Tuesday, June 9, 2015

Live Anthrax Sent to 66 Labs in 19 States, Washington DC and 3 Countries: But Nobody Discovered This for Years? It Strains Credulity.

I will be writing more about the US DoD assertion that up to 66 laboratories in 19 states, the District of Columbia and three countries  (Australia, South Korea and Canada) received live anthrax spores by mistake from an Army lab at Dugway Proving Ground in Utah. The June 4 WSJ (behind a paywall) says the problem has existed for "over a decade".

The story seems improbable to me, since the mistake should have been caught fairly quickly, not many years later.  Scientists who work with potentially deadly organisms generally don't take claims of their inactivation by others, elsewhere, on faith:  they plate them out and incubate them and check whether there is growth, to be sure there are no live spores, before exposing themselves to a deadly agent.  To imply that scientists at 66 laboratories all failed to perform this procedure, which is SOP, strains credulity.

In 2014 it was found that CDC had a similar issue: it was reported that anthrax spores had been 'inactivated' using a chemical procedure that was sufficient for anthrax in its vegetative form, but not in its spore form. This was said to have exposed about 80 people at CDC to potentially aerosolized spores.  The mistake was discovered when plates were left by chance for a week in an incubator, and late growth was discovered.

The story was that after the chemical treatment, spore growth on plates (into vegetative cells) was delayed but not prevented.  The incident exposed several levels of incompetence. Any idiot working with anthrax should have understood that anthrax' niche as a bioweapon stems from its ability to retain growth potential even after being exposed to extremes of pressure, temperature and dryness (dropped from bombs or airplanes), while in spore form. A spore is anthrax in 'suspended animation.' But during active growth, in the vegetative stage, anthrax is very easily killed.  (Though you must be sure none of it forms spores before it is killed.) As a spore, anthrax is inactivated only with great difficulty.  Which is reflected by how it can start to grow, given the right conditions, even after spending one hundred years or more in spore form.

The CDC mishap informed researchers that they should be alert for delayed growth, when anthrax has been exposed to a procedure intended to kill or damage it, so plates testing anthrax growth should be incubated for longer than normal.  

Below are excerpts from 2 abstracts pointing out that irradiation of spores may not always kill them.  Many factors influence the dose of radiation required to kill all spores in a bundle, including whether spores are wet or dry, the type of diluent, the geometry of how spores are packaged together during irradiation, type of packaging and spore concentration. These factors are only partly understood.  Furthermore, bacteria and spores undergo log killing (as opposed to an all-or-nothing process): a low radiation dose allows, for example, 1/1,000 spores to survive, while a higher dose only allows 1/1,000,000 to survive.

But that is why you test your batch to ensure it is fully inactivated before you expose yourself to it. And Dugway would have been required to do so before shipping it out to others.  Dugway produced most of the anthrax that was stored in Bruce Ivins' flask RMR1029.  Dugway has been making large production runs of anthrax for many years. It is not a newcomer to this enterprise.

Also note that when spores are shipped, their packaging within packaging is supposed to prevent any leakage of contents, no matter what type of indignities the package may be subjected to, en route. (There are federal rules that were enacted after leakage many decades ago.) And packages must be tracked carefully as well.

For example:  here is part of an SOP published by University of Pittsburgh investigators on what it takes to approve a method for inactivation of select agents (those microbes, like anthrax, designated as high threat agents for bioterrorism):

Standard Operating Procedure for Obtaining Approval and Safety Testing of a Sample Inactivation Method
  • 1. For all sample inactivation procedures, the investigator  and  his  or  her  staff must  notify the  Team  of the intent to  inactivate  biological  materials  for  removal  from  the  RBL  BSL­3/registered Select Agent facility. 
  • 2. The proposed inactivation procedure must be discussed  either at a strategy meeting prior to initiation of a project or  at  the  investigator’s  standing  monthly  meeting  with  the  Team. 
  • 3. Each proposed inactivation method must be described  in  detail,  along  with  corresponding  safety  testing  procedures that demonstrate the lack of viable infectious material  after the inactivation procedure. 
  • 4. An investigator may use an original inactivation procedure, or  he  or  she may  use an existing inactivation  procedure published elsewhere or developed by another investigator and listed in this SOP. 
  • 5. Once  the  Team  has  been  notified  of  the  inactivation  procedure,  the  investigator  may  proceed  with  performing  safety testing on the inactivated materials. Safety testing is  required  for  both  original  inactivation  procedures  and  for  existing inactivation procedures developed by other investigators or published in the literature. 
  • 6. If  an inactivation method is to  be  used  for more than  one pathogen, safety testing data must be provided for each  pathogen or type of pathogen. 
  • 7. It is  recommended to  use  a  high­ titer  or  concentrated stock of infectious agent in the safety testing to provide the  most rigorous challenge to the inactivation procedure...
1.    2006 Sep;101(3):514-25. 
Spores of Bacillus subtilis: their resistance to and killing by radiation, heat and chemicals.
Setlow P1.
A number of mechanisms are responsible for the resistance of spores of Bacillus species to heat, radiation and chemicals and for spore killing by these agents... Both UV and gamma-radiation also kill spores via DNA damage. The mechanism of spore resistance to gamma-radiation is not well understood, although the alpha/beta-type SASP are not involved...  Given the importance of the killing of spores of Bacillus species in the food and medical products industry, a deeper understanding of the mechanisms of spore resistance and killing may lead to improved methods for spore destruction.

2.    2008 Jul;74(14):4427-33. doi: 10.1128/AEM.00557-08. Epub 2008 May 30

Gamma irradiation can be used to inactivate Bacillus anthracis spores without compromising the sensitivity of diagnostic assays.

Dauphin LA1Newton BRRasmussen MVMeyer RFBowen MD

The use of Bacillus anthracis as a biological weapon in 2001 heightened awareness of the need for validated methods for the inactivation of B. anthracis spores. This study determined the gamma irradiation dose for inactivating virulent B. anthracis spores in suspension and its effects on real-time PCR and antigen detection assays. Strains representing eight genetic groups of B. anthracis were exposed to gamma radiation, and it was found that subjecting spores at a concentration of 10(7) CFU/ml to a dose of 2.5 x 10(6) rads resulted in a 6-log-unit reduction of spore viability... 

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