4
Diagnostics, Therapeutics, and Other Technologies to Control SARS

OVERVIEW

The strong possibility that SARS will return is being addressed by multiple sectors, including public health planners preparing for a broad range of challenges and contingencies (see also Chapter 1); researchers developing clinical diagnostics and technologies for infection control, as well as antiviral drugs and vaccines; and epidemiologists searching for clues from the recent SARS epidemic that could prevent a future outbreak or reduce its impact. Each of these perspectives is discussed in this chapter.

The development of a diagnostic test to rapidly detect SARS in its early stages is a top research priority. Because researchers do not know which tissues contain the highest concentrations of virus in the presymptomatic stages of infection, this task is particularly challenging. Reverse-transcription polymerase chain reaction (RT-PCR), a method to detect viral nucleic acids, is considered to be a likely platform for early SARS testing due to its high analytical sensitivity and speed. An evaluation of two RT-PCR protocols presented in this chapter found them to be highly specific for the SARS coronavirus; however, the tests were determined to be insufficiently sensitive to reliably detect the virus in respiratory specimens. Without a clinical diagnostic test, suspected cases of SARS must be confirmed in the laboratory, using RT-PCR or slower methods of detection—involving serology or viral culture, isolation, and identification by electron microscopy—thereby causing a significant increase in the time required for an accurate diagnosis.



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Learning From Sars: Preparing for the Next Disease Outbreak - Workshop Summary 4 Diagnostics, Therapeutics, and Other Technologies to Control SARS OVERVIEW The strong possibility that SARS will return is being addressed by multiple sectors, including public health planners preparing for a broad range of challenges and contingencies (see also Chapter 1); researchers developing clinical diagnostics and technologies for infection control, as well as antiviral drugs and vaccines; and epidemiologists searching for clues from the recent SARS epidemic that could prevent a future outbreak or reduce its impact. Each of these perspectives is discussed in this chapter. The development of a diagnostic test to rapidly detect SARS in its early stages is a top research priority. Because researchers do not know which tissues contain the highest concentrations of virus in the presymptomatic stages of infection, this task is particularly challenging. Reverse-transcription polymerase chain reaction (RT-PCR), a method to detect viral nucleic acids, is considered to be a likely platform for early SARS testing due to its high analytical sensitivity and speed. An evaluation of two RT-PCR protocols presented in this chapter found them to be highly specific for the SARS coronavirus; however, the tests were determined to be insufficiently sensitive to reliably detect the virus in respiratory specimens. Without a clinical diagnostic test, suspected cases of SARS must be confirmed in the laboratory, using RT-PCR or slower methods of detection—involving serology or viral culture, isolation, and identification by electron microscopy—thereby causing a significant increase in the time required for an accurate diagnosis.

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Learning From Sars: Preparing for the Next Disease Outbreak - Workshop Summary This chapter also includes a description of an alternative diagnostic platform—the mass spectroscopic identification of microbial nucleic acid signatures—that can be adapted to detect the SARS coronavirus. Using technology originally designed for the environmental surveillance of biowarfare agents, this platform could potentially identify the SARS virus directly from a patient sample, obviating the need for time-consuming viral culture. This method is designed to distinguish between SARS and other coronaviruses, and perhaps even between genetic variants of the SARS virus; however, direct comparisons of sensitivity between this and other SARS detection systems using patient samples have yet to be conducted. Several workshop participants expressed concern about the limited capacity in health care systems—particularly related to workforce and facilities shortages—that present a significant barrier to preparations for SARS and other threats to public health. It was suggested at the workshop by Jerome Schentag that this situation might be mitigated in some degree through the use of flexible approaches to isolating SARS patients. One such approach, discussed in this chapter, is a mobile technology that destroys viral particles and droplets in the air. These mobile units, by isolating individual patients being transported to and within hospitals, potentially could be used to protect staff during high-risk procedures such as intubation or bronchoscopy, to decontaminate larger areas such as hospital waiting rooms or airplanes, and to create air exchange systems for isolation facilities or areas within hospitals. Importantly however, it was noted during the workshop that the technologies described here must be thoroughly evaluated to determine their suitability for containing SARS in a variety of clinical settings before they are recommended for use. Research has proceeded rapidly to develop antiviral drugs and vaccines to combat SARS. Previous antiviral discovery efforts by researchers at Pfizer on the human rhinovirus protease 3C—a functional, genetic, and structural analog to a key SARS coronavirus protease that has therefore been named “3C-like” (3CL)—are recounted in this chapter. This knowledge has aided in a search for 3CL protease inhibitors, a project undertaken by Pfizer in collaboration with scientists at the National Institute of Allergy and Infectious Diseases and the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID). Several candidate inhibitors have been selected by bioassay and are currently being evaluated for clinical development, while others are being sought through alternative strategies such as structure-based design and combinatorial chemistry. A vaccine for SARS—even if steered along a highly streamlined route to development—might still postdate a return of SARS, perhaps by several years. Nevertheless, because the medical need for developing such a vaccine and/or effective antiviral drugs is perceived to be acute, several pharmaceutical and biotechnology companies have taken up this challenge.

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Learning From Sars: Preparing for the Next Disease Outbreak - Workshop Summary EVALUATION OF REVERSE TRANSCRIPTION-PCR ASSAYS FOR RAPID DIAGNOSIS OF SEVERE ACUTE RESPIRATORY SYNDROME ASSOCIATED WITH A NOVEL CORONAVIRUS W.C. Yam, K.H. Chan, L.L.M. Poon, Y. Guan, K.Y. Yuen, W.H. Seto, and J.S.M. Peiris Department of Microbiology, Queen Mary Hospital, The University of Hong Kong, Hong Kong, People’s Republic of China Reprinted with permission, American Society for Microbiology. Copyright 2003, American Society for Microbiology. All Rights Reserved. The reverse transcription (RT)-PCR protocols of two World Health Organization (WHO) severe acute respiratory syndrome (SARS) network laboratories (WHO SARS network laboratories at The University of Hong Kong [WHO-HKU] and at the Bernhard-Nocht Institute in Hamburg, Germany [WHO-Hamburg]) were evaluated for rapid diagnosis of a novel coronavirus (CoV) associated with SARS in Hong Kong. A total of 303 clinical specimens were collected from 163 patients suspected to have SARS. The end point of both WHO-HKU and WHO-Hamburg RT-PCR assays was determined to be 0.1 50 percent tissue culture infective dose. Using seroconversion to CoV as the “gold standard” for SARS CoV diagnosis, WHO-HKU and WHO-Hamburg RT-PCR assays exhibited diagnostic sensitivities of 61 and 68 percent (nasopharyngeal aspirate specimens), 65 and 72 percent (throat swab specimens), 50 and 54 percent (urine specimens), and 58 and 63 percent (stool specimens), respectively, with an overall specificity of 100 percent. For patients confirmed to have SARS CoV and from whom two or more respiratory specimens were collected, testing the second specimen increased the sensitivity from 64 and 71 percent to 75 and 79 percent for the WHO-HKU and WHO-Hamburg RT-PCR assays, respectively. Testing more than one respiratory specimen will maximize the sensitivity of PCR assays for SARS CoV. A global outbreak of a new emerging illness, severe acute respiratory syndrome (SARS), was associated with a novel coronavirus, SARS CoV (Lee et al., 2003; Peiris et al., 2003a; Tsang et al., 2003). By the end of April 2003, more than 1,500 patients were diagnosed with SARS in Hong Kong. Transmission within hospitals was a major contributor to disease amplification. Rapid laboratory confirmation of SARS CoV infection was important for managing patient care and for preventing nosocomial transmission. While serological testing was reliable as a retrospective diagnostic method, diagnosis of the infection in the early phase of the illness was important for patient care. The identification of the etiological agent and its partial gene sequence data made it possible to develop molecular diagnostic methods for SARS CoV (Drosten et al., 2003; Peiris et al., 2003b). The protocols were made available through the WHO website (http://www.who.int/csr/sars/primers/en). This study evaluates two of the first-generation reverse transcription (RT)-PCR assays that were used during this outbreak.

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Learning From Sars: Preparing for the Next Disease Outbreak - Workshop Summary Materials and Methods Patients and Specimen Collection Specimens were available for 163 patients who presented with clinically suspected SARS according to the WHO definition (WHO, 2003) and who were admitted to three acute regional hospitals in Hong Kong between 26 February and 17 April 2003. For each patient, paired acute- and convalescent-phase serum samples and at least one respiratory specimen were collected for study. A total of 303 specimens (124 nasopharyngeal aspirate specimens, 65 throat swab specimens, 95 urine specimens, and 19 stool specimens) were available for study. Respiratory specimens were collected between days 1 and 5 after admission, whereas urine and stool specimens were collected between days 5 and 10. The acute-phase sera were collected in the first week of illness, and the convalescent-phase sera were collected 21 days after the onset of clinical symptoms. Nasopharyngeal aspirate specimens were assessed by rapid direct immunofluorescent antigen detection for influenza virus A and B, para-influenza virus types 1, 2, and 3, respiratory syncytial virus (RSV), and adenovirus as described previously (Chan et al., 2002). Paired serum samples were assayed for increasing titer against CoV. Nasopharyngeal aspirate and stool specimens from patients suffering from unrelated diseases were collected as controls. Extraction of CoV RNA Nasopharyngeal aspirate and throat swab specimens were suspended in viral transport medium. Urine specimens were transported in sterile containers. Stool specimens were mixed in viral transport medium (diluted 1:10) and microcentrifuged at 10,000 × g for 1 min, and supernatant was collected. Viral RNA was extracted from 140 μl samples using a Qiagen viral RNA mini kit (Qiagen, Hilden, Germany). The initial processing of specimens was performed under biohazard level 2 containment conditions. After lysis of the sample by the lysing buffer, the mixture was applied to a spin column as described by the manufacturer. The extracted RNA was eluted in a total volume of 50 μl of RNase-free water before RT-PCR amplification. RT-PCR Amplification The RT-PCR protocols of two WHO SARS network laboratories (Table 4-1) were evaluated in this study. The WHO SARS network laboratory at the University of Hong Kong (WHO-HKU) used a single RT step to synthesize cDNA, followed by subsequent PCR amplification with specific primers in another reaction tube (Peiris et al., 2003a). The WHO SARS network laboratory at the Bernhard-Nocht Institute in Hamburg, Germany (WHO-Hamburg) used a single

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Learning From Sars: Preparing for the Next Disease Outbreak - Workshop Summary TABLE 4-1 RT-PCR Protocols for Rapid Diagnosis of CoV Associated with SARSa Characterisitic or component of protocol RT WHO-HKU PCR RT-PVR Second PCR Primer sequences   TACACACCIFCAGCGTTG CACGAACGTIGACGAAT ATGAATTACCAAGTCAATGGTTAC CATAACCAGTCGGTACAGCTAC GAAGCTATfCGTCACG CTGTAGAAAATCCTAGCTGGAG Sense   Antisense Reagent formulation Superscript II RTA ( Invitrogen) (i) 4 μl of 5x first-strand buffer (ii) 10 mM DTT (iii) 500 μMdNTP (iv) 0.15 μg of random primer (v) 200 U of Superscript II (vi) 12 μl of RNA extract (vii) Make up total volume of 20 μl AmpliTaq Gold (Roche) (i) 5 μl of 10x reaction buffer (ii) 200 μM dlU (iii) 2.5 μM MgSO4 (iv) 250 nM (each) primer (v) 2 U of AmpliTaq Gold (vi) 2 μl of RT product (vii) Make up total volume of 50 μl Superscript II RT-PCR (Invitrogen) (i) 10 μl of 2x reaction buffer (ii) 2.45 mM MgSO4 (iii) 500 μM (each) primer (iv) 0.4 μl of RTA-Taq mixture (v) 2 μl of RNA extract (vi) Make up total volume of 20 μl AmpliTaq Gold (Roche) (i) 5 μl of 10x reaction buffer (ii) 200 μM dNTP (iii) 2.5 μM MgSo4 (iv) 200 nM (each) primer (v) 2 U of AmpliTaq Gold (vi) 1 μl of RT-PCR product (vii) Make up total volume of 50 μl Thermal cycling profile (i) 25°C, 10 min (ii) 42°C, 50 min (iii) 94°C, 3 min (i) 94°C, 10 min (ii) 40 cycles (a)94°C, 30 s (b) 50°C, 40 s (c) 72°C, 15 s (iii) 72°C, 10 min (i) 45°C, 30 min (ii) 95°C, 3 min (iii) 10 cycles (a) 95°C, 10 s (b)60°C, 10 s (decrease by 1°C/cycle) (c) 72°C, 20 s (iv) 40 cycles (a) 95°C, 10 s (b) 56°C, 10 s (decrease by 1°C/cycle) (c) 72°C, 20 s (i) 95°C, 5 min (ii) 10 cycles (a)95°C, 10 s (b) 60°C, 10 s (decrease by 1°C/cycle) (c) 72°C, 30 s (iii) 20 cycles (a) 95°C, 10 s (b) 56°C, 10 s (c) 72°C, 30 Expected PCR product size (bp)   182 189 108 aThe RT-PCR protocols of two WHO SARS network laboratories. WHO-HKU (Peiris et al., 2003a) and WHO-Hamburg (Drosten et al., 2003) are also available online (http://www/who.int/esr/sars/primers/en). Abbreviations: RTA, reverse transcriptase; DDT, dithiotheritol; dNTP, deoxynucleoside triphosphate.

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Learning From Sars: Preparing for the Next Disease Outbreak - Workshop Summary RT-PCR step, followed by transfer of the initial PCR products to the nested PCR amplification mixture (Drosten et al., 2003). Positive and negative controls were included in each run, and all precautions to prevent cross-contamination were observed. For nested PCR, RT-PCR amplicon tubes were spun (in pulses) before the tubes were opened using separate Eppendorf tube openers for transferring RT-PCR products to the nested PCR mix. Negative control was incorporated for every five nested PCRs to monitor cross-contamination. Amplified products were electrophoresed through a 2 percent agarose gel in Tris-borate buffer. Target bands were visualized by staining with ethidium bromide. CoV Immunoglobulin G Serology Smears of CoV-infected Vero cells were prepared, fixed in acetone for 10 min, and stored at –80°C before use (Peiris et al., 2003a). Each batch of SARS CoV-infected cell smears with 60 to 70 percent infected cells was prepared and tested with a high-titer, positive-control serum sample from a confirmed SARS patient as a standard to assess sensitivity and batch-to-batch variations. Serial twofold dilutions starting with a 1:10 dilution of each patient serum sample were added to the smears and incubated for 30 min at 37°C. After two 5-min washes in phosphate-buffered saline, fluorescein isothiocyanate-conjugated goat anti-human immunoglobulin G (INOVA Diagnostics, Inc., San Diego, California) was added to the smears, and the smears were incubated for 30 min at 37°C. Acute- and convalescent-phase serum samples from each patient were assayed for SARS CoV antibodies in the same experiment to minimize experimental variations. The titer was determined as the highest dilution of serum exhibiting fluorescence of the infected cells. A weakly positive patient serum sample was included as a control in each run. A sample was scored as a positive result if the fluorescent intensity was equal to or higher than that of the positive control. Determination of the End Points of the RT-PCR Assays A 96-well microtiter plate containing 0.1 ml of confluent Vero cells was used to determine the 50 percent tissue culture infective dose (TCID50) of SARS CoV under biohazard level 3 containment conditions. Tenfold serial dilutions of a cell-adapted SARS CoV strain from 10–1 to 10–8 were prepared. One hundred microliters of each dilution were added to each well of four replicate wells and incubated at 37°C for 2 to 3 days to observe cytopathic effect. TCID50s were determined by the Kärber method (Ballew, 1992). For the same serial dilutions of virus, 100-μl samples were subjected to RNA extraction, and the end points of the two RT-PCR assays were determined.

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Learning From Sars: Preparing for the Next Disease Outbreak - Workshop Summary Results Of 303 specimens from clinically suspected SARS cases (see Table 4-2), 145 were positive by one or both PCR assays and more than 87 percent of PCR-positive samples were identified by both PCR assays. Common respiratory viral pathogens, including influenza virus A and B, parainfluenza virus types 1, 2, and 3, RSV, and adenovirus, were not detected in the 124 nasopharyngeal aspirate specimens. The end point for both WHO-HKU and WHO-Hamburg RT-PCR methods was determined to be 0.1 TCID50. The acute-phase serum samples from all patients were seronegative for SARS CoV. Eighty-six patients were confirmed to have SARS CoV infections on the basis of seroconversion. Using seroconversion as the gold standard for SARS diagnosis, the sensitivities of the WHO-HKU and WHO-Hamburg RT-PCR assays were found to be 61 and 68 percent (nasopharyngeal aspirate specimens), 65 and 72 percent (throat swab specimens), 50 and 54 percent (urine specimens), and 58 and 63 percent (stool specimens). A specificity of 100 percent was exhibited by both RT-PCR assays, as none of the seronegative patient samples and control samples gave a positive PCR result. Among the 163 patients, two or more respiratory specimens (nasopharyngeal aspirate or throat swab specimens) were available from 41 patients. Of the 41 patients, 28 were subsequently confirmed to have SARS CoV on the basis of seroconversion. In these 28 patients, the numbers of first specimens positive for WHO-HKU and WHO-Hamburg RT-PCR were 18 and 20, respectively, but testing a second specimen increased the overall sensitivity from 64 and 71 percent to 75 and 79 percent, respectively. Discussion In Hong Kong, SARS is a serious respiratory illness that led to significant morbidity and mortality (Donnelly et al., 2003). The diagnosis depends mainly on the clinical findings of an atypical pneumonia not attributed to another cause and a history of exposure to a suspect or probable case of SARS or to the respiratory secretions and other bodily fluids of individuals with SARS. Definitive diagnosis of this novel CoV relies on classic tissue culture isolation, followed by electron microscopy studies to identify the virus on cell culture, which is technically very demanding. Serological testing for increasing titer against SARS-associated CoV was shown to be highly sensitive and specific (Peiris et al., 2003a) but was not suitable for rapid laboratory diagnosis. The rapid isolation and characterization of the novel CoV associated with SARS allowed for the timely development of diagnostic tests (Marra et al., 2003; Rota et al., 2003). RT-PCR protocols of two WHO SARS network laboratories were evaluated for rapid diagnosis of SARS-associated CoV in Hong Kong. The end point for the novel CoV by both RT-PCR assays was similar to the previous finding for human CoV (Vabret et al., 2001), yet sufficient diagnostic sensitivity was not achieved, despite attaining a

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Learning From Sars: Preparing for the Next Disease Outbreak - Workshop Summary TABLE 4-2 Performance of RT-PCR Assays for Rapid Detection of CoV Associated with SARS   No. of specimens positive by RT-PCR assay Specimens (no.) No. of specimens tested Seroconversiona WHO-HKU WHO-Hamburg Both WHO-HKU and WHO-Hamburg Clinically suspected SARS   Nasopharygneal aspirate specimens (124) 72 + 44 49 43   52 – 0 0 0 Throat swab specimens (65) 54 + 35 39 33 Urine specimens (19) 78 + 39 42 39 Stool specimens 19 + 11 12 11 Controls   Nasopharygneal aspirate specimens 22b ND 0 0 0 Stool specimen 21c ND 0 0 0 aA fourfold rise of more in antibody titer against CoV was considered seroconversion (+). ND, not done. bSamples positive for other viral pathogens included nine samples positive for influenza virus A, one sample positive for influenxa virus B, six samples positive for adenovirus, and six samples positive for RSV by immunoflourescence (Chan et al., 2002). cNo intestinal pathogens detected.

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Learning From Sars: Preparing for the Next Disease Outbreak - Workshop Summary specificity of 100 percent. A recent study using real-time RT-PCR revealed that the viral load in nasopharyngeal aspirate specimens peaked in the second week of the illness (Peiris et al., 2003b). Results indicated a more sensitive RT-PCR assay is essential for rapid diagnosis of SARS CoV during the early stage of disease. Due to the nature of respiratory specimens with inconsistent pathogen loads at various sample times, testing of multiple specimens has been shown to increase the sensitivity of laboratory diagnosis for Mycobacterium tuberculosis (Nelson et al., 1998). Testing a second respiratory specimen by RT-PCR increased the sensitivity of diagnosis for SARS CoV. The examination of more than one respiratory specimen is necessary to maximize the sensitivity of RT-PCR assays for SARS CoV. As molecular characterization of this novel CoV is ongoing, targeting genomic segments of the virus for diagnostic application is still unclear. Amplification of a second genome region may further increase test specificity. In this study, the high specificity and concordance of both RT-PCR assays verified that the amplified genomic segments for both protocols are suitable for diagnostic application. Incorporation of internal probe hybridization will probably increase the sensitivity of the WHO-HKU RT-PCR assay. In this global outbreak of SARS, prompt communication and exchange of information among the WHO collaborating laboratories facilitate development of rapid diagnostic assays with shortened turnaround time. The availability of the protocols on the WHO website was helpful to diagnostic laboratories. The collaborative approach can be invaluable in our efforts to understand and control emerging pathogens in the future. Acknowledgments We thank Christian Drosten of the Bernhard-Nocht Institute (Hamburg, Germany) and TIB-MOLBIOL (Hamburg, Germany) for providing DNA primers used in the WHO-Hamburg RT-PCR protocol. We also thank the staff of the Department of Microbiology, Queen Mary Hospital, The University of Hong Kong for their technical assistance. NOVEL BIOSENSOR FOR INFECTIOUS DISEASE DIAGNOSTICS Rangarajan Sampath and David J. Ecker Ibis Therapeutics, a division of Isis Pharmaceuticals We describe a novel approach for surveillance of emerging infectious diseases that can be used for rapid and broad identification of infectious disease causative agents. The premise of our technology is that we can provide rapid, sensitive, and cost-effective detection of a broad range of “normal” pathogenic organisms and simultaneously also diagnose disease caused by a biological weapon or an unexpected emerging infectious organism. This broad-function

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Learning From Sars: Preparing for the Next Disease Outbreak - Workshop Summary technology may be the only practical way to rapidly diagnose diseases caused by a bioterrorist attack or emerging infectious diseases that otherwise might be missed or mistaken for a more common infection. According to a recent review (Taylor et al., 2001), more than 1,400 organisms are infectious to humans. These numbers do not include numerous strain variants of each organism, bioengineered versions, or pathogens that infect plants or animals. Paradoxically, most of the new technology being developed for detection of infectious agents incorporates a version of quantitative PCR, which is based on the use of highly specific primers and probes designed to selectively detect specific pathogenic organisms. This approach requires assumptions about the type and strain of bacteria or virus. Experience has shown that it is very difficult to anticipate where the next emerging infectious agent might come from, as was the case with the outbreak of SARS early in 2003. An alternative to single-agent tests is to do broad-range consensus priming of a gene target conserved across groups of organisms (Kroes et al., 1999; Oberste et al., 2000, 2001, 2003). The drawback of this approach for unknown agent detection and epidemiology is that analysis of the PCR products requires the cloning and sequencing of hundreds to thousands of colonies per sample, which is impractical to perform rapidly or on a large number of samples. New approaches to the parallel detection of multiple infectious agents include multiplexed PCR methods (Brito et al., 2003; Fout et al., 2003) and microarray strategies (Wang et al., 2002, 2003; Wilson et al., 2002). Microarray strategies are promising because undiscovered organisms might be detected by hybridization to probes on the array that were designed to bind conserved regions of previously known families of bacteria and viruses. Here we present an alternative, a universal pathogen-sensing approach for high-throughput detection of infectious organisms that is capable of identifying previously undiscovered organisms (see Figure 4-1). Our strategy is based on the principle that, despite the enormous diversity of microbes, all forms of life on earth share sets of essential common features in the biomolecules encoded in their genomes. Bacteria, for example, have highly conserved sequences in a variety of locations on their genomes. Most notable is the universally conserved region of the ribosome, but there are also conserved elements in other noncoding RNAs, including RNAse P and the signal recognition particle, among others. There are also conserved motifs in essential protein-encoding genes, in bacteria as well as viruses. Use of such broad-range priming targets across the broadest possible grouping of organisms for PCR, followed by electrospray ionization mass spectrometry for accurate mass measurement, enables us to determine the base composition (numbers of A, G, C, and T nucleotides) of the PCR amplicons. The measured base compositions from strategically selected locations of the genome are used as a signature to identify and distinguish the organisms present in the original sample. An important feature of the primer design strategy used in our approach is the positioning of propynylated

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Learning From Sars: Preparing for the Next Disease Outbreak - Workshop Summary FIGURE 4-1 Overview of the universal pathogen sensor for the detection of a diverse mixture of microbial organisms present in a sample. Genomic DNA, or cDNA obtained by batch reverse-transcription of RNA, from each sample are amplified using broad range PCR primers to generate a complex mixture of PCR products. This mixture of DNA is directly sprayed into a mass spectrometer that essentially weighs each intact nucleic acid strand in the mixture at the same time, This measurement is done at high mass accuracy, which enables us to calculate the exact number of A’s, C’s, G’s, and T’s that make up the DNA in our sample. This count serves as a base-composition fingerprint that can be mapped back to specific organisms. Examination of multiple base-count fingerprints for each organism generated by multiple pairs of broad-range primers to conserved sites distributed across the microbial genome (not shown) allows discrimination of microbial species and subspecies with great accuracy.

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Learning From Sars: Preparing for the Next Disease Outbreak - Workshop Summary FIGURE 4-6 The Model 07. Coverage during bronchoscopy or other aerosol-generating procedures. Removal of toxic smoke or fumes. The FASS Medical Isolation Units offer the following benefits: Minimal set-up time to respond immediately to an emergency situation.

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Learning From Sars: Preparing for the Next Disease Outbreak - Workshop Summary Dual-use flexibility to provide isolation containment (negative pressure enclosure) at any place at any time. A system that does not alter the infrastructure within the enclosed protective area. A cost-effective solution to emergency isolation. Clean air for extended use. FASS Applications The FASS Medical Isolation Units are fume hoods on wheels that combine the proven HEPA filter capacity of 99.97 percent capture at 0.1 microns with ultraviolet light. This toxic microbial capture and containment system builds on years of proven studies specifically involving Bacillus anthracis (anthrax) and smallpox, and can readily be applied to infection control of SARS-related incidents. These units are approved by the Food and Drug Administration (FDA) and satisfy CDC guidelines for isolation. They are the only FDA-approved portable isolation units currently on the market. SARS Response: Deployment Considerations FailSafe Medical Isolation Units can be deployed in several ways as a response to a suspected SARS incident: Immediate isolation and evacuation of a suspected SARS patient. Transport of infected patients through crowded population (e.g., airports, train stations). Transport to hospital or triage area. Transport within hospital (from emergency room to SARS isolation floor). Emergency workers can provide isolation and unrelated medical treatment to suspected SARS patients within the confines of the Medical Isolation Units while protecting caregivers and the healthy population. Bedridden patients showing symptoms of SARS can be quarantined immediately without having to be moved to another room or facility. System Description Both of these FASS Isolation Units combine HEPA filtration with UVGI irradiation. The units consist of a mobile platform that allows the patient to sit in a mobile chair or a bed that is surrounded by a plastic curtain. The outside air is drawn under the curtain, across the patient, and then up into the air-purifying system that consists of a HEPA filter and a UVGI lamp, thereby reducing infectious aerosols such as tuberculosis and SARS.

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Learning From Sars: Preparing for the Next Disease Outbreak - Workshop Summary The FailSafe Mobile Containment System is a patented process (U.S. Patent No. 6,162,118 [18 December 2000] entitled “Portable Isolation Device and Method”) that integrates the technologies of filtration, ultraviolet germicidal irradiation, and ozone oxidation. The FailSafe process primary technology is based on high-efficiency filtration using a glass fiber HEPA filtration media that collects and traps particles greater than 0.1 micron with an efficiency greater than 99.97 percent. The filtration will collect most biological pathogens, including fungi, bacteria, and encapsulated viruses. To ensure that the pathogens collected and trapped on the HEPA filter are neutralized, the HEPA filter media surface face is illuminated with ultraviolet germicidal irradiation. Another advantage of illuminating both faces of the HEPA filter is that viruses smaller than 0.1 micron will be neutralized by irradiation. FailSafe Mobile Containment Systems (NOT the medical Model 77 or 07 units) also incorporate ozone generation capability as a third technology. Ozone is generated with the use of ultraviolet (UV) lamps that will convert atmospheric oxygen into ozone. At concentrations below NIOSH limits, the ozone will chemically react with volatile organic compounds or odor. The FailSafe Mobile Containment Systems also have the capability of generating very high ozone levels that can be used for neutralizing pathogens on surfaces such as walls, ceilings, and floors. Setup and Operation The Medical Isolation Units for health care are designed with operational simplicity to make it a “turnkey” operation and to allow health providers to focus on the individual patient and the biological contamination itself. The units are designed for easy use with three switches, and the controls are simple, as follows: Power up the system. Check to see that the system is working properly and that the operation light is on. Turn the FASS system ON and select the appropriate fan speed to begin air scrubbing, treatment, and capture. Identify suspected infected patient. Place patient in Model 07 chair, or encompass sickbed under Model 77 unit. Place plastic curtains around patient. Preliminary Efficacy Testing Laboratory testing: FDA 510k application. The HEPA filtration and UVGI irradiation components used in the FASS units are incorporated in Model 07 and Model 77 to protect medical personnel transporting TB and other infectious patients. Preliminary laboratory testing was performed on these units by an independent laboratory for FDA Class II certification.

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Learning From Sars: Preparing for the Next Disease Outbreak - Workshop Summary Discussion of Biological Efficacy Filtration HEPA filters. The safety and health protection offered by HEPA (High-Efficiency Particulate Air) filtered fume hoods has long been established by the FDA, CDC, Environmental Protection Agency (EPA), NIOSH, ASTM, and JCAHO. HEPA Filtration is the “Best Available Control Technology” at 99.99 percent at 0.3-micron efficiency level and is “Generally Accepted Control Technology” at 99.97 percent at 0.1-micron efficiency level. The added feature of the new 0.1-micron advanced filters is the “gel” seal and micro fiberglass construction that allows combining these filters with UV light disinfection. HEPA filters combined with charcoal and prefilters are the highest approved filters available for NIOSH-certified respirators. There are no adverse safety, health, or environmental aspects to HEPA filters. HEPA filters are now the primary filtration media for electronic clean room assembly, hospital surgery rooms, bioengineering, pharmaceutical processes, and any applications where maximum reduction or removal of submicron particulates is required. Air from HEPA filters is free of 99.99 percent of all particles larger than 0.3 microns (including bacterial, fungal, and other opportunistic microbiological organisms) according to the size exclusion as described in Table 4-5. Generally, HEPA filters belong to the “interception” family of filters and are variously referred to as “absolute” or “super interception.” Such filters have a deep bed of randomly positioned fibers in which the total bed depth is very large in comparison to the average fiber diameter and effective pore or free-path cross-sectional area. Even though the media may be only 1/16 thick, this is an enormous distance compared to the 0.3- to 1.0-micron fiber diameter. The passage through which air must flow is not straight, but full of twists and turns. As particulates impact on the fibers, they adhere. Thus the pore size becomes increasingly smaller, resulting in the filter efficacy increasing. New HEPA filters, used by FailSafe in Models 77 and 07, provide efficiency down to 0.1-micron particles at a removal efficiency of 99.97 percent. HEPA filter bed media manufactured from glass fibers are reflective to ultraviolet irradiation, allowing the UVGI irradiation to partially penetrate the filter bed. The result of the combination of UVGI with ozone generation and the HEPA TABLE 4-5 Relative Size of Fungus, Bacteria, and Viruses Microbe Size Range (diameter–micron) Fungus 0.2–80 Bacteria 0.2–2.0 Viruses 0.02–0.3 CDC guideline cutoff 0.3 FASS unit cutoff 0.1

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Learning From Sars: Preparing for the Next Disease Outbreak - Workshop Summary filter is that the bacteria, fungi, and viruses that are trapped in the filter media will be exposed to sufficient irradiation and ozone concentration to disinfect the filter. The advantage of this antimicrobial treatment combination is that the air stream is inhibited from becoming recontaminated from any growth on the filter media resulting in particle breakthrough. Ultraviolet UV irradiation can cause eye damage and surface burns on unshielded human skin, eyes, and other organs. Therefore the UV lights used in the FASS units are sealed inside and not visible to the operator or other personnel. Ultraviolet radiation, in the wavelength range of 2,250 to 3,020 angstroms as used for air/surface disinfection and sterilization, is referred to as ultraviolet germicidal irradiation or UVGI. Ultraviolet germicidal radiation was first applied to disinfect water systems in 1909. Its use in air purification was first evaluated in the laboratory in the 1920s, in an operating room in the 1930s to sterilize the air in an operating room (Sharp, 1939), and in a school ventilation system to reduce measles infection (Riley, 1972). It is also common practice to use to disinfect medical equipment. UVGI is currently being employed to control bacteria, fungus, and algae growth on surfaces. European breweries have been using UVGI to control microbial growth on cooling coils since 1975. The use of UVGI can control microbial growth on filter surfaces that are subject to moisture or high humidity that will allow for natural fungal growth. Figure 4-7 illustrates a filter with natural fungal growth and a filter that was irradiated with UVGI at a rated intensity of 100 micro/cm at a distance of 1m from the midpoint of the filter (Kowalski and Bahnfleth, 2000). This surface disinfection protects the air stream from being recontaminated due to bacterial, fungus, or viruses that are collected by the filter media. FIGURE 4-7 (left) Microbial growth on nonirradiated filters. (right) Microbe-free UV irradiated filters (Kowalski and Bahnfleth, 2000).

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Learning From Sars: Preparing for the Next Disease Outbreak - Workshop Summary Microbial Response to Ultraviolet Radiation The FASS system is an integration of room recirculation to rid the air of biological threats and surface disinfection to kill the biothreat that is collected on the HEPA filters. The primary target of UV radiation is the microorganism DNA molecule with the predominant injury of strand breakage and the formation of photo-induced byproducts such as thymine diamers. This damaged DNA cannot be used for cell reproduction or for proper mRNA templates that is required for the formation of all cellular toxic products. Viruses are especially susceptible to UVGI, more so than bacteria, and are also difficult to filter because of their size. However, viruses are more susceptible to ultraviolet radiation at wavelengths slightly above the normal UVGI broadband wavelength of 253.7 nm. Microorganisms, when exposed to UVGI irradiation, will be killed or decreased in population at a rate according to a first order equation: S(t) = e–kIt where k = standard decay-rate constant, cm2/microW-s I = Intensity of UVGI irradiation, microW/cm2 t = time of exposure (sec) The rate constant [k] is unique to each microorganism and defines its sensitivity of each microorganism to UVGI intensity. The dose of ultraviolet radiation that an airborne microbe receives depends on the amount of time the microbe is being irradiated and the UV intensity. The upper limit of kill rate is obtained by mixing the air within the UVGI exposure chamber. This mixed airflow will have an average velocity that will determine the exposure time required for all microbes in the air stream. If the air is not mixed, then the flow will be partial laminar resulting in the microbes receiving different dosages of UV radiation. Microbes nearest the UV lamp will get the highest dosages and those near the wall of the chamber will have significantly less exposure to the UV radiation. Laboratory experiments can be used to determine the upper limit of Kill Rate Constant (mixed air) and lower limit of Kill Rate Constant (unmixed air). Ozone Ozone, an allotropic form of oxygen, possesses unique properties when it oxidizes or interacts with chemical and biological systems. Ozone, best known for its protective role in the earth’s ecological environment and its interaction with industrial pollutants, has bactericidal, virucidal, and fungicidal actions that have been used in water treatment, odor control, and medicinal applications. Ozone [O3], a powerful oxidant reacting with organic molecules containing double or triple bonds, yields many complex byproducts. It is this property of ozone that has been applied as a disinfectant and sterilant against bacteria, viruses, and fungi.

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Learning From Sars: Preparing for the Next Disease Outbreak - Workshop Summary Although the inhibitory and lethal effects of ozone on pathogenic organisms have been observed since the latter part of the 19th century, the mechanisms for these actions have not yet been satisfactorily highlighted. The most often cited explanation for ozone’s bactericidal effects centers on disruption of envelope integrity through peroxidation of phospholipids. There is also evidence for interaction with proteins (Mudd et al., 1969). In one study (Ishizaki et al., 1987) exploring the effect of ozone on E. coli, investigators found cell membrane penetration with ozone, subsequent reaction with cytoplasmic substances, and conversion of the closed circular plasmid DNA to open circular DNA. It is notable that higher organisms have enzymatic mechanisms to stabilize disrupted DNA and RNA, which could provide a partial explanation for why, in clinical treatment, ozone appears to be toxic to infecting organisms and not to the patient (Cech, 1986). Ozone possesses fungicidal effects, although the mechanism is poorly understood. In one study, Candida utilis cell growth inhibition with ozone was greatly dependent on phases of their growth, budding cells exhibiting the most sensitivity to its presence (Matus et al., 1981). Interestingly, low doses of ozone stimulated the growth and development of Monilia fructagen and Phytophtora infestans, while higher doses were inhibitory (Matus et al., 1982). Thus, high concentrations of ozone are required for effective antimicrobial activity. Viruses have been studied during their interaction with ozone (Roy et al., 1981). After 30 seconds of exposure to ozone, 99 percent of the viruses were inactivated and demonstrated damage to their envelope proteins, which could result in failure of attachment to normal cells and breakage of the single-stranded RNA. The Occupational Safety and Health Administration (OSHA) has set Public Health Air Standards of 0.1 ppm for 8 hours or 0.3 ppm for 15 minutes as the limit of the amount of ozone to which people can be safely exposed. Air cleaners based on ozone must not generate ozone levels above the Public Health Standards, which are far below any antimicrobial activity or effective odor control. Low ozone concentrations, below the EPA-acceptable indoor limit, have been used as air cleaners, but their effectiveness has been questioned by many studies (Dyas et al., 1983; Foard et al., 1997). At high ozone concentration, ozone has been used to decontaminate unoccupied spaces of some chemical and biological contaminants and odors such as smoke. Air Flow The Center for Disease Control and Prevention’s guidelines for air flow into an isolation room state that there shall be greater than 12 air changes per hour (ACH). However, a higher ACH means more efficiency in removing any airborne infectious materials. There are two settings on the air flow volumes. The number of ACH obtained is a function of room volume, as illustrated in Table 4-6, which is color coded based on obtaining 12 ACH as the minimal level required for meeting CDC guidelines for isolation precautions.

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Learning From Sars: Preparing for the Next Disease Outbreak - Workshop Summary TABLE 4-6 ACH as a Function of Isolation Room Volume and FASS Capabilities (calculated on a 10 percent reduction in air flow capability) Room Size L × W × H Room Volume (cu ft) FASS 700 (ACH) FASS 1000 (ACH) FASS 2000 (ACH) 9' × 12' × 8' 864 43.8 62.5 125.0 12' × 12' × 8' 1,152 32.8 46.9 93.8 15' × 12' × 8' 1,440 26.3 37.5 75.0 15' × 20' × 8' 2,400 15.8 22.5 45.0 20' × 20' × 8' 3,200 11.8 16.9 33.8 20' × 30' × 8' 4,800 7.9 11.3 22.5 30' × 30' × 8' 7,200 5.3 7.5 15.0 Summary The described FASS Medical Isolation Units are available in the United States, Canada, and Asia from FailSafe Air Safety Systems Corporation of Tonawanda, NY. They may offer the best opportunity to increase the numbers of isolation rooms in hospitals and especially in emergency rooms. By doing this, they provide a cost-effective solution to the challenge of new viral pathogen outbreaks. It must be emphasized that these units will only control respiratory transmissions, and are not a substitute for contact precautions or for treatment of the infection itself. Traditional measures still must be instituted to deal with surface contamination. For cleanup of biological contamination, the FASS Mobile Containment Systems also generate ozone to eradicate pathogens from surfaces. These units should be used in conjunction with the Models 77 and 07 for additional remediation of the hospital or emergency room environment. REFERENCES American Institute of Architects. 2001. Guidelines for Design and Construction of Hospital and Health Care Facilities, AIA. Anand K, Palm GJ, Mesters JR, Siddell SG, Ziebuhr J, Hilgenfeld R. 2002. Structure of coronavirus main proteinase reveals combination of a chymotrypsin fold with an extra alpha-helical domain. EMBO Journal 21(13):3213-24. Anand K, Ziebuhr J, Wadhwani P, Mesters JR, Hilgenfeld R. 2003. Coronavirus main proteinase (3clpro) structure: basis for design of anti-SARS drugs. Science 300(5626):1763-7. Ballew HC. 1992. Neutralization. In: Specter S, Lancz G, eds. Clinical Virology Manual. New York: Elsevier. Pp. 229-41. Barnes TW 3rd, Turner DH. 2001a. C5-(1-Propynyl)-2'-deoxy-pyrimidines enhance mismatch penalties of DNA:RNA duplex formation. Biochemistry 40(42):12738-45. Barnes TW 3rd, Turner DH. 2001b. Long-range cooperativity due to C5-propynylation of oligopyrimidines enhances specific recognition by uridine of ribo-adenosine over ribo-guanosine. Journal of the American Chemical Society 123(37):9186-7. Brito DA, Ramirez M, de Lencastre H. 2003. Serotyping streptococcus pneumoniae by multiplex PCR. Journal of Clinical Microbiology 41(6):2378-84. Centers for Disease Control and Prevention. 1994. Guidelines for preventing the transmission of mycobacterium tuberculosis in health-care facilities, 1994. Morbidity & Mortality Weekly Report Recommendations & Reports 43(RR-13):1-132.

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Learning From Sars: Preparing for the Next Disease Outbreak - Workshop Summary Poutanen SM, Low DE, Henry B, Finkelstein S, Rose D, Green K, Tellier R, Draker R, Adachi D, Ayers M, Chan AK, Skowronski DM, Salit I, Simor AE, Slutsky AS, Doyle PW, Krajden M, Petric M, Brunham RC, McGeer AJ, National Microbiology Laboratory Canada, Canadian Severe Acute Respiratory Syndrome Study Team. 2003. Identification of severe acute respiratory syndrome in Canada. New England Journal of Medicine 348(20):1995-2005. Riley RL. 1972. Airborne Infections. New York: Macmillan. Rota PA, Oberste MS, Monroe SS, Nix WA, Campagnoli R, Icenogle JP, Penaranda S, Bankamp B, Maher K, Chen MH, Tong S, Tamin A, Lowe L, Frace M, DeRisi JL, Chen Q, Wang D, Erdman DD, Peret TC, Burns C, Ksiazek TG, Rollin PE, Sanchez A, Liffick S, Holloway B, Limor J, McCaustland K, Olsen-Rasmussen M, Fouchier R, Gunther S, Osterhaus AD, Drosten C, Pallansch MA, Anderson LJ, Bellini WJ. 2003. Characterization of a novel coronavirus associated with severe acute respiratory syndrome. Science 300(5624):1394-9. Roy D, Wong PK, Engelbrecht RS, Chian ES. 1981. Mechanism of enteroviral inactivation by ozone. Applied Environmental Microbiology 41:718-23. Sadowski A, Gasteiger J. 1993. From atoms and bonds to three-dimensional atomic coordinates: automatic model builders. Chemical Reviews 93:2567-81. Sali A, Blundell TL. 1993. Comparative protein modelling by satisfaction of spatial restraints. Journal of Molecular Biology 234(3):779-815. Sharp G. 1939. The lethal action of short ultraviolet rays on several common pathogenic bacteria. Journal of Bacteriology 37:447-59. Swaminathan B, Barrett TJ, Hunter SB, Tauxe RV, CDC PulseNet Task Force. 2001. Pulsenet: the molecular subtyping network for foodborne bacterial disease surveillance, United States. Emerging Infectious Diseases 7(3):382-9. Taylor LH, Latham SM, Woolhouse ME. 2001. Risk factors for human disease emergence. Philosophical Transactions of the Royal Society of London—Series B: Biological Sciences 356(1411):983-9. Tsang KW, Ho PL, Ooi GC, Yee WK, Wang T, Chan-Yeung M, Lam WK, Seto WH, Yam LY, Cheung TM, Wong PC, Lam B, Ip MS, Chan J, Yuen KY, Lai KN. 2003. A cluster of cases of severe acute respiratory syndrome in Hong Kong. New England Journal of Medicine 348(20):1977-85. Vabret A, Mouthon F, Mourez T, Gouarin S, Petitjean J, Freymuth F. 2001. Direct diagnosis of human respiratory coronaviruses 229e and Oc43 by the polymerase chain reaction. Journal of Virological Methods 97(1-2):59-66. Wagner RW, Matteucci MD, Lewis JG, Gutierrez AJ, Moulds C, Froehler BC. 1993. Antisense gene inhibition by oligonucleotides containing C-5 propyne pyrimidines. Science 260(5113):1510-3. Wang D, Coscoy L, Zylberberg M, Avila PC, Boushey HA, Ganem D, DeRisi JL. Microarray-based detection and genotyping of viral pathogens. Proceedings of the National Academy of Sciences of the United States of America 99(24):15687-92. Wang D, Urisman A, Liu YT, Springer M, Ksiazek TG, Erdman DD, Mardis ER, Hickenbotham M, Magrini V, Eldred J, Latreille JP, Wilson RK, Ganem D, DeRisi JL. 2003. Viral discovery and sequence recovery using DNA microarrays. PLOS Biology 1(2):257. Wilson KH, Wilson WJ, Radosevich JL, DeSantis TZ, Viswanathan VS, Kuczmarski TA, Andersen GL. 2002. High-density microarray of small-subunit ribosomal DNA probes. Applied & Environmental Microbiology 68(5):2535-41. WHO. 2003. Severe acute respiratory syndrome (SARS). Weekly Epidemiological Record 78(12):81-3. Ziebuhr J, Snijder EJ, Gorbalenya AE. 2000. Virus-encoded proteinases and proteolytic processing in the Nidovirales. Journal of General Virology 81(Pt 4):853-79.