screening of blood donations and transfusion-associated transmission --- United States, Available from

30. Recommended Additional Reading
Biggerstaff, B. J., Petersen, L. R. (2003). Estimated risk of transmission of the West Nile virus through blood transfusion in the US, Transfusion, 43:8, doi: /j x.
Complete list of donor screening assays for infectious agents and HIV diagnostic assays. Available from
Diamond, M.S., Klein, R.S. (2004). West Nile virus: crossing the blood-brain barrier. National Medicine, 10(12),
Harrington, T., Kuehnert, M. J., Kamel, H., Lanciotti, R. S., Hand, S., Currier, M., Chamberland, M. E., Petersen, L. R., Marfin, A. A. (2003). West Nile virus infection transmitted by blood transfusion. Transfusion, 43(8), doi: /j x
Lanciotti, R. S., Roehrig, J. T., Deubel, V., Smith, J., Parker, M., Steele, K., et al. (1999). Origin of the West Nile Virus responsible for an outbreak of encephalitis in the Northeastern United States. Science, 286, 2333 – doi: /science
Macedo de Oliveira, A., Beecham, B. D., Montgomery, S. P., Lanciotti, R. S., Linnen, J. M., et al. (2004). West Nile Virus blood transfusion-related infection despite nucleic acid testing. Transfusion, 44(12),
MMWR. (2005). West Nile Virus infections in organ transplant recipients --- New York and Pennsylvania, August--September, Available from
Montgomery, S. P., Brown, J. A., Kuehnert, M., Smith, T. L., Crall, N., Lanciotti, R. S., et al. (2006). Transfusion-associated transmission of West Nile virus, United States 2003 through Transfusion, 46(12), doi: /j x

31. Recommended Additional Reading
Pealer, L. N., Marfin, A. A., Petersen, L. R., Lanciotti, R. S., Page, P.L., Stramer, S.L., et al. (2003). Transmission of West Nile Virus through blood transfusion in the United States in New England Journal of Medicine, 349,
Weiss, D., Carr, J., Kellachan, C., Tan, M., Phillips, E., Bresnitz, M., et al. (2001). Clinical findings of West Nile virus infection in hospitalized patients, New York and New Jersey, Emerging Infectious Diseases, 7(4): 654–658.