This set of breakout sessions sought to (1) describe the public health needs lab workers are attempting to meet, the range of tools being utilized, and the challenges regularly encountered and (2) examine the spectrum of molecular, immunological, and culture-based assays available as well as their associated costs, effectiveness, and biosafety requirements. To inform the discussion, participants in each session first listened to several talks that described examples of the diagnostics currently employed.
BREAKOUT SESSION 1: HUMAN DISEASES PART 1
Chair: Ingegerd Kallings
Rapporteur: Alison Hottes
This session opened with talks that both illustrated the application of current biosafety recommendations to diagnostics and surveillance and looked ahead to the type of detailed, protocol-specific biosafety guidance that might be available in the future. The first talk portrayed the changing set of diagnostic tools available for clinical work and the ability of a BSL-3 lab to allow a clinical group to perform complimentary research. The second talk described a large-scale surveillance operation that seeks to understand influenza strain dynamics. The final talk gave an example of the potential for a detailed risk assessment to provide more nuanced biosafety guidance than that available using the standard World Health Organization (WHO) or “Biosafety in Microbiological and Biomedical Laboratories” (BMBL) four level systems. After the talks concluded, Ingegerd Kallings (Swedish Institute for Communicable Disease Control, Sweden) led a discussion.
BREAKOUT SESSION PRESENTATIONS
Preparedness for the Detection of Emerging and Re-emerging Pathogens in Croatia
Alemka Markotić (University Hospital for Infectious Diseases, Croatia) described the diagnostic tools she uses at University Hospital for Infectious Diseases which includes both the largest hospital in the region and a reference center with a new BSL-3 lab.
Dr. Markotić explained that she does both clinical and research work on hantaviruses, Dengue fever virus, tick-borne encephalitis, West Nile virus, Chikungunya virus, and influenza A using several methods including point-of-care (POC) tests, enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) tests, sequencing, and cell culture. She and the other members of her lab staff generally follow United States Centers for Disease Control and Prevention (CDC) recommendations except those that pertain to the United States Select Agents program, meaning that serology is done in BSL-2 conditions in a biological safety cabinet (BSC) and that molecular tests start in BSL-2 conditions and then move to a molecular lab. Cell culture and virus isolation, which are typically part of research, not clinical work, are done in BSL-3 conditions except for small quantities of Dengue virus that are grown and handled in the BSL-2 lab.
Dr. Markotić indicated variable levels of satisfaction with POC tests. When she initially tried using POC tests to detect Puumala and Dobrava hantaviruses, she experienced too many false positives; recently, however, the quality of commercial tests has improved and she has resumed using them. She also uses POC tests to look for influenza A, Dengue fever virus, and Chikungunya. Dr. Markotić emphasized that while POC tests in conjunction with a clinical evaluation can serve as a valuable early indicator, she always proceeds with additional confirmatory testing.
Dr. Markotić also compared the costs of various techniques. For example, given the short shelf life of many ELISA reagents and the frequency with which she does the tests for clinical work, she indicated that PCR tests are comparatively cheaper for her lab. She reported mainly using ELISA tests for research projects when she knows the reagents will be used before they expire. Although University Hospital for Infectious Diseases has in-house sequencing capabilities, she normally uses the equipment at the Institute for Biological Services, which is cheaper. She noted, however, that the cheapest option is to send the samples to South Korea. Dr. Markotić pointed out that, in general, costs in Croatia are extremely high, and reagents often cost 5-6 times more than they would in the United States.
She explained that her research uses a number of culture-based techniques that are performed in BSL-3 conditions and that she would like to pursue more immunopathogenesis research. In particular, she developed a research model for hantaviruses using 293-HEK cells (Markotic et al., 2003). Resource limitations have, however, put this research on hold. Her lab, however, is currently working to develop molecular tests for Crimean-Congo hemorrhagic fever (CCHF), other hemorrhagic fevers, poxvirus, and rickettsia. As the Croatian Ministry of Health has limited opportunities that support research, Dr. Markotić indicated that she relies on international collaborations and foreign research grants for support.
Biorisks Connected with Wild Birds: Results of Avian Influenza Virus Surveillance in Southwest Siberia (Russia) in 2010
Alexander Shestopalov (State Research Center of Virology and Biotechnology, Russia), whose division monitors influenza in humans, wild birds, and poultry and contributes their data to WHO, described his lab’s efforts to understand the dynamics of highly pathogenic avian influenza (HPAI) in Eurasia.
Dr. Shestopalov started by explaining that southwest Siberia contains many lakes, three major flyways for migrating birds, and played an important role in the 2005 expansion of influenza A, H5N1 in Eurasia. He believes that the lakes and rivers in the region play an important role in the circulation of avian influenza (AI) and that by monitoring influenza activity in migratory and resident bird there, they could provide an early warning system for HPAI outbreaks in birds in Eurasia.
In 2010, his lab isolated 32 AI viruses from 743 samples collected in western Siberia. Phylogenetic subtyping using PCR and sequencing indicated that 10 were H3N8, 4 were H3N6, and 8 were H4N6, while 10 could not be typed.
Another of the lab’s datasets demonstrated that the H5N1 viruses present on Uvs–Nuur Lake in Mongolia near the Russian border shifted from clade 2.2 to clade 2.3.2 (evolutionarily distinct groups) between 2006 and 2009-2010. Hemagglutination inhibition assays using sera from ferrets showed no cross-reactivity between either virus and the opposite sera, suggesting that the viruses produce distinct immunological responses and display distinct antigens. As such, Dr. Shestopalov proposed that the current circulation of clade 2.3.2 and the disappearance of clade 2.2 could be explained by antigenic drift of the hemagglutinin under the pressure of population immunity in the natural host species. Clade 2.3.2 viruses were subsequently found in Russian in 2009, Bulgaria and Romania in 2010, and Japan in 2011, illustrating the potential for the region to function as an early warning system.
Biosafety Recommendations for Laboratory Testing for TB
Thomas Shinnick (Division of TB Elimination, United States CDC) described the Global Laboratory Initiative’s (GLI) soon-to-be-finalized consensus guidance for tuberculosis (TB) lab procedures.
Dr. Shinnick started by explaining that TB lab workers are at a 3-fold to 9-fold higher risk of becoming infected with Mycobacterium tuberculosis than other lab workers and that infection often results from the unrecognized production of infectious, bacteria-containing aerosols, although infection can also occur in other ways, such as through needle sticks or contact with broken skin. He also reminded the audience that while TB is classified as a risk group 3 organism, different protocols for a single agent may necessitate the use of precautions from different biosafety levels.1
Dr. Shinnick explained that GLI performed a systems-based risk analysis and divided TB procedures into one of three sets depending on whether they posed a limited, moderate, or high risk of generating infectious aerosols.
GLI classified direct acid-fast bacillus (AFB) smear microscopy, which involves few cells in a viscous material that impedes aerosolization, as a limited risk procedure.2 As such, GLI feels AFB smear microscopy can be done on an open bench in a laboratory with restricted access. The laboratory should have adequate ventilation, meaning 6-12 air changes per hour (ACH) from either natural or mechanical ventilation with directional airflow from the worker to the sample to the exterior. Infectious material should be disposed of properly.
In general, however, GLI regards sputum specimen manipulation for smear, culture, or molecular tests as a moderate-risk activity and recommends precautions like separating labs from public areas and restricting access. Surfaces should be impermeable for easy cleaning, infectious material should be disposed of properly, windows should be closed, and air (6-12 ACH) should flow into the lab without recirculation to non-lab areas. Properly installed and annually certified Class I or II BSCs should be used for all open manipulations of agents. Exhaust should be directed outside through a thimble fitting (preferred) or a hard duct; exhaust from a properly functioning BSC may be recirculated into the room. Aerosol-containment rotors should be used for centrifugation and opened in a BSC.
Finally, GLI classified work with cultures, which includes virtually all research, as high-risk due to the extreme likelihood of generating infectious aerosols while manipulating liquid suspensions. The recommendations are similar to those for moderate risk, except an autoclave should be available on site, the lab should have a double door entry (not necessarily an airlock), and windows should be sealed, not just closed. The ability to seal the lab for fumigation, however, was not deemed necessary by GLI; once TB settles it is difficult to re-aerosolize, so making the room amenable to surface decontamination was judged sufficient.
Dr. Shinnick reflected that in general, the new guidance does not map perfectly to any of the four generic BSL levels and represents an effort to move away from the traditional biosafety level boxes.
He closed by mentioning that the GLI expert committee is also considering suitable lab floor plans as well as specific recommendations for appropriate respirator usage, for human immunodeficiency virus (HIV)-positive technicians, and for samples from known or highly suspected extremely drug-resistant (XDR) TB patients.
1 Current guidance on TB work is available in the BMBL (United States HHS, 2009; see pp. 145-147).
2 GLI also considers Cepheid’s Xpert® MTB/RIF molecular test limited risk.
BREAKOUT SESSION 2: HUMAN DISEASES PART 2
Chair: Peter Palese
Rapporteur: Michael Callahan
This session opened with a talk that described the recent outbreak of Escherichia coli in northern Germany and the associated epidemiological and diagnostic work. While containment labs are not ordinarily used to process E. coli specimens, the experience illustrates many of the challenges associated with emerging infectious diseases, including the need to increase hospital capacity during an outbreak, characterize the causative agent, and identify the outbreak’s source. The second talk described a diagnostic network for influenza and emerging infectious diseases. The final talks presented the range of diagnostic tests available and the associated precautions routinely employed for two diseases: severe acute respiratory syndrome (SARS) and Crimean-Congo hemorrhagic fever (CCHF). Following the talks, Peter Palese (Mount Sinai School of Medicine, United States) led a discussion.
BREAKOUT SESSION PRESENTATIONS
EHEC O104:H4 in Germany 2011: Large Outbreak of Bloody Diarrhea and Haemolytic Uraemic Syndrome by Shiga Toxin-Producing E. coli via Contaminated Food
Reinhard Burger (Robert Koch Institute [RKI], Germany) described Germany’s recent outbreak of enterohaemorrhagic E. coli (EHEC) and the associated public health response.
Dr. Burger started by updating the audience on the then on-going outbreak, which included an unusually high incidence of haemolytic uraemic syndrome (HUS). He indicated that as of 7 July 2011, Germany had recorded 3,322 cases of EHEC gastroenteritis that did not include HUS and 859 cases that did. By that time, the outbreak had resulted in 49 deaths. He also noted that Sweden, Denmark, and France had reported multiple cases and that 14 additional countries had reported single cases. He then walked the audience through a timeline of RKI’s response to the outbreak (Box 6-1).
Once the outbreak was recognized, Dr. Burger indicated that RKI enhanced EHEC/HUS surveillance. They instituted daily transfer of normal surveillance data, which they augmented with laboratory surveillance of positive EHEC tests and by requesting reports of emergency cases with bloody diarrhea admitted to hospitals.
Dr. Burger then elaborated on the epidemiological investigation, which involved over 85 staff members. The group’s early analyses, which were later found to be erroneous, suggested that Spanish cucumbers were the source, and the resulting decrease in consumption caused between 80 and 200 million Euros in losses. Later analyses used novel techniques to obtain more accurate information, including using billing data and digital photographs to remind cases and controls what they ate at a restaurant.
Initial Events in RKI’s Response to Germany’s 2011 EHEC Outbreak
May 19: RKI received a call from the Hamburg health department and notified the German Federal Institute for Risk Assessment and the Federal Ministry of Health.a
May 20: RKI visited Hamburg and interviewed patients and local public health authorities.
May 21: RKI passed information about the qualitative role of vegetables in the outbreak to the Food Safety Authorities.
May 22: RKI warned WHO and local public health authorities and informed the German Press Agency about the possible involvement of uncooked vegetables.
May 23: RKI began publishing information on their website. Polymerase chain reaction (PCR) tests for the responsible pathogen became available.
May 24: The first official International Health Regulations (IHR) notification took place. The pathogen’s serotype was identified as O104.
May 25: RKI identified the pathogen and advised northern Germany to avoid uncooked tomatoes, cucumbers, and lettuce.
a German authorities had tracked an increased incidence of HUS since May 2, 2011. See: Outbreak Notice. Shiga toxin-producing E. coli O104:H4 infections in Germany. June 3, 2011. Available at: http://www.vdh.state.va.us/clinicians/pdf/06-03-11%20Clinicians%27%20Letter%20-%20CDC%20Health%20Alert%20Shiga%20Toxin.pdf.
Dr. Burger indicated that the case control studies ultimately pinpointed bean sprouts as the source of the German outbreak, and subsequent cluster and traceback analysis linked the sprouts to a single farm. Bacteriological screening of over 10,000 samples of sprouts, seeds, and materials from the production site, however, produced only a single positive result. Additional work attributed the French outbreak to Egyptian fenugreek seeds and indicated that the German sprout producer and the French seed distributer had a common supplier. As a result of the investigation, imports of Egyptian seeds and beans were temporarily banned.
In addition to surveillance and epidemiology, Dr. Burger indicated that RKI also contributed communications and microbiology expertise to the effort. Seven staff members oversaw communications efforts that included press conferences, outreach to public health authorities, and dissemination of accurate information through the RKI website. Over 10 staff members engaged in primary and reference laboratory typing, and microbiological characterization of the strain indicated that the organism was unusually adherent to the intestine, produced Shigatoxin 2, and had an unusual antibiotic resistance profile. Dr. Burger noted that the strain was likely of natural origin.
In conclusion, Dr. Burger mentioned that retrospective analysis of case reports indicated that it took about two weeks to detect the outbreak and that identifying the source then took another three weeks. By way of comparison, he noted that investigations of similar outbreaks in Japan in 1996 (E. coli in radish sprouts) and in the United States in 2008 (Salmonella in chili peppers) took longer to accomplish each of those milestones.
Biosafety and the Southeast Asian Clinical Infectious Diseases Network
Rogier van Doorn (Oxford University Clinical Research Unit, Netherlands) described the present and future work of the Oxford University Clinical Research Unit’s Ho Chi Minh City, Vietnam (OUCRU-HCM) facility.
Dr. van Doorn opened by saying that the OUCRU-HCM is part of the Southeast Asian Clinical Infectious Diseases Network (SEAICRN), which also has facilities in Thailand, Indonesia, and Singapore. He explained that SEAICRN works to advance the scientific knowledge and clinical management of influenza and emerging infectious diseases through integrated, collaborative clinical research and to produce evidence that will inform health policies and clinical practice. As part of its pandemic preparedness efforts, the network also builds public health capacity for the world.
He noted that OCURU-HCM receives core funding from the Wellcome Trust and started external collaborations in 1991. The facility has BSL-2 diagnostic labs for bacteriology, serology, and molecular virology; BSL-2 research labs for virology culture and molecular work; an insectary; BSL-3 labs for mycobacterial culture and virology; and a U.K. Specified Animal Pathogens Order 4 (SAPO4) containment lab for virology. The facilities were audited and certified by WHO and use closed circuit television, signage, and restricted access for security.
While the existing BSL-3 facilities opened in 2002, Dr. van Doorn explained that in 2008, the lab began the design, construction, and certification of a multifunctional BSL-3 suite that is expected to open in 2011. The new lab is funded by the United States National Institutes of Health (NIH) National Institute of Allergies and Infectious Diseases (NIAID) through SEAICRN and complies with United Kingdom laws and guidelines. An independent party, Oxford University, and Vietnam’s Ministry of Health will all certify the lab for operation. He indicated that they are also considering applying to the United States Centers for Disease Control and Prevention (CDC) for Select Agent certification. The lab plans to work on hand, foot, and mouth disease and viral encephalitis and run outbreak investigations, a pathogen discovery program, and a zoonoses program.
In closing, Dr. van Doorn described the OUCRU-HCM’s virology reference lab, which works to build state-of-the-art molecular diagnostic units and to disseminate low-biorisk diagnostics. The lab provides a centralized facility for developing assays and standard operating procedures, ordering reagents, and performing high-risk diagnostics involving culture. The reference lab is particularly experienced in influenza diagnostics and offers a range of molecular diagnostics including reverse transcription polymerase chain reaction (RT-PCR), in-house tests for resistance-associated mutations, Sanger sequencing, and pyrosequencing. The reference lab also performs culture-based susceptibility testing and microneutralization assays. The lab trains its staff on site and participates in external quality assessment programs.
SARS-Coronavirus: Diagnosis, Antibody Responses, and Biosafety Concerns
Cheng Cao (Beijing Institute of Biotechnology, China) characterized a range of diagnostic tests for SARS as well as related biosafety and biosecurity precautions.
Dr. Cao started by reminding the audience that the SARS epidemic that started in Guangdong Province in November of 2002 was the first time China had fought against a communicable disease caused by an initially, unidentified pathogen. In spite of intensive public health measures to identify and isolate patients, the outbreak caused more than 8000 cases (more than half in China) and over 700 deaths. Once the etiological agent was confirmed in early April 2003, he and others began developing fast, accurate laboratory diagnostics. Dr. Cao indicated that while China currently has around 30 BSL-3 labs, the small number of BSL-3 labs in China at the time and the lack of an accreditation system for the labs made the initial work more difficult.
Dr. Cao then described a number of the diagnostics that are currently available for SARS:
• RT-PCR assays convert viral RNA into complementary DNA, which is then amplified and detected. The technique requires specialized equipment, often suffers from low sensitivity, and contamination may cause false positives (Di et al., 2005). Since the technique looks for the virus directly instead of waiting for an immune response, it can be used earlier in an infection than other methods, but it cannot be used for retrospective tests.
• Enzyme-linked immunosorbent assays (ELISA) using monoclonal antibodies can look for antigen to the SARS coronavirus nucleocapsid protein (Di et al., 2005). As antigen appears before antibodies, the test is useful for early diagnosis. However, as the antigen becomes undetectable 20 days post onset of symptoms, the test, which does not require viral culture, cannot be used for retrospective work.
• Dr. Cao showed that immunofluorescence assays (IFA) could detect SARS antibodies in 90 percent of the patients 15 days after infection.
• ELISA assays using whole viral lysate as the antigen, like IFA assays, can detect SARS antibodies in serum. Dr. Cao reported that the technique has a higher false positive rate than the comparatively more difficult IFA.
• ELISA assays using recombinant nucleocapsid protein as the antigen can detect SARS in 68 percent of patients 6-10 days after the start of infection and in 90 percent of patients after 10-61 days (Shi et al., 2003). Unlike ELISA with whole viral lysate or IFA, viral culture is not required as the needed protein can be expressed in E. coli. Dr. Cao developed the technique in April 2003, and China’s State Food and Drug Administration certified it soon after. Antibodies to nucleocapsid protein persist for months (Liu et al., 2004), allowing the assay to be used for retrospective work.
Dr. Cao also shared some biosafety and biosecurity concerns and advice. First, as SARS-coronavirus-like viruses are circulating in the Chinese and Slovenian horseshoe bat populations and the Nigerian leaf-nosed bat population, disease reemergence is a concern. Second, the 2004 laboratory leak in Beijing that resulted in 8 cases of SARS, including one death, and the SARS LAIs in laboratory workers in Singapore and Taiwan illustrate the importance of rigorously following safety procedures. To that end, Dr. Cao argued that SARS coronavirus and infected tissues must be manipulated only in BSL-3 labs by well-trained scientists and technicians. While serum tests can be done in BSL-2 labs in BSCs, he cautioned that sera from SARS patients should be incubated at 56°C for 30 minutes to inactivate the virus.
Dr. Cao also presented data showing the role of nucleocapsid protein in the immune response to SARS coronavirus.
Crimean-Congo Hemorrhagic Fever: Pakistan’s Perspective
Birjees Mazhar Kazi (National Institute of Health, Islamabad-Pakistan) described the tests and the accompanying precautions the National Institute of Health in Islamabad, Pakistan uses for CCHF diagnosis.
Dr. Kazi explained that due to the prevalence of the tick vector, Pakistan experiences CCHF outbreaks and that the National Institute of Health, Pakistan tracks reported cases. Current diagnostic tools include immunoglobulin M (IgM) capture ELISA using a kit from Biological Diagnostic Supplies Limited, RT-PCR using a published protocol (Schwarz et al., 1996), and genetic sequencing. Dr. Kazi noted that although the National Institute of Health, Pakistan has the infrastructure and trained personnel for DNA sequencing, reagents for this
protocol are currently limited. Past techniques that are no longer used include electron microscopy, inoculation into suckling mice, and fluorescent microscopy.
When handling CCHF samples, the National Institute of Health, Pakistan employs a number of safety precautions. In general, separate designated areas with restricted personnel access are used for sample receipt, processing, and testing. Diagnostic samples are inactivated at receiving prior to moving them to the lab. The work itself is then done in BSL-2+ laboratories using enhanced personal protective equipment. Finally, waste is destroyed by autoclaving and incineration.
In the future, the lab plans to establish standard guidelines for diagnosis and to establish a separate core laboratory facility.
BREAKOUT SESSION 3: ANIMAL AND LIVESTOCK DISEASES
Chair: David Franz
Rapporteur: Fran Sharples
This session began with presentations describing the work of three institutions that provide surveillance and diagnostic services to both their home country as well as neighboring countries. The first two talks detailed the operations of networks that include high-containment facilities and are responsible for monitoring a wide range of animals for signs of many different diseases. In contrast, the final talk described the diagnostic tools at a lab developed to detect endemic risk group 4 agents without a BSL-4 lab. Following the talks, David Franz (MRIGlobal, United States) led a discussion.
BREAKOUT SESSION PRESENTATIONS
The Work and Capabilities of the High Security Animal Disease Laboratory at Bhopal, India
Gaya Prasad (Indian Council of Agricultural Research, India) described the range of diagnostics the High Security Animal Disease Laboratory (HSADL) is developing and using to detect and combat emerging and exotic diseases in India. He noted that the HSADL lab also serves Bangladesh, Nepal, Bhutan, and other nearby nations without analytical lab capabilities on a case-by-case basis subject to government authorization.
Dr. Prasad started by explaining that while India only has one veterinarian for every 20,000 animals, the government runs all veterinary hospitals allowing unusual livestock diseases to be detected and rapidly brought to government notice. The HSADL has operated as a BSL-3+ facility since its commissioning in 2000. With the help of the Food and Agriculture Organization of the United Nations (FAO) and the United Nations Development Program, the facility was recently upgraded to BSL-4 including an animal biosafety level (ABSL)-4 facility with isolators. The new BSL-4 lab allows the facility to work on Nipah virus and other dangerous pathogens.
Dr. Prasad noted that in its capacity as a World Organisation for Animal Health (OIE) reference lab for avian influenza, HSADL has tested over 450,000 samples since 2006 when HPAI H5N1 was first detected in domestic poultry. In addition to molecular and phylogenetic analyses of HPAI isolates, the lab also characterizes the pathogenicity of influenza isolates and engages in surveillance of migratory birds. HSADL developed an avian influenza database and a reverse transcription polymerase chain reaction (RT-PCR) test for the diagnosis of avian influenza H5N1.
HSADL has also been active in the field of swine influenza research and surveillance. The lab conducts antibody detection by hemagglutination inhibition and enzyme-linked
immunosorbent assays (ELISA) and detects strains and subtypes using RT-PCR and sequencing. Overall, sera from 401 pigs have been tested, and 76 were positive for H1N1 antibody. Additionally, 656 nasal swabs were tested, and H1N1 viruses were isolated from two samples.
In addition to its influenza work, HSADL also regularly tests cattle, buffalo, sheep, and goat samples for bovine viral diarrhoea (BVD) and border disease (BD) viruses. The lab isolated and characterized the BD virus using nucleotide sequencing, antigenic analysis, and transmission electron microscopy, and developed a monoclonal antibody-based ELISA test for the diagnosis of BVD virus in cattle and an immunoperoxidase-linked neutralization assay for the detection of neutralizing antibodies to BVD virus. The Department of Animal Husbandry, Dairying, and Fisheries’ proposal for OIE reference lab status for BVD virus for HSADL is under the final stages of consideration by OIE.
Dr. Prasad also described a number of other diagnostics that HSADL has developed including a recombinant nucleoprotein-based ELISA test for the detection of porcine reproductive and respiratory syndrome virus antibodies in pigs and real-time PCR tests for the diagnosis of pseudorabies, porcine parvovirus infection, and malignant catarrhal fever. He concluded by mentioning that HSADL also works with international agencies such as FAO, the World Health Organization (WHO), and the International Consortium on Anti-Virals; domestic agencies; leading national pharmaceutical, research and development, and vaccine production firms; and national and international biosafety organizations.
Diagnostic Capabilities for Exotic and Emerging Animal Diseases of Mexico’s Official Laboratory Network
Marco Antonio Rico Gaytán (Mexico-US Commission for the Prevention of Foot and Mouth Disease and Other Exotic Animal Diseases [CPA], Mexico) described the organization and diagnostic capabilities of CPA’s laboratory network whose mission is to protect terrestrial and aquatic animals from exotic and emerging diseases.
Dr. Gaytán explained that the CPA network contains 13 molecular biology labs, 7 regional labs, and one BSL-3 lab that is located in Cuajimalpa in Mexico City. The molecular biology labs, which are all of the same design, are BSL-2 labs with some enhancements and run serology and molecular biology-based diagnostic tests including standard polymerase chain reaction (PCR), real-time PCR, sequencing (3 labs only), and ELISA tests using commercial kits. He noted that the National Services of Health, Safety, and Food Quality operate an additional three molecular biology labs and that each regional lab has specialized equipment and reagents and performs specific diagnostic tests. The BSL-3 lab was certified in 2006 by the University of Texas Medical Branch, which, in the absence of specific Mexican regulations, checked for compliance to the United States Department of Agriculture Manual and the Containment Standard for Veterinary Facilities from the Canadian Food Inspection Agency. The BLS-3 lab, which was renovated from a lab originally built in 1949, contains a number of biosecurity measures including fingerprint-based access control. The labs, particularly the molecular biology labs, are distributed throughout Mexico.
The network is currently responsible for the diagnosis of 33 diseases and performs a total of 110 tests of 20 different types. As such, the network has multiple ways to diagnose most diseases. The most common techniques are PCR (24 diseases), virus isolation in cell culture (19 diseases), ELISA (13 diseases), and virus isolation in laboratory animals (12 diseases). The CPA network also has a large number of people in the field who perform surveillance and technical training.
While noting that exact costs are difficult to establish, Dr. Gaytán estimated that the network, which receives funding from the Mexican government and CPA and offers free services to its clients, probably requires about $27 million U.S. annually. He explained that over
95% of the laboratory equipment and over half of the laboratory materials and reagents are imported, which increases costs. To provide a frame of reference for participants, he noted that in U.S. dollars the price of diagnostic tests (materials and reagents only) varies from about $120 for sequencing tests to $5 for ELISA tests to $1 for hemmaglutination inhibition assays.
Coping with Deadly Viruses
Supaporn Wacharapluesadee (Chulalongkorn University, Thailand) described diagnostics and procedures she developed for monitoring and diagnosing viral zoonoses in wildlife and humans.
Dr. Wacharapluesadee started by describing the history of Chulalongkorn University, which has been studying encephalitis viruses and serving as a referral hospital for patients with encephalitis since 1989. In 2002, Chulalongkorn University started active wildlife surveillance of Nipah, Lyssa, rabies, and other viruses in Thailand in a project sponsored largely by the Thailand Research Fund. In recognition of this work, the lab was designated as a WHO Collaborating Centre for Research and Training on Viral Zoonoses in 2009. Then, in 2011, the work expanded under the One Health program to include more human and domestic animal components. She noted that the lab partners with the Thai Red Cross, the United States Defense Advanced Research Projects Agency (DARPA), the United States Naval Health Research Center, EcoHealth Alliance, and FAO.
She explained that much of the group’s work involves surveillance for Nipah virus, a risk group 4 pathogen that causes encephalitis in humans with a 40-70% mortality rate and severe respiratory disease in pigs.3 Bats are a natural reservoir for the virus, and human infection can result from drinking palm juice from trees where bats roost. As Thailand has no BSL-4 labs, her group developed a duplex nested RT-PCR test to detect low amounts of viral RNA in bat specimens including urine, saliva, serum, and blood without the need for a containment lab (Wacharapluesadee and Hemachudha, 2007). She described how specimens are taken from urine collected on plastic sheets under trees and from bats captured with mist nets and subsequently released. The samples are put into lysis buffer containing guanidine thiocyanate, which inactivates the virus and protects the RNA until the samples can be transported on ice to the lab for testing. She noted that Nipah RT-PCR tests are also used as a diagnostic tool for encephalitis patients.
Dr. Wacharapluesadee then described how her group performs rabies diagnoses using brain samples obtained during human and animal post-mortems. In the rabies test protocol, she places animal brain samples on Flinders Technology Associates (FTA) cards, which inactivate the virus, allowing safe handling and transport (Picard-Meyer et al., 2007). She noted that FTA cards also stabilize virus nucleic acids, allowing accurate analysis after even months of storage (Wacharapluesadee et al., 2003).
She also mentioned that her group has started using high-throughput sequencing methods and metagenomics to analyze the microorganisms present in bat saliva.
SYNTHESIS BREAKOUT GROUP DISCUSSIONS
Following the presentations, each session was asked to consider a number of questions:
1. As they are performed, what commonly used procedures/assays cause the highest risk for workers and/or the community?
3 The BMBL recommends that Nipah virus, including diagnostic specimens, be handled at BSL-4 (United States HHS, 2009; see pages 201-202).
2. To what extent can molecular diagnostic tests replace traditional techniques that necessitate working with larger quantities of pathogens?
3. Are an appropriate number of reference pathogen collections available?
4. How do biosafety/biosecurity considerations impact your ability to perform necessary analyses for clinical diagnosis or outbreak investigations?
In addition to elaborating on the opportunities and limitations of molecular diagnostics, including point-of-care tests, participants also discussed how transport restrictions and the availability of reference pathogen collections impact diagnostic and research work.
While generally enthusiastic about the potential for molecular tests, which use inactivated pathogens and can safely be performed in BSL-2 labs, multiple people stressed that molecular tests will never completely eliminate the need to isolate and characterize pathogens in high-containment labs. Several people also indicated that molecular tests are best suited for answering routine questions about normal specimens and that only culture offered the flexibility needed to deal with novel situations and outbreaks. As an example of a case where molecular tests are increasing capabilities, one person mentioned that some countries such as India that have historically avoided TB culture capabilities due to biosafety concerns are now embracing TB molecular tests. While some expressed frustration with the low accuracy of some molecular tests, one person countered that some of the tests being replaced, such as direct smear microscopy, which detects 55 percent of TB cases, also have low accuracy. Several people indicated that they were looking forward to the opportunities that will accompany the increasing availability of high-throughput DNA sequencing.
Several participants observed that since diagnostics are developing quickly, biosafety and biosecurity systems must keep pace. TB was discussed as a case in point. As BSL-3 facilities are no longer necessary for most common TB testing (i.e., everything except resistance testing for drugs other than rifampicin), countries with a low incidence of multidrug-resistant tuberculosis (MDR-TB) may need many TB labs but only a single BSL-3 lab for drug-resistance testing. The Global Fund to Fight AIDS, Tuberculosis, and Malaria, however, is just starting to be open to using non-BSL-3 labs for TB work, and many previously approved requests for BSL-3 labs are proceeding.
A number of participants elaborated on the strengths and limitations of point-of-care (POC) tests, which are usually performed in a BSC using blood or sera and typically take 5-30 minutes. Several people remarked that while the quality of individual POC tests is highly variable and the tests are relatively expensive, there is a need for good POC tests in both medical and veterinary practices. One person mentioned that while POC tests cannot be used to rule out an infection, they serve as a useful orientation for clinicians and could reduce the need to transport potentially high-risk patients. Someone else noted that people do tend to believe the positives and that even low-quality tests can be informative about an outbreak. Another pointed out that as POC tests are neither 100% sensitive nor specific, clinicians in the field should be trained to recognize diseases of concern.
During a number of these sessions, some participants expressed frustration with what they perceived as unnecessarily restrictive transport, import, and export regulations. Individuals
complained about burdensome paperwork, precautions out of proportion to the risk, long delays to obtain permission, and multiple levels of bureaucracy that could block a transfer. They noted that some countries will not allow the import of drug-resistant TB strains while in other cases intellectual property restrictions block pathogen export. One mentioned that most African countries will not allow samples to be transported by air. Another person recounted that even when regulations permit transfer of samples awaiting diagnostics, inadequate transportation infrastructure in some regions may cause life-threatening delays. One person did acknowledge, however, that sample transport, particularly for pathogen collections, does present risks.
Various participants also identified consequences of the current situation:
• Labs that aspire to become international reference labs, which must be able to receive unknown samples, typically need to engage in detailed conversations with regulators to put transfer agreements in place. Additionally, regulations effectively bar some labs from ever serving as reference labs.
• Difficulty in obtaining samples when needed encourages facilities to create or retain their own reference collections.
• An inability to ship samples for timely diagnostics encourages countries and localities to build their own laboratories.
• Particularly burdensome transport regulations, such as the United States Select Agent rules that require individuals who ship select agents within or into the United States to obtain a United States security clearance, discourages potential, foreign collaborators.
To ameliorate some of these problems, several participants suggested continuing to engage the International Air Transport Association (IATA), the U.N. Committee on Dangerous Goods Transport, and national governments in a dialog to better define the requirements for safe transport and to accurately characterize the associated risks. One suggested that TB, due to the large potential number of samples and the comparatively well-characterized risks might be an ideal starting point for a conversation, particularly as transporting TB samples is much less risky than transporting TB patients.
One person indicated that countries are increasingly relying on in-country DNA sequencing capabilities rather than sample export for diagnoses. While valuable for public health independence and IHR compliance, the individual worried that such a model might stymie international characterization of emerging threat pathogens.
As reference collections present security risks, participants discussed both the demand for pathogen collections as well as the ethics of destroying collections. Many indicated that research, particularly pathogenesis studies, almost always requires viable organisms that are typically obtained from collections. In contrast, many felt that diagnostics, particularly for organisms like TB where the tests are standardized and use internal controls, created much less demand.
Opinions were mixed concerning whether pathogen collections should ever be destroyed, and one person cited that the recent eradication of rinderpest and the accompanying debate over whether or not to destroy all samples as a topical example. One viewpoint maintained that dangerous pathogens are inherently interesting and that we should retain and study them to understand the source of the pathogenicity. Another viewpoint argued for destroying samples when transporting them to more secure, centralized locations represented an unacceptably high risk, particularly when the samples are unlabeled or without context.