1. UNCLASSIFIED LTC John M. Scherer/(301) 619-8837/john.scherer@us.army.mil 1 March 2010 A Historical Look at Anthrax: Facts, Misperceptions, and the Importance of Diagnostics LTC John M. Scherer, Ph.D., M.T. (ASCP) U.S. Army

2. UNCLASSIFIED LTC John M. Scherer/(301) 619-8837/john.scherer@us.army.mil 1 March 2010 Slide 2 Annals New York Acad Sci 1970; 174: 577-582 Anthrax vaccine trials begin

3. UNCLASSIFIED LTC John M. Scherer/(301) 619-8837/john.scherer@us.army.mil 1 March 2010 Fact or Fiction Clinical Disease = Mortality Only particles < 5um in size are important Subclincal infections do not exist Inhalation Anthrax = Widened Mediastinum Diagnosis of anthrax is easy Diagnostics only provide post-mortem confirmation Slide 3

4. UNCLASSIFIED LTC John M. Scherer/(301) 619-8837/john.scherer@us.army.mil 1 March 2010 Agenda Impact of particle size on infection Subclinical infections Inhalation anthrax cases from the Anthrax letters Diagnostics –Why is it so hard? –Are there sufficient bacteria to detect? Temporal influence of antibiotic use on survival of inhalation anthrax Conclusion Slide 4

5. UNCLASSIFIED LTC John M. Scherer/(301) 619-8837/john.scherer@us.army.mil 1 March 2010 Particle Size Alters Infectious Dose Slide 5 J of Hyg 1953; 51 359-371 In NHPs 12 um particles are14X less effective than single cell particles

6. UNCLASSIFIED LTC John M. Scherer/(301) 619-8837/john.scherer@us.army.mil 1 March 2010 Aerosolized Bacillus anthracis in Goat Hair Processing Mills Slide 6 Am J Hyg 1960, Vol 72: 24-31

7. UNCLASSIFIED LTC John M. Scherer/(301) 619-8837/john.scherer@us.army.mil 1 March 2010 Do Subclinical Infections Occur? Slide 7

8. UNCLASSIFIED LTC John M. Scherer/(301) 619-8837/john.scherer@us.army.mil 1 March 2010 The case against Subclinical Infection No increase in protection based on length of employment in hair processing mills (Anal New York Acad Sci 1958; 70: 574-583) –Does subclinical infection correlate with protection? No asymptomatic cases found following sero-surveys of potentially exposed individuals of anthrax letters (n=66) (Clinical Infectious Diseases 2005; 41:991–7) –Antibiotic use? Slide 8

9. UNCLASSIFIED LTC John M. Scherer/(301) 619-8837/john.scherer@us.army.mil 1 March 2010 The case for Subclincal Infection Slide 9 Norman, PS, Am J Hyg 1960; 72: 32-37. Albrink WS. Am J Path 1959; 35: 1055- 1065 “The first two animals (John and Melvin) exhibited no positive physical disorder after their initial exposure and survived despite the fact that organisms were demonstrated in the blood of one on the second through the tenth days and of the other from the third through the eleventh days. The animals maintained their appetites and their vigorous protestations to physical examination in unabated manner. Although a bacteremia was apparent in each, it was of low grade and exhibited no progression. The temperature varied little from normal (100 to 101 F).”

10. UNCLASSIFIED LTC John M. Scherer/(301) 619-8837/john.scherer@us.army.mil 1 March 2010 2001 Inhalation Anthrax Cases

11. UNCLASSIFIED LTC John M. Scherer/(301) 619-8837/john.scherer@us.army.mil 1 March 2010 Inhalation anthrax from Anthrax letters 11 people infected with Bacillus anthracis by aerosol route 55% (6/11) of the inhalation anthrax cases survived Average time from exposure to symptoms (when known) – 4.5 days (SD 0.8 days) Average time of symptoms before treatment with antibiotics – 3.8 days (SD 1.6 days) On average it was ~8 days from the initial exposure before therapy was initiated

12. UNCLASSIFIED LTC John M. Scherer/(301) 619-8837/john.scherer@us.army.mil 1 March 2010 Inhalation anthrax from Anthrax letters On average it was ~8 days from the initial exposure before therapy was initiated Observations consistent with historical cases of inhalation anthrax Only 7 of 11 (64%) had a widened mediastinum

13. UNCLASSIFIED LTC John M. Scherer/(301) 619-8837/john.scherer@us.army.mil 1 March 2010 Technical Memorandum, Medical Aspects of Anthrax, AD 801-504, 1966 (date scanned) United States Army Biological Laboratories, Fort Detrick

14. UNCLASSIFIED LTC John M. Scherer/(301) 619-8837/john.scherer@us.army.mil 1 March 2010 Inhalation anthrax from Anthrax letters On average it was ~8 days from the initial exposure before therapy was initiated Observations consistent with historical cases of inhalation anthrax Only 7 of 11 (64%) had a widened mediastinum In the post 2001 sero-survey, ~10% (n=6/66) hade a widened mediastinum that was not attributed to B. anthracis exposure 3 of the 11 individuals (27%) were sent home after seeking health care

15. UNCLASSIFIED LTC John M. Scherer/(301) 619-8837/john.scherer@us.army.mil 1 March 2010 Diagnostics

16. UNCLASSIFIED LTC John M. Scherer/(301) 619-8837/john.scherer@us.army.mil 1 March 2010 Why is Bacillus anthracis so difficult to diagnose? Physician –Uncommon –Generic flu-like illness Laboratory –Uncommon –Culture contamination with Bacillus species is “common” –Looks like other non-pathogenic Bacillus species –Clinical labs reluctant to report contaminants

17. UNCLASSIFIED LTC John M. Scherer/(301) 619-8837/john.scherer@us.army.mil 1 March 2010 Why is Bacillus anthracis so difficult for clinical labs to identify? Study conducted by Connecticut Depart of Public Health (EID 2005; 11: 1583-1486) 33 of 34 of Connecticut's clinical labs participated Mar to Dec 2003 (10 months) GPRs in blood or CSF isolated < 32hours 623 isolates reported (average 62/month) 195 of the isolates were Bacillus species (not anthracis) Additional workload ~0.3 FTEs Slide 17

18. UNCLASSIFIED LTC John M. Scherer/(301) 619-8837/john.scherer@us.army.mil 1 March 2010 In animal models, is there sufficient bacteremia to detect? Some basic assumption that are supported: –Higher doses decrease incubation period –There is bacteraemia when animals are symptomatic –Levels vary but typically are > 1000 org/ml –Toxin levels are also detectable However, it is extremely difficult to provide a precise number because the studies use different strains, doses, animal models, and methods for determining bacteremia

19. UNCLASSIFIED LTC John M. Scherer/(301) 619-8837/john.scherer@us.army.mil 1 March 2010 Sample data Technical Memorandum 19, Pathogenesis of Anthrax- A Progress Report, November 1962 United States Army Biological Laboratories, Fort Detrick

20. UNCLASSIFIED LTC John M. Scherer/(301) 619-8837/john.scherer@us.army.mil 1 March 2010 Clinical Cases

21. UNCLASSIFIED LTC John M. Scherer/(301) 619-8837/john.scherer@us.army.mil 1 March 2010 Reported as a minimum value Technical Memorandum 19, Pathogenesis of Anthrax- A Progress Report, November 1962 United States Army Biological Laboratories, Fort Detrick 1958 Ft Detrick Case

22. UNCLASSIFIED LTC John M. Scherer/(301) 619-8837/john.scherer@us.army.mil 1 March 2010 Inhalation anthrax from Anthrax letters Inhalation anthrax letter cases - All blood samples tested before administration of antibiotics were positive by PCR –Assay sensitivity 1 pg or 167 org (EID 2002; 8: 1178- 1181).........1 org ~6 fg –Sample volume 5 ul –Equates to ~30,000 org/ml of eluate –Specimen processing should “concentrate” sample by a factor of 10-100x Therefore, predicted levels in the blood would be at a minimum between 300-3,000 org/ml Time-to-positive estimates for blood cultures supports an estimate of >1000 org/ml (ave 14.5hrs from collection, n=7)

23. UNCLASSIFIED LTC John M. Scherer/(301) 619-8837/john.scherer@us.army.mil 1 March 2010 Clinical Infectious Diseases 2007; 44:968–71 Legend is incorrect in manuscript, should be ng/ml

24. UNCLASSIFIED LTC John M. Scherer/(301) 619-8837/john.scherer@us.army.mil 1 March 2010 Temporal influence of antibiotic use on survival of inhalation anthrax

25. UNCLASSIFIED LTC John M. Scherer/(301) 619-8837/john.scherer@us.army.mil 1 March 2010 Anthrax survival predictions Wilkening, PNAS 2006;103: 7589–7594 Holty, Ann Intern Med. 2006;144: 270-280 Note: Left graph X axis is day symptoms develop, right graph is day of exposure For left graph symptoms arise at a mean of 4 days following exposure

26. UNCLASSIFIED LTC John M. Scherer/(301) 619-8837/john.scherer@us.army.mil 1 March 2010 Conclusion Diagnosis of inhalation anthrax does not equal death (with appropriate therapy, individuals who are symptomatic for 4 days still have ~50% survival rate) Initiating therapy each day before the onset of fulminate anthrax improves survival by ~10-20% Increasing the particle size increases the ID50, it does not render the material non-infective Widened-mediastinum is present in only approximately 50% of cases and has been observed in non-anthrax cases Individuals seeking medical care are predicted to have detectable levels bacteremia Anthrax toxins are readily detectable at the same time as bacteria appear in the blood

27. UNCLASSIFIED LTC John M. Scherer/(301) 619-8837/john.scherer@us.army.mil 1 March 2010 LTC John M. Scherer Ph.D., M.T. (ASCP) Voice: 301-619-8837 / DSN 343-8837 Email: john.scherer@us.army.mil LTC John M. Scherer Ph.D., M.T. (ASCP) Voice: 301-619-8837 / DSN 343-8837 Email: john.scherer@us.army.mil