[ActionGreens] West Nile Virus Autopsies: 1999

[Guest guest]

Also see Jim West's website........http://www.geocities.com/noxot/TOC.htm

from Meryl Dorey, AVN, Australia.....

Fw: [ActionGreens] West Nile Virus Autopsies: 1999

>From Arnold Gore:

It now appears that the people who were said to have died from WNV may not

have had this as the cause and we were sprayed for " other " reasons. see

below an extract from the medical examiners report on the brains of the

people who died of WNV encephalitis.

The neuropathological examination of each West Nile encephalitis-related

fatality revealed a grossly normal brain.

arnold

[ActionGreens] West Nile Virus Autopsies: 1999

Lederman forwarded the link to this book,

http://www.annalsnyas.org/content/vol951/issue1/#WEST_NILE_VIRUS__DETECTION_

SURVEILLANCE__AND_CONT

and I pulled up the chapter on the autopsies that started all the West Nile

debate, which I post below.

Please note the following (and more experienced researchers should add to my

list, or tell me if I've leapt to improper conclusions.):

1) There is no isolation of West Nile Virus from the brains of any of those

autopsied. The diagnosis of West Nile Viral-encephalitis was based on

ANTIGEN tests, not on any finding of West Nile Virus in the brain tissue

extracted. Having been exposed to WNV at ANY point in one's life could lead

to the development of antibodies/antigens. That does not make it the CAUSE

of death at this time, in this place.

2) Four people thought to have died from WNV-encephalitis were autopsied,

but only TWO were " fully " done! (Two others had religious objections.) Can

we know fully the underlying ailments afflicting these people that may have

contributed to their deaths, or to this diagnosis, since they had not been

fully autopsied?

3) Seven people, not four, had died from what was claimed to be West Nile

Viral encephalitis in 1999. No autopsies were done on the other three.

4) The spinal cords were not examined in ANY of the four autopsies (which

could possibly lead to different explanations for the symptoms or deaths).

5) As the report states: " The average age of the four decedents was 81.5

years. They each had coexistent medical problems. " To what extent did these

coexistent medical problems contribute to their deaths, or at least make

them susceptible to ailments easily rebuffed by a healthy immune system?

6) If the latter, would it not be most propitious to figure out ways of

bolstering people's immune systems and to eliminate those environmental

factors that are attacking it, instead of resorting to indiscriminate

spraying of toxic insecticides such as Malathion and Pyrethroids?

7) We still have not seen the actual autopsy reports themselves -- only the

investigators conclusions from them. Why are these kept secret? Shouldn't

trained researchers be provided access to the reports, as well as the

biological material under investigation, so that independent investigators

could confirm or reject these conclusions?

All of these ASIDE from methodological challenges to the use of PCR

technology, which deserves its own separate enunciation.

- Mitchel Cohen

From: ls of the New York Academy of Sciences 951:172-178 (2001) © 2001

New York Academy of Sciences

West Nile Encephalitis

The Neuropathology of Four Fatalities

BARBARA A. SAMPSON and VERNON ARMBRUSTMACHER

Office of Chief Medical Examiner of the City of New York, 520 First Avenue,

New York, New York 10016, USA

Address for correspondence: Barbara A. Sampson, M.D., Ph.D., Office of Chief

Medical Examiner of the City of New York, 520 First Ave., New York, NY

10016.

Voice: 212-447-2335; fax: 212-447-2716.

basampson@...

West Nile virus was identified by immunohistochemistry (IHC) and polymerase

chain reaction (PCR) as the etiologic agent in four encephalitis fatalities

in New York City in the late summer of 1999. Fever and profound muscle

weakness were the predominant symptoms. Autopsy disclosed encephalitis in

two instances and meningoencephalitis in the remaining two. The inflammation

was mostly mononuclear and formed microglial nodules and perivascular

clusters in the white and gray matter. The brain stem, particularly the

medulla, was involved most extensively. In two brains, cranial nerve roots

had endoneural mononuclear inflammation. In addition, one person had acute

pancreatitis. On the basis of our experience, we offer recommendations for

the autopsy evaluation of suspected WNV fatalities.

------------------------------------------------------------------------

West Nile virus, a member of the Flaviviridae family, was first isolated

from the blood of a febrile Ugandan woman in 1937.1 It is a single-stranded

RNA virus, usually transmitted by Culex species mosquitoes, with wild birds

as hosts. Humans and domestic animals are incidental hosts.2 Although it is

found throughout Africa, the Middle East, and parts of Europe and Western

Asia, little is known about the pathological changes caused by this virus in

humans. In endemic areas, such as Egypt, a seroprevalence of 40% has been

reported in adults.3

The incubation period is 5-15 days. Infections are usually asymptomatic or

may cause mild, nonspecific symptoms.2 In more serious cases, pancreatitis,4

myocarditis,5 and hepatitis6 have been reported. Treatment is currently

nonspecific and supportive. Fatalities generally occur in older people,

although child fatalities have been documented.7,8

We report the autopsy results of four fatalities from West Nile virus

infection that occurred during an outbreak in New York City in the late

summer of 1999. This is the first West Nile virus outbreak reported on this

continent.9 On the basis of our experience, we offer recommendations for

autopsy procedures to best evaluate and study cases of suspected West Nile

virus infection.

Case Selection

The Office of Chief Medical Examiner of the City of New York accepted under

our jurisdiction all suspected cases of West Nile virus infection because of

the obvious public health implications. Four decedents were confirmed to be

positive for West Nile virus by immunohistochemistry (IHC) and polymerase

chain reaction (PCR) of brain tissue by the Centers for Disease Control. Two

of the four decedents had limited autopsies due to religious objection,

which are empowered by law in New York State. The limited autopsies included

gross inspections of all organs, but only the most necessary tissue samples

were taken for routine histology, IHC, and PCR. The other two decedents had

full autopsies, with all organs examined grossly and by routine histology;

tissue also was obtained for IHC and PCR.

Protocol for Tissue Collection

Tissue for IHC and PCR was collected following guidelines provided by the

New York City Department of Health and by the Centers for Disease Control.

Two tubes of CSF and one container with sections of fresh brain were

submitted on dry ice. Two purple-top tubes of blood and formalin-fixed

tissues were submitted in a separate container without dry ice. We recommend

submission of pancreas, liver, heart, lung, spleen, and kidney for routine

histologic exam and special studies. Brain sections should include several

from the brain stem, particularly from the medulla and the spinal cord,

including dorsal and ventral roots.

Immunohistochemistry

Sections of formalin-fixed, paraffin-embedded tissue were cut onto

positively charged glass slides and stained using a modified avidin-biotin

immunoperoxidase technique10 and an automated staining system (Ventana,

Tucson, AZ). Briefly, sections were dewaxed in xylene and hydrated through

graded alcohols to deionized water. Pretreatment of slides for optimum

antigen retrieval was performed as follows: for CD20 (L26), slides were

microwave heated on medium-low power for 10 minutes in citrate buffer, pH

6.0; for CD4, slides were boiled in 0.01M EDTA, pH 8.0 for 20 minutes; for

CD8, the slides were microwave heated on medium-low power in citrate buffer,

pH 8.0 for 20 minutes. Following inhibition of endogenous peroxidase,

primary antibodies CD20 (mouse monoclonal, Dako, Carpinteria, CA) and CD4

(mouse monoclonal, Novacastra Laboratories, Ltd., United Kingdom) were

applied at a 1:250 dilution, and CD8 (mouse monoclonal, Dako, Carpinteria,

CA) was used at 1:50 dilution. The reaction was visualized using a

commercially prepared detection system (Basic DAB Detection Kit, Ventana,

Tucson, AZ) using biotinylated secondary antibody and the standard

avidin-biotin-complex technique with copper-enhanced 3,3' diaminobenzidine

(DAB) and hematoxylin counterstain. Slides were dehydrated in ascending

grades of alcohol, fixed in xylene, and mounted.

The average age of the four decedents was 81.5 years. They each had

coexistent medical problems. Their clinical course has been described

elsewhere.11 General autopsy showed no myocarditis or hepatitis. One patient

had an acute, hemorrhagic pancreatitis.

Histology

The neuropathological examination of each West Nile encephalitis-related

fatality revealed a grossly normal brain. Histologically all showed

microglial nodules composed mainly of lymphocytes and histiocytes. Some

nodules included degenerating neurons. These were present predominantly in

the grey matter but also in the white matter (Fig. 1A). Variable mononuclear

perivascular inflammation was present (Fig. 1B). The medulla and thalamus

were involved most consistently, but similar pathological changes were also

present in the frontal lobe, temporal lobe, parietal lobe, hippocampus, and

cerebellum in some instances. Scattered mononuclear inflammatory infiltrate

was present in the leptomeninges in two of the four cases (Fig. 1C). Two

autopsies revealed encephalitis with involvement of cranial nerve roots.

Focal mononuclear inflammation was present in cranial nerve roots of the

medulla (Fig. 1D).

------------

FIGURE 1A. Hematoxylin and eosin-stained section of medulla, showing a

microglial nodule composed of histiocytes and occasional lymphocytes (70x

magnification). (From Sampson et al.18 Reproduced by permission.)

------------

FIGURE 1B. Hematoxylin and eosin-stained section of medulla, showing a

perivascular collection of lymphocytes (50x magnification). (From Sampson et

al.18 Reproduced by permission.)

------------

FIGURE 1C. Hematoxylin and eosin-stained section of medulla, showing

extension of the perivascular inflammation into the leptomeninges (50x

magnification). (From Sampson et al.18 Reproduced by permission.)

------------

FIGURE 1D. Hematoxylin and eosin-stained section, showing a cranial nerve

as it exits from the medulla with endoneural lymphocytic inflammation (50x

magnification). (From Sampson et al.18 Reproduced by permission.)

------------

Immunohistochemistry

Immunohistochemistry was performed to analyze the populations of lymphocytes

prominent in this infection. CD8+ T cells were numerous and were present

within the microglial nodules, in the perivascular infiltrates, in the

meninges, and in the cranial nerve roots.

CD4+ T cells also were present in these areas but were far fewer. In the

most

heavily involved areas of the medulla, CD8+ T cells were found scattered

throughout the parenchyma. CD20+ B cells were scattered and most prominent

around the blood vessels.

These are the first fatalities due to complications of West Nile virus

encephalitis in North America. A detailed description of the clinical course

of three of these patients, as well as that of five people who survived WNV

infection has recently been published.11 WNV also caused the deaths of many

New York City birds, particularly crows, and several exotic birds at the

Bronx Zoo. Necropsies of the birds revealed meningoencephalitis, which was

much more pronounced than in humans, and marked myocarditis.12

In 1954 a study using West Nile virus for the treatment of advanced cancer

was published.13 Eleven percent of their patients developed encephalitis.14

The neuropathology of the 1999 New York City fatalities was significant for

scattered microglial nodules composed of lymphocytes and histiocytes and

mononuclear perivascular inflammatory infiltrate. These findings were most

common in the brain stem but were also found in the thalamus, cerebellum,

and cerebral cortex. Leptomeningitis was present in two instances, and

mononuclear inflammation was seen around cranial nerve roots in two

instances. The predominant T cell was CD8+.

These pathological changes are quite different from the more fulminant

arbovirus encephalitides, such as Eastern and Western equine encephalitis.

In West Nile virus encephalitis the inflammatory infiltrate is focal and

slight. There is no vasculitis. This difference probably results from the

less fulminant course of this infection with prolonged survival with maximal

medical support after infection. In St. Louis encephalitis the distribution

of lesions is predominantly cerebral, while with West Nile virus the brain

stem is involved most consistently.

The most striking clinical manifestation of West Nile virus encephalitis

present in over half of the confirmed WNV encephalitis cases (including

nonfatal and fatal cases) in this outbreak is profound muscle weakness,

often with axonal neuropathy and requiring mechanical ventilation.

Clinically Guillain-Barré syndrome was considered a possible diagnosis.

Instances have been described with chronic neurologic sequelae, in

particular persistent muscle weakness (M. Layton, personal communication).

Our examinations revealed a mononuclear cranial nerve root infiltrate in two

brains with a nerve root included incidentally in the examination of the

medulla. The spinal cord was not examined in any of the four fatalities.

Examination of the entire spinal cord and nerve roots in a confirmed death

due to WNV infection might help explain the clinical presentation and

emphasizes the need for autopsies in all deaths suspected or confirmed to be

due to West Nile virus. Although an instance of acute anterior myelitis

complicating WNV encephalitis has been reported,15 the type of severe axonal

neuropathy observed in our study population usually is not associated with

the epidemic encephalitides and has not been documented pathologically.

It appears that West Nile virus is now endemic in the New York area. West

Nile virus RNA was detected in overwintering mosquitoes.16 Moreover, in

2000, 21 cases of West Nile virus associated illness were reported in New

York, New Jersey, and Connecticut.17 One fatality in New York City in 2000

has been attributed to West Nile virus. No autopsy was performed.

The threshold for suspecting WNV infection should be low, because the

symptoms are common and nonspecific. Also, the scope of the possible

manifestations of this infection are not yet clearly established. The New

York City Department of Heath requested that any patients be reported to

them with fever > 38.0°, altered mental status, CSF pleocytosis with

predominant lymphocytes and/or elevated protein, and muscle weakness

(especially flaccid

paralysis) confirmed by neurologic exam or EMG. Also to be reported are any

patients with a presumed diagnosis of viral encephalitis or with focal CNS

findings and fever, any patients with fever and presumed Guillain-Barré

syndrome or acute flaccid paralysis, or any patients with aseptic

meningitis. At autopsy, particular attention should be paid to complete

neuropathologic examination, including the spinal cord and nerve roots. If

autopsy is limited for any reason, the brain stem, particularly the medulla,

should be sampled. We recommend submission of pancreas, liver, heart, lung,

spleen, and kidney for routine histologic exam and special studies.

We would like to thank Drs. R. Lanciotti (PCR) and S. Zaki (IHC). In

addition we thank Drs. M. Layton and D. Asnis for many thought-provoking

discussions.

1. burn, K.C., T.P. , A.W. Burke, et al. 1940. A neurotropic

virus isolated from the blood of a native of Uganda. Am. J. Trop. Med. 20:

471-492.

2. Monath, T. & F.X. Heinz. 1996. Flaviviruses. In Virology. B.N. Fields,

D.M. Knipe & P.M. Howley, Eds.: 1004-1006. Raven Press. New York.

3. Corwin, A., M. Habib, D. Watts, et al. 1993. Community-based prevalence

profile of arboviral, rickettsial and Hantaan-like viral antibody in the

Nile River delta of Egypt. Am. J. Trop. Med. Hyg. 48: 776-783.[Medline]

4. Perelman, A. & J. Stern. 1974. Acute pancreatitis in West Nile fever. Am.

J. Trop. Med. Hyg. 23(6): 1150-1152.

5. Albagalic, C.R. 1959. A case of West Nile myocarditis. J. Med. Assoc.

Isr.

57: 274-275.

6. s, A.J., J.L. Lesbordes, M.C. s-Courbot, et al. 1987. Fatal

hepatitis from West Nile virus. Ann. Inst. Pasteur/Virol. 138: 237-244.

7. , S., M. Gourie-Devi, J.A. Rao, et al. 1984. Isolation of West Nile

virus from the brains of children who died of encephalitis. Bull. W.H.O.

62(6): 879-882.

8. Le Guenno, B., A. Bougermouh, T. Azzam & R. Bouakaz. 1996. West Nile: a

deadly virus? Lancet 348: 1315.

9. Centers for Disease Control. 1999. Outbreak of West Nile-like viral

encephalitis-New York. Morb. Mortal. Wkly. Rep. 48: 845-849.

10. Hsu, S-M., L. Raine & H. Fanger. 1981. Use of avidin-biotin-peroxidase

complex (ABC) in immunoperoxidase technique: a comparison between ABC and

unlabeled antibody (PAP) procedures. J. Histochem. Cytochem. 29:

577-580.[Abstract]

11. Asnis, D.S., R. Conetta, A.A. Teixeira, et al. 2000. The West Nile virus

outbreak of 1999: The Flushing Hospital Experience. Clin. Infect. Dis. 30:

413-418.[Medline]

12. Steele, K.E., M.J. Linn, R.J. Schoepp, et al. 2001. Pathology of Fatal

West Nile virus infections in native and exotic birds during the 1999

outbreak in New York City, New York. Vet. Pathol. 37: 208-224.

13. Newman, W. & C.M. Southam. 1954. Virus treatment in advanced cancer: a

pathological study of fifty-seven cases. Cancer 7: 106-118.

14. Southam, C.M. & E. . 1954. Induced virus infections in man by the

Egypt isolates of West Nile virus. Am. J. Trop. Med. 3: 19-50.

15. Gadoth, N., S. Weitzman & E. Lehmann. 1979. Acute anterior myelitis

complicating West Nile fever. Arch. Neurol. 36: 172-173.[Medline]

16. Centers for Disease Control. 2000. Update surveillance for West Nile

virus in overwintering mosquitoes-New York, 2000. Morb. Mortal. Wkly. Rep.

49: 178-179.7

17. Centers for Disease Control. 2001. Serosurveys for West Nile virus

infection-New York and Connecticut counties, 2000. Morb. Mortal. Wkly. Rep.

50: 37-39.

18. Sampson, B.A., C. Ambrosi, A. Charlot, et al. 2000. The pathol

--------------------------------------------------------

Sheri Nakken, R.N., MA, Hahnemannian Homeopath

Vaccination Information & Choice Network, Nevada City CA & Wales UK

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