Before October 12, the NYC Public Health Laboratory (PHL) processed one or two suspected bioterrorism environmental samples per month, utilizing a small Biosafety Level 2 (BSL-2) room with two dedicated personnel. In the year before the attack, the PHL received approximately 10 samples, all of which were hoaxes. The laboratory was set up according to Centers for Disease Control and Prevention (CDC) protocols, and staff were trained by CDC on methods for isolating and identifying bioterrorism agents.

The bioterrorism laboratory consisted of a 400–square-foot area designed at BSL-2+ as described in Biosafety in Microbiological and Biomedical Laboratories (4). Entrance to the laboratory was controlled by proximity card access and monitored 24 hours a day by video cameras. In the space were a biosafety cabinet, a fluorescence/phase-contrast microscope, incubators, freezers and refrigerators, a Wallach/Perkin Elmer Victor Time Resolved Fluorescence instrument (The Perkin-Elmer Corp., Norwalk, CN), computers, and necessary laboratory supplies. This configuration provided a comfortable and controlled access space for sample preparation and analysis. Because of the low sample volume, each sample was treated uniquely, and a generalized method for handling numbers of environmental specimens was not considered necessary.

Before October 12, all specimens submitted to NYC BTRL were tested for four priority bioterrorism agents: B. anthracis (anthrax), Francisella tularensis (tularemia), Yersinia pestis (plague), and Brucella species (brucellosis). Protocols defined and validated by CDC were used to isolate and identify these agents (5). All specimens tested during that time were culture negative for the four priority bioterrorism agents according to the validated protocols.

Before B. anthracis was identified in letter C from media outlet 1, two other letters (A and B) were received and tested by BTRL. Letter A came from media outlet 1, and letter B came from media outlet 2. Letters A and B were tested for the four priority bioterrorism agents and were negative. At the time, the negative result for letter A was somewhat surprising because the patient diagnosed with cutaneous anthrax was employed by media outlet 1. When letter C later arrived at BTRL, it was tested and found to contain a powdery substance that was positively identified as spores of B. anthracis. The discrepancy involving the positive results of letters A and C was soon resolved when it was determined that letter C was actually received before letter A at media outlet 1 but was inadvertently placed in a corporate “hate-mail” file and was thus recovered after letter A.

A number of important events took place almost simultaneously after letter C tested positive for B. anthracis: a) BTRL was contaminated with B. anthracis spores during the sampling process and three BTRL laboratory employees were exposed; b) the news media and U.S. Attorney General John Ashcroft broadcast a message to Americans asking them to report all suspicious mail to their local law enforcement authorities (6); and c) as a result of this increased attention, the sample volume surged and did not abate for another 6 weeks.

These events worked synergistically to complicate NYC DOH’s ability to contend with bioterrorism testing on the scale needed during this crisis. At this time, CDC contacted NYC DOH to offer support and aid. On learning of the situation developing in NYC and the events surrounding the contamination of BTRL, including exposure of employees, the PHL, in conjunction with CDC, instituted several important policies: 1) A Bioterrorism Response Laboratory Command Center was established at PHL to direct and coordinate all bioterrorism laboratory activities and communications; 2) A secure and separate entryway was set up so bioterrorism specimens could enter the PHL building without jeopardizing the safety of PHL building personnel; 3) A separate specimen-receiving area containing a decontamination site was established, and all specimens were doubled bagged and externally decontaminated (sprayed with a bleach solution) before being brought to the testing laboratory for analysis; 4) All environmental bioterrorism specimens were tested by using strict and secure BSL-3 containment and BSL-3 protocols; 5) BTRL personnel exposed in the contaminated laboratory were treated with ciprofloxacin HCl; 6) Extensive infection control and environmental monitoring procedures were set up throughout the PHL building to monitor for B. anthracis spores; 7) Security was extensively increased throughout the building’s interior and exterior; 8) During the transition to the new BSL-3 testing facility, samples received for bioterrorism testing were shipped to offsite level C laboratories for analysis; 9) A dedicated database was developed for accepting and tracking bioterrorism specimens and testing results; and 10) CDC and NYC DOH requested a Department of Defense (DOD) Microbiology Response Team to assist with rapid testing of bioterrorism specimens.

After the initial evaluation, the NYC PHL facility was configured to operate 24 hours a day, accepting, processing, and testing samples. Additional laboratory space was identified, consisting of three separate areas for handling and testing bioterrorism samples (two polymerase chain reaction [PCR] units and an enzyme immunoassay [EIA] rapid screening unit). The BTRL coordinator was also appointed to work in conjunction with CDC and DOD teams. Staffs from other units were also redeployed to further assist in the bioterrorism response effort.

Within days of the initial event on October 12, all the essential elements of BTRL were in place. Table 1 describes the transition before and after October 12. Both the types of laboratory activities and their scale changed dramatically. The sample volume increased approximately 3,000 times for both environmental and clinical testing. Not surprisingly, the number of laboratories and ancillary spaces BTRL required increased almost twentyfold, and 25 times more personnel than originally envisioned staffed these additional areas. New instrumentation (i.e., the PCR rapid assays) was brought into BTRL to attempt to process the sample volume more quickly. To supply this dramatic surge, six tons of equipment and supplies was needed. The scale of the operation and the tracking needs threatened to overwhelm the support staff, and a hastily constructed but workable database system was put into place.

Figure 1

Figure 1. Depiction of the algorithm used to determine the priority of items received for testing at the New York City Bioterrorism Response Laboratory. One of the salient features of the surge was...

Figure 2

Figure 2. Diagrammatic tracking of an environmental sample through the various units and laboratories as it was processed and tested for anthrax at the New York City Bioterrorism Response Laboratory. The first level...

This sample volume surge was expected to be specimens of human origin (clinical specimens); the need for large-scale environmental sampling and testing had not been anticipated. The clinical laboratories experienced exponential increases in volume but had enough latent capacity to handle the increased workload. Increases in coverage and overtime, plus additional reagents, sufficed to contain the testing volume within manageable limits. Clinical sample processing and tracking were not adversely affected, but environmental sampling was severely hampered. The original testing laboratory was never designed to handle more than perhaps a few samples per day. On the first day of the surge, the laboratory received 34 samples that were considered high priority (Figure 1). Figure 2 shows the flow of a sample as it enters the BTRL. The laboratory can be divided into three main functional entities: 1) a receiving area, which contains a decontamination site in processing area and a secured temporary storage facility; 2) two sampling areas (one each of BSL-2 and BSL-3), containing facilities to unwrap and examine environmental samples and retrieve samples for further analysis (BSL-3), clinical microbiology laboratories, and the PCR laboratories; and 3) locked and guarded storage for samples that had completed the testing protocol and were ready for subsequent distribution as waste, returnable property, or evidence.

Figure 3

Figure 3. Depiction of the data flow at the New York City Bioterrorism Response Laboratory adopted soon after the surge of isolates after the bioterrorism attack. An access database was developed, and a...

Samples were tracked through the system by a specially designed database that reflected the testing status of the sample and its final report status. A large portion of the database was devoted to description and demographics (Figure 3). Table 2 shows a section of a typical spreadsheet.

All environmental samples entered the building through the designated bioterrorism intake area. The main function of this area was to provide decontamination, documentation, and security. Samples would be accepted only from designated first responders and law enforcement personnel. Although standard protocols now ensure that the samples brought in for laboratory testing are not externally contaminated with a bioterrorism agent (7), as a prudent preventive measure the outer packaging still needed to be decontaminated in the intake area. A breach in any procedure could compromise the laboratory.

Chain-of-custody documentation was maintained in the intake unit as well as initial entry into the database. All packages came with a test request/manifest document with the data entered and manually maintained at the intake area. Security (provided by NYC DOH Police Department) were present in the area continuously. After passing through decontamination and receiving, packages were held in a nearby temporary storage area until requested by the sampling or testing laboratories.

Testing Protocols and Reporting Algorithm<H2>

All testing protocols were adapted from established protocols (8). In short, samples were analyzed by using a rapid screening assay (PCR) to provide preliminary information to health-care providers and law enforcement. However, final disposition of samples was only made after exhaustive identification according to recommended microbiology protocols.

Figure 4

Figure 4. Chart tracking the time needed to report the status of a sample brought in for classical Bacillus anthracis testing at the New York Bioterrorism Response Laboratory. Negative samples with no suspicious...

Figure 4 outlines the workflow through the analytical units. Clinical samples were generally directly plated onto SBA. The environmental samples often were simultaneously transferred into BHIB and heat shocked to kill nonsporulating organisms and enrich for B. anthracis spores. A sample was reported as positive only if it had all the following phenotypes: nonmotile; penicillin sensitive; γ-phage positive; and positive by both cell wall and capsule direct fluorescent-antibody assay. Extensive environmental monitoring was performed on the reports before they were released. All negative clinical reports were compiled into a manifest and sent to the Mayor’s Office of Emergency Management, where they were distributed to the appropriate parties.

At the peak of the surge, BTRL was testing 60–100 samples per 24-hour shift. Each sample required, at a minimum, duplicate PCR and an SBA culture. Any growth required the phenotypic testing described above. Most samples were also transferred to BHIB; growth in that medium required phenotypic analysis. The average sample, even if it resulted in a negative finding, required at least 14 separate testing procedures to determine its status.

Processing specimens sampled in the BSL-3 required 30–60 minutes and limited the flow to the microbiology laboratories. Despite any effort to speed testing through the microbiology laboratories, the limiting factor remained the maximal BSL-3 throughput of approximately 40 samples per 24 hours.

The events of September 11, 2001, placed New York City on high alert immediately (11). On the heels of this tragedy, the City became the target of a bioterrorism attack (12). NYC DOH, as part of the city’s emergency response network, was extensively involved with the mitigation of both these catastrophes. The laboratory had recent experience in public health emergencies such as the West Nile virus outbreak (13) and the 1999 bottled-water scare.

Although PHL had chain-of-custody experience through its Toxicology and Environmental Laboratories and outbreak testing during the West Nile outbreak, nothing could have prepared the laboratory for the events of October 2001. Nevertheless, staff outfitted the laboratory within days to accept, test, report, store, and return evidence from thousands of environmental and clinical samples.

In the months after the crisis, BTRL still receives about five suspicious samples per week. Samples are now routinely tested for the four priority agents, and plans have been finalized for dedicated laboratory space designed by using the lessons learned from October 2001.

Nevertheless, before October 2001, we thought we were prepared to confront an event on the scale of this bioterrorism attack. An important lesson from this experience is that, despite all additional precautions and enhancements made to the laboratory and the response network, another attack, if and when it occurs, will present further surprises. While the laboratory has now institutionalized weapons of mass destruction testing to be performed as part of routine surveillance (e.g., testing of drinking water), potential means and targets for future attacks cannot be perfectly forecast. Vigilance and continued emphasis on flexibility, creativity, and the ability to rapidly expand our response, as needed, to bioterrorism events and the surprises they present will determine our effectiveness and ultimate success.