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1.  Triage and Management of Accidental Laboratory Exposures to Biosafety Level-3 and -4 Agents 
The recent expansion of biocontainment laboratory capacity in the United States has drawn attention to the possibility of occupational exposures to BSL-3 and -4 agents and has prompted a reassessment of medical management procedures and facilities to deal with these contingencies. A workshop hosted by the National Interagency Biodefense Campus was held in October 2007 and was attended by representatives of all existing and planned BSL-4 research facilities in the U.S. and Canada. This report summarizes important points of discussion and recommendations for future coordinated action, including guidelines for the engineering and operational controls appropriate for a hospital care and isolation unit. Recommendations pertained to initial management of exposures (ie, immediate treatment of penetrating injuries, reporting of exposures, initial evaluation, and triage). Isolation and medical care in a referral hospital (including minimum standards for isolation units), staff recruitment and training, and community outreach also were addressed. Workshop participants agreed that any unit designated for the isolation and treatment of laboratory employees accidentally infected with a BSL-3 or -4 pathogen should be designed to maximize the efficacy of patient care while minimizing the risk of transmission of infection. Further, participants concurred that there is no medically based rationale for building care and isolation units to standards approximating a BSL-4 laboratory. Instead, laboratory workers accidentally exposed to pathogens should be cared for in hospital isolation suites staffed by highly trained professionals following strict infection control procedures.
doi:10.1089/bsp.2009.0002
PMCID: PMC2749272  PMID: 19634998
2.  Preparing a Community Hospital to Manage Work-related Exposures to Infectious Agents in BioSafety Level 3 and 4 Laboratories 
Emerging Infectious Diseases  2010;16(3):373-378.
Training increased willingness of healthcare workers to care for patients with all types of communicable diseases.
Construction of new BioSafety Level (BSL) 3 and 4 laboratories has raised concerns regarding provision of care to exposed workers because of healthcare worker (HCW) unfamiliarity with precautions required. When the National Institutes of Health began construction of a new BSL-4 laboratory in Hamilton, Montana, USA, in 2005, they contracted with St. Patrick Hospital in Missoula, Montana, for care of those exposed. A care and isolation unit is described. We developed a training program for HCWs that emphasized the optimal use of barrier precautions and used pathogen-specific modules and simulations with mannequins and fluorescent liquids that represented infectious body fluids. The facility and training led to increased willingness among HCWs to care for patients with all types of communicable diseases. This model may be useful for other hospitals, whether they support a BSL-4 facility, are in the proximity of a BSL-3 facility, or are interested in upgrading their facilities to prepare for exotic and novel infectious diseases.
doi:10.3201/eid1603.091485
PMCID: PMC3322039  PMID: 20202409
Viral hemorrhagic fevers; occupational exposure; curriculum; biosafety level; viruses; community hospital; perspective
3.  Biosafety Training and Incident-reporting Practices in the United States: A 2008 Survey of Biosafety Professionals 
Concern over the adequacy of biosafety training and incident-reporting practices within biological laboratories in the United States has risen in recent years due to the increase in research on infectious diseases and the concomitant rise in the number of biocontainment laboratories. Reports of laboratory-acquired infections and delays in reporting such incidents have also contributed to the concern. Consequently, biosafety training and incident-reporting practices are being given considerable attention by both the executive branch and Congress. We conducted a 51-question survey of biosafety professionals in June 2008 to capture information on methods used to train new laboratory workers within biosafety level 2 (BSL-2) laboratories, animal biosafety level 2 (ABSL-2) laboratories, biosafety level 3 (BSL-3) laboratories, and animal biosafety level 3 (ABSL-3) laboratories. The survey results suggest nearly all senior scientists, faculty, staff, and students working in these biocontainment laboratories are required to have biosafety training, and three-quarters of respondents indicated a biosafety or environmental health and safety professional provides explicit instructions on reporting incidents to each new lab worker. Only half of the respondents with BSL-2/ABSL-2 laboratories at their institution and 59% of respondents from institutions with BSL-3/ABSL-3 laboratories indicated custodial or maintenance workers are required to receive biosafety training at the BSL-2/ABSL-2 and BSL-3/ABSL-3 levels, respectively. Opportunities for targeted improvement such as providing training to non-traditional laboratory workers (e.g., custodians, maintenance workers) and posting laboratory incident-reporting protocols on institutional environmental health and safety websites may exist. Variations in biosafety training requirements, incident-reporting practices, and attitudes towards laboratory safety revealed through this survey of biosafety professionals also support the development of core competencies in biosafety practice that could lead to more uniform practices and robust safety cultures.
PMCID: PMC2947438  PMID: 20890389
4.  On the implementation of the Biological Threat Reduction Program in the Republic of Uzbekistan 
Objective
To review the implementation of the Biological Threat Reduction Program (BTRP) of the U.S. Defense Threat Reduction Agency in the Republic of Uzbekistan since 2004.
Introduction
The Biological Threat Reduction Program (BTRP) has been being implemented in the Republic of Uzbekistan since 2004 within the framework of the Agreement between the Government of the Republic of Uzbekistan and the Government of the United States of America Concerning Cooperation in the Area of the Promotion of Defense Relations and the Prevention of Proliferation of Weapons of Mass Destruction of 06.05.2001. Threat agent detection and response activities that target a list of especially dangerous pathogens are being carried out under the BTRP within the health care system of Uzbekistan. This presentation reviews some of the achievements of the program to date.
Results
BTRP, in partnership with the Government of Uzbekistan, has funded the establishment of five Regional Diagnostic Laboratories (RDL) and ten Epidemiological Support Units (ESU), operated by the Ministry of Health of Uzbekistan, which are intended to improve the diagnosis of quarantine and especially dangerous infections, and to ensure timely preventive and anti-epidemic measures.
RDLs provide a high level of biosafety and biosecurity to conduct rapid laboratory diagnostics (PCR, ELISA) of especially dangerous infections. RDLs are equipped with up-to-date diagnostic laboratory equipment that conforms to internationals standards, as well as with all necessary consumables.
Personnel of RDLs have been appropriately trained in epidemiology, clinical and diagnostic techniques for especially dangerous infections, including such state-of-the-art techniques as rapid PCR and ELISA diagnostics, as well as in work and equipment operation safety regulations.
Epidemiological Support Units (ESU) have been established on the basis of the Especially Dangerous Infections Divisions of Oblast, city and Rayon Centers for State Sanitary and Epidemiological Surveillance (SES) of the Ministry of Health. The BTRP ESU efforts include renovation activities, supply and installation of appropriate equipment for rapid laboratory diagnostics, and vehicles.
ESUs are meant to ensure emergency notification in cases of suspected occurrence of quarantine and especially dangerous infections, for timely implementation of anti-epidemic and preventive measures.
Three Cooperative Biological Research projects on quarantine and especially dangerous infections have been implemented within BTRP with the financial support of the US Civilian Research and Development Foundation (CRDF).
To ensure the sustainability of training and availability of a pool of skilled personnel, a training laboratory is to be established at the Tashkent Institute for Post-Graduate Medical Education (TIPME) to train personnel of RDLs and ESUs. The training laboratory will fully replicate the setup of a BSL-2 Regional Diagnostic Laboratory, but will maintain no operations with live pathogens.
Conclusions
The implementation of BTRP within the health care system of the Republic of Uzbekistan contributes to the stable and sustainable well-being in the population of the country.
PMCID: PMC3692828
Biological Threat Reduction Program; Especially dangerous pathogens; Resource-poor setting
5.  Framework for Leadership and Training of Biosafety Level 4 Laboratory Workers 
Emerging Infectious Diseases  2008;14(11):1685-1688.
One-sentence summary for table of contents: Training should include theoretical consideration of biocontainment principles, practical hands-on training, and mentored on-the-job experience.
Construction of several new Biosafety Level 4 (BSL-4) laboratories and expansion of existing operations have created an increased international demand for well-trained staff and facility leaders. Directors of most North American BSL-4 laboratories met and agreed upon a framework for leadership and training of biocontainment research and operations staff. They agreed on essential preparation and training that includes theoretical consideration of biocontainment principles, practical hands-on training, and mentored on-the-job experiences relevant to positional responsibilities as essential preparation before a person’s independent access to a BSL-4 facility. They also agreed that the BSL-4 laboratory director is the key person most responsible for ensuring that staff members are appropriately prepared for BSL-4 operations. Although standardized certification of training does not formally exist, the directors agreed that facility-specific, time-limited documentation to recognize specific skills and experiences of trained persons is needed.
doi:10.3201/eid1411.080741
PMCID: PMC2630756  PMID: 18976549
BSL-4 laboratory; containment laboratories; training; perspective
6.  Biocontainment in Gain-of-Function Infectious Disease Research 
mBio  2012;3(5):e00290-12.
ABSTRACT
The discussion of H5N1 influenza virus gain-of-function research has focused chiefly on its risk-to-benefit ratio. Another key component of risk is the level of containment employed. Work is more expensive and less efficient when pursued at biosafety level 4 (BSL-4) than at BSL-3 or at BSL-3 as modified for work with agricultural pathogens (BSL-3-Ag). However, here too a risk-to-benefit ratio analysis is applicable. BSL-4 procedures mandate daily inspection of facilities and equipment, monitoring of personnel for signs and symptoms of disease, and logs of dates and times that personnel, equipment, supplies, and samples enter and exit containment. These measures are not required at BSL-3 or BSL-3-Ag. Given the implications of inadvertent or deliberate release of high-threat pathogens with pandemic potential, it is imperative that the World Health Organization establish strict criteria for biocontainment that can be fairly applied in the developing world, as well as in more economically developed countries.
doi:10.1128/mBio.00290-12
PMCID: PMC3484385  PMID: 23047747
7.  The Impact of Regulations, Safety Considerations and Physical Limitations on Research Progress at Maximum Biocontainment 
Viruses  2012;4(12):3932-3951.
We describe herein, limitations on research at biosafety level 4 (BSL-4) containment laboratories, with regard to biosecurity regulations, safety considerations, research space limitations, and physical constraints in executing experimental procedures. These limitations can severely impact the number of collaborations and size of research projects investigating microbial pathogens of biodefense concern. Acquisition, use, storage, and transfer of biological select agents and toxins (BSAT) are highly regulated due to their potential to pose a severe threat to public health and safety. All federal, state, city, and local regulations must be followed to obtain and maintain registration for the institution to conduct research involving BSAT. These include initial screening and continuous monitoring of personnel, controlled access to containment laboratories, accurate and current BSAT inventory records. Safety considerations are paramount in BSL-4 containment laboratories while considering the types of research tools, workflow and time required for conducting both in vivo and in vitro experiments in limited space. Required use of a positive-pressure encapsulating suit imposes tremendous physical limitations on the researcher. Successful mitigation of these constraints requires additional time, effort, good communication, and creative solutions. Test and evaluation of novel vaccines and therapeutics conducted under good laboratory practice (GLP) conditions for FDA approval are prioritized and frequently share the same physical space with important ongoing basic research studies. The possibilities and limitations of biomedical research involving microbial pathogens of biodefense concern in BSL-4 containment laboratories are explored in this review.
doi:10.3390/v4123932
PMCID: PMC3528297  PMID: 23342380
biocontainment; biosafety level 4 (BSL-4); biological select agents and toxins (BSAT); positive pressure suit; biodefense; biosecurity; ebola virus; highly pathogenic viruses; limitations; collaboration
8.  Characterization of systemic and pneumonic murine models of plague infection using a conditionally virulent strain 
Yersinia pestis causes bubonic and pneumonic plague in humans. The pneumonic infection is the most severe and invariably fatal if untreated. Because of its high virulence, ease of delivery and precedent of use in warfare, Y. pestis is considered a potential bioterror agent. No licensed plague vaccine is currently available in the US. Laboratory research with virulent strains requires appropriate biocontainment (i.e., Biosafety Level 3 (BSL-3) for procedures that generate aerosol/droplets) and secure facilities that comply with federal select agent regulations. To assist in the identification of promising vaccine candidates during the early phases of development, we characterized mouse models of systemic and pneumonic plague infection using the Y. pestis strain EV76, an attenuated human vaccine strain that can be rendered virulent in mice under in vivo iron supplementation. Mice inoculated intranasally or intravenously with Y. pestis EV76 in the presence of iron developed a systemic and pneumonic plague infection that resulted in disease and lethality. Bacteria replicated and severely compromised the spleen, liver and lungs. Susceptibility was age dependent, with younger mice being more vulnerable to pneumonic infection. We used these models of infection to assess the protective capacity of newly developed Salmonella-based plague vaccines. The protective outcome varied depending on the route and dose of infection. Protection was associated with the induction of specific immunological effectors in systemic/mucosal compartments. The models of infection described could serve as safe and practical tools for identifying promising vaccine candidates that warrant further potency evaluation using fully virulent strains in BSL-3 settings.
doi:10.1016/j.cimid.2012.10.005
PMCID: PMC3594127  PMID: 23195858
Yersinia pestis; plague infection model; EV76; plague vaccines
9.  A Burkholderia pseudomallei ΔpurM Mutant Is Avirulent in Immunocompetent and Immunodeficient Animals: Candidate Strain for Exclusion from Select-Agent Lists▿  
Infection and Immunity  2010;78(7):3136-3143.
Burkholderia pseudomallei causes the disease melioidosis in humans and is classified as a category B select agent. Research utilizing this pathogen is highly regulated in the United States, and even basic studies must be conducted in biosafety level 3 (BSL-3) facilities. There is currently no attenuated B. pseudomallei strain available that is excluded from select-agent regulations and can be safely handled at BSL-2 facilities. To address this need, we created Bp82 and Bp190, which are ΔpurM derivatives of B. pseudomallei strains 1026b and K96243 that are deficient in adenine and thiamine biosynthesis but replication competent in vitro in rich medium. A series of animal challenge studies was conducted to ensure that these strains were fully attenuated. Whereas the parental strains 1026b and K96243 and the complemented mutants Bp410 and Bp454 were virulent in BALB/c mice following intranasal inoculation, the ΔpurM mutants Bp82 and Bp190 were avirulent even when they were administered at doses 4 logs higher than the doses used for the parental strains. Animals challenged with high doses of the ΔpurM mutants rapidly cleared the bacterium from tissues (lung, liver, and spleen) and remained free of culturable bacteria for the duration of the experiments (up to 60 days postinfection). Moreover, highly susceptible 129/SvEv mice and immune incompetent mice (IFN-γ−/−, SCID) were resistant to challenges with ΔpurM mutant Bp82. This strain was also avirulent in the Syrian hamster challenge model. We concluded that ΔpurM mutant Bp82 is fully attenuated and safe for use under BSL-2 laboratory conditions and thus is a candidate for exclusion from the select-agent list.
doi:10.1128/IAI.01313-09
PMCID: PMC2897367  PMID: 20404077
10.  Identification of Virulence Determinants within the L Genomic Segment of the Pichinde Arenavirus 
Journal of Virology  2013;87(12):6635-6643.
Several arenaviruses are responsible for causing viral hemorrhagic fevers (VHF) in humans. Lassa virus (LASV), the causative agent of Lassa fever, is a biosafety level 4 (BSL4) pathogen that requires handling in BSL4 facilities. In contrast, the Pichinde arenavirus (PICV) is a BSL2 pathogen that can cause hemorrhagic fever-like symptoms in guinea pigs that resemble those observed in human Lassa fever. Comparative sequence analysis of the avirulent P2 strain of PICV and the virulent P18 strain shows a high degree of sequence homology in the bisegmented genome between the two strains despite the polarized clinical outcomes noted for the infected animals. Using reverse genetics systems that we have recently developed, we have mapped the sequence changes in the large (L) segment of the PICV genome that are responsible for the heightened virulence phenotype of the P18 strain. By monitoring the degree of disease severity and lethality caused by the different mutant viruses, we have identified specific residues located within the viral L polymerase gene encoded on the L segment essential for mediating disease pathogenesis. Through quantitative reverse transcription-PCR (RT-PCR) analysis, we have confirmed that the same set of residues is responsible for the increased viral replicative potential of the P18 strain and its heightened disease severity in vivo. Our laboratory findings serve to reinforce field observations that a high level of viremia often correlates with severe disease outcomes in LASV-infected patients.
doi:10.1128/JVI.00044-13
PMCID: PMC3676128  PMID: 23552411
11.  Generation of VSV Pseudotypes Using Recombinant ΔG-VSV for Studies on Virus Entry, Identification of Entry Inhibitors, and Immune Responses to Vaccines 
Journal of virological methods  2010;169(2):365-374.
Vesicular stomatitis virus (VSV) is a prototypic enveloped animal virus that has been used extensively to study virus entry, replication and assembly due to its broad host range and robust replication properties in a wide variety of mammalian and insect cells. Studies on VSV assembly led to the creation of a recombinant VSV in which the glycoprotein (G) gene was deleted. This recombinant (rVSV-ΔG) has been used to produce VSV pseudotypes containing the envelope glycoproteins of heterologous viruses, including viruses that require high-level biocontainment; however, because the infectivity of rVSV-ΔG pseudotypes is restricted to a single round of replication the analysis can be performed using biosafety level 2 (BSL-2) containment. As such, rVSV-ΔG pseudotypes have facilitated the analysis of virus entry for numerous viral pathogens without the need for specialized containment facilities. The pseudotypes also provide a robust platform to screen libraries for entry inhibitors and to evaluate the neutralizing antibody responses following vaccination. This manuscript describes methods to produce and titer rVSV-ΔG pseudotypes. Procedures to generate rVSV-ΔG stocks and to quantify virus infectivity are also described. These protocols should allow any laboratory knowledgeable in general virological and cell culture techniques to produce successfully replication-restricted rVSV-ΔG pseudotypes for subsequent analysis.
doi:10.1016/j.jviromet.2010.08.006
PMCID: PMC2956192  PMID: 20709108
Pseudotypes; VSV; transfection; envelope glycoprotein; virus entry; receptor identification; high-throughput screening; vaccines
12.  The Use of NanoTrap Particles as a Sample Enrichment Method to Enhance the Detection of Rift Valley Fever Virus 
Background
Rift Valley Fever Virus (RVFV) is a zoonotic virus that is not only an emerging pathogen but is also considered a biodefense pathogen due to the threat it may cause to public health and national security. The current state of diagnosis has led to misdiagnosis early on in infection. Here we describe the use of a novel sample preparation technology, NanoTrap particles, to enhance the detection of RVFV. Previous studies demonstrated that NanoTrap particles lead to both 100 percent capture of protein analytes as well as an improvement of more than 100-fold in sensitivity compared to existing methods. Here we extend these findings by demonstrating the capture and enrichment of viruses.
Results
Screening of NanoTrap particles indicated that one particle, NT53, was the most efficient at RVFV capture as demonstrated by both qRT-PCR and plaque assays. Importantly, NT53 capture of RVFV resulted in greater than 100-fold enrichment from low viral titers when other diagnostics assays may produce false negatives. NT53 was also capable of capturing and enhancing RVFV detection from serum samples. RVFV that was inactivated through either detergent or heat treatment was still found bound to NT53, indicating the ability to use NanoTrap particles for viral capture prior to transport to a BSL-2 environment. Furthermore, both NP-40-lysed virus and purified RVFV RNA were bound by NT53. Importantly, NT53 protected viral RNA from RNase A degradation, which was not observed with other commercially available beads. Incubation of RVFV samples with NT53 also resulted in increased viral stability as demonstrated through preservation of infectivity at elevated temperatures. Finally, NanoTrap particles were capable of capturing VEEV and HIV, demonstrating the broad applicability of NanoTrap particles for viral diagnostics.
Conclusion
This study demonstrates NanoTrap particles are capable of capturing, enriching, and protecting RVFV virions. Furthermore, the use of NanoTrap particles can be extended to a variety of viruses, including VEEV and HIV.
Author Summary
There is a dire need for fast and efficient diagnosis of many viral diseases. Our research specifically looked at RVFV, a virus that can only be worked with in biosafety level 3 (BSL-3) laboratories, and its capture with NanoTrap particles. NanoTrap particles are hydrogel particles that contain internal affinity baits. They have previously been used in the capture of several analytes, but never in the capture of whole virus particles. We were not only able to capture and detect RVFV at very low titers from both media and serum, but we were also able to inactivate the virus, which allows for its safe transport to BSL-2 laboratories. While there are other commercially available beads that can also capture virus, NanoTrap particles are the only beads that can protect the viral RNA from enzymatic degradation. Furthermore, we demonstrated that whole virus detection with NanoTrap particles is not limited to only RVFV, but that NanoTrap particles can be used to detect other viruses such as Human Immunodeficiency Virus (HIV) and Venezuelan Equine Encephalitis Virus (VEEV).
doi:10.1371/journal.pntd.0002296
PMCID: PMC3701711  PMID: 23861988
13.  Laboratory-associated infections and biosafety. 
Clinical Microbiology Reviews  1995;8(3):389-405.
An estimated 500,000 laboratory workers in the United States are at risk of exposure to infectious agents that cause disease ranging from inapparent to life-threatening infections, but the precise risk to a given worker unknown. The emergence of human immunodeficiency virus and hantavirus, the continuing problem of hepatitis B virus, and the reemergence of Mycobacterium tuberculosis have renewed interest in biosafety for the employees of laboratories and health care facilities. This review examines the history, the causes, and the methods for prevention of laboratory-associated infections. The initial step in a biosafety program is the assessment of risk to the employee. Risk assessment guidelines include the pathogenicity of the infectious agent, the method of transmission, worker-related risk factors, the source and route of infection, and the design of the laboratory facility. Strategies for the prevention and management of laboratory-associated infections are based on the containment of the infectious agent by physical separation from the laboratory worker and the environment, employee education about the occupational risks, and availability of an employee health program. Adherence to the biosafety guidelines mandated or proposed by various governmental and accrediting agencies reduces the risk of an occupational exposure to infectious agents handled in the workplace.
PMCID: PMC174631  PMID: 7553572
14.  Automated Detection of Infectious Disease Outbreaks in Hospitals: A Retrospective Cohort Study 
PLoS Medicine  2010;7(2):e1000238.
Susan Huang and colleagues describe an automated statistical software, WHONET-SaTScan, its application in a hospital, and the potential it has to identify hospital infection clusters that had escaped routine detection.
Background
Detection of outbreaks of hospital-acquired infections is often based on simple rules, such as the occurrence of three new cases of a single pathogen in two weeks on the same ward. These rules typically focus on only a few pathogens, and they do not account for the pathogens' underlying prevalence, the normal random variation in rates, and clusters that may occur beyond a single ward, such as those associated with specialty services. Ideally, outbreak detection programs should evaluate many pathogens, using a wide array of data sources.
Methods and Findings
We applied a space-time permutation scan statistic to microbiology data from patients admitted to a 750-bed academic medical center in 2002–2006, using WHONET-SaTScan laboratory information software from the World Health Organization (WHO) Collaborating Centre for Surveillance of Antimicrobial Resistance. We evaluated patients' first isolates for each potential pathogenic species. In order to evaluate hospital-associated infections, only pathogens first isolated >2 d after admission were included. Clusters were sought daily across the entire hospital, as well as in hospital wards, specialty services, and using similar antimicrobial susceptibility profiles. We assessed clusters that had a likelihood of occurring by chance less than once per year. For methicillin-resistant Staphylococcus aureus (MRSA) or vancomycin-resistant enterococci (VRE), WHONET-SaTScan–generated clusters were compared to those previously identified by the Infection Control program, which were based on a rule-based criterion of three occurrences in two weeks in the same ward. Two hospital epidemiologists independently classified each cluster's importance. From 2002 to 2006, WHONET-SaTScan found 59 clusters involving 2–27 patients (median 4). Clusters were identified by antimicrobial resistance profile (41%), wards (29%), service (13%), and hospital-wide assessments (17%). WHONET-SaTScan rapidly detected the two previously known gram-negative pathogen clusters. Compared to rule-based thresholds, WHONET-SaTScan considered only one of 73 previously designated MRSA clusters and 0 of 87 VRE clusters as episodes statistically unlikely to have occurred by chance. WHONET-SaTScan identified six MRSA and four VRE clusters that were previously unknown. Epidemiologists considered more than 95% of the 59 detected clusters to merit consideration, with 27% warranting active investigation or intervention.
Conclusions
Automated statistical software identified hospital clusters that had escaped routine detection. It also classified many previously identified clusters as events likely to occur because of normal random fluctuations. This automated method has the potential to provide valuable real-time guidance both by identifying otherwise unrecognized outbreaks and by preventing the unnecessary implementation of resource-intensive infection control measures that interfere with regular patient care.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Admission to a hospital is often a life-saving necessity—individuals injured in a road accident, for example, may need immediate medical and surgical attention if they are to survive. Unfortunately, many patients acquire infections, some of which are life-threatening, during their stay in a hospital. The World Health Organization has estimated that, globally, 8.7% of hospital patients develop hospital-acquired infections (infections that are identified more than two days after admission to hospital). In the US alone, 2 million people develop a hospital-acquired infection every year, often an infection of a surgical wound, or a urinary tract or lung infection. Infections are common among hospital patients because increasing age or underlying illnesses can reduce immunity to infection and because many medical and surgical procedures bypass the body's natural protective barriers. In addition, poor infection control practices can facilitate the transmission of bacteria—including meticillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE)—and other infectious agents (pathogens) between patients.
Why Was This Study Done?
Sometimes, the number of cases of hospital-acquired infections increases unexpectedly or a new infection emerges. Such clusters account for relatively few health care–associated infections, but, because they may arise from the transmission of a pathogen within a hospital, they need to be rapidly identified and measures implemented (for example, isolation of affected patients) to stop transmission if an outbreak is confirmed. Currently, the detection of clusters of hospital-acquired infections is based on simple rules, such as the occurrence of three new cases of a single pathogen in two weeks on the same ward. This rule-based approach relies on the human eye to detect infection clusters within microbiology data (information collected on the pathogens isolated from patients), it focuses on a few pathogens, and it does not consider the random variation in infection rates or the possibility that clusters might be associated with shared facilities rather than with individual wards. In this study, the researchers test whether an automated statistical system can detect outbreaks of hospital-acquired infections quickly and accurately.
What Did the Researchers Do and Find?
The researchers combined two software packages used to track diseases in populations to create the WHONET-SaTScan cluster detection tool. They then compared the clusters of hospital-acquired infection identified by the new tool in microbiology data from a 750-bed US academic medical center with those generated by the hospital's infection control program, which was largely based on the simple rule described above. WHONET-SaTScan found 59 clusters of infection that occurred between 2002 and 2006, about three-quarters of which were identified by characteristics other than a ward-based location. Nearly half the cluster alerts were generated on the basis of shared antibiotic susceptibility patterns. Although WHONET-SaTScan identified all the clusters previously identified by the hospital's infection control program, it classified most of these clusters as likely to be the result of normal random variations in infection rates rather than the result of “true” outbreaks. By contrast, the hospital's infection control department only identified three of the 59 statistically significant clusters identified by WHONET-SaTScan. Furthermore, the new tool identified six previously unknown MRSA outbreaks and four previously unknown VRE outbreaks. Finally, two hospital epidemiologists (scientists who study diseases in populations) classified 95% of the clusters detected by WHONET-SaTScan as worthy of consideration by the hospital infection control team and a quarter of the clusters as warranting active investigation or intervention.
What Do These Findings Mean?
These findings suggest that automated statistical software should be able to detect clusters of hospital-acquired infections that would escape detection using routine rule-based systems. Importantly, they also suggest that an automated system would be able to discount a large number of supposed outbreaks identified by rule-based systems. These findings need to be confirmed in other settings and in prospective studies in which the outcomes of clusters detected with WHONET-SaTScan are carefully analyzed. For now, however, these findings suggest that automated statistical tools could provide hospital infection control experts with valuable real-time guidance by identifying outbreaks that would be missed by routine detection methods and by preventing the implementation of intensive and costly infection control measures in situations where they are unnecessary.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1000238.
The World Health Organization's Prevention of Hospital-Acquired Infections, A Practical Guide contains detailed information on all aspects of hospital-acquired infections
MedlinePlus provides links to information on infection control in hospitals (in English and Spanish)
The US Centers for Disease Control and Prevention also provides information on infectious diseases in health care settings (in English and Spanish)
The WHONET/Baclink software and the SatScan software, the two components of WHONET-SaTScan are both available on the internet (the WHONET-SaTScan cluster detection tool is freely available as part of the version of WHONET/BacLink released June 2009)
doi:10.1371/journal.pmed.1000238
PMCID: PMC2826381  PMID: 20186274
15.  Evaluation of Chimeric Japanese Encephalitis and Dengue Viruses for Use in Diagnostic Plaque Reduction Neutralization Tests▿  
The plaque reduction neutralization test (PRNT) is a specific serological test used to identify and confirm arbovirus infection in diagnostic laboratories and monitor immunological protection in vaccine recipients. Wild-type (wt) viruses used in the PRNT may be difficult to grow and plaque titrate, such as the dengue viruses (DENV), and/or may require biosafety level 3 (BSL3) containment, such as West Nile virus (WNV), St. Louis encephalitis virus (SLEV), and Japanese encephalitis virus (JEV). These requirements preclude their use in diagnostic laboratories with only BSL2 capacity. In addition, wt JEV falls under the jurisdiction of the select-agent program and can be used only in approved laboratories. The chimeric vaccine viruses ChimeriVax-WNV and -SLEV have previously been shown to elicit antibody reactivity comparable to that of parental wt WNV and SLEV. ChimeriVax viruses provide advantages for PRNT, as follows: they grow more rapidly than most wt flaviviruses, produce large plaques, require BSL2 conditions, and are not under select-agent restrictions. We evaluated the ChimeriVax-DENV serotype 1 (DENV1), -DENV2, -DENV3, -DENV4, and -JEV for use in PRNT on sera from DENV- and JEV-infected patients and from JEV vaccine recipients. Serostatus agreement was 100% between the ChimeriVax-DENV serotypes and wt prototype DENV and 97% overall with ChimeriVax-JEV compared to prototype Nakayama JEV, 92% in a subgroup of JEV vaccine recipients, and 100% in serum from encephalitis patients naturally infected with JEV. ChimeriVax-DENV and -JEV plaque phenotype and BSL2 requirements, combined with sensitive and specific reactivity, make them good substitutes for wt DENV and JEV in PRNT in public health diagnostic laboratories.
doi:10.1128/CVI.00095-09
PMCID: PMC2708403  PMID: 19458204
16.  Air Cleaning Technologies 
Executive Summary
Objective
This health technology policy assessment will answer the following questions:
When should in-room air cleaners be used?
How effective are in-room air cleaners?
Are in-room air cleaners that use combined HEPA and UVGI air cleaning technology more effective than those that use HEPA filtration alone?
What is the Plasmacluster ion air purifier in the pandemic influenza preparation plan?
The experience of severe acute respiratory syndrome (SARS) locally, nationally, and internationally underscored the importance of administrative, environmental, and personal protective infection control measures in health care facilities. In the aftermath of the SARS crisis, there was a need for a clearer understanding of Ontario’s capacity to manage suspected or confirmed cases of airborne infectious diseases. In so doing, the Walker Commission thought that more attention should be paid to the potential use of new technologies such as in-room air cleaning units. It recommended that the Medical Advisory Secretariat of the Ontario Ministry of Health and Long-Term Care evaluate the appropriate use and effectiveness of such new technologies.
Accordingly, the Ontario Health Technology Advisory Committee asked the Medical Advisory Secretariat to review the literature on the effectiveness and utility of in-room air cleaners that use high-efficiency particle air (HEPA) filters and ultraviolet germicidal irradiation (UVGI) air cleaning technology.
Additionally, the Ontario Health Technology Advisory Committee prioritized a request from the ministry’s Emergency Management Unit to investigate the possible role of the Plasmacluster ion air purifier manufactured by Sharp Electronics Corporation, in the pandemic influenza preparation plan.
Clinical Need
Airborne transmission of infectious diseases depends in part on the concentration of breathable infectious pathogens (germs) in room air. Infection control is achieved by a combination of administrative, engineering, and personal protection methods. Engineering methods that are usually carried out by the building’s heating, ventilation, and air conditioning (HVAC) system function to prevent the spread of airborne infectious pathogens by diluting (dilution ventilation) and removing (exhaust ventilation) contaminated air from a room, controlling the direction of airflow and the air flow patterns in a building. However, general wear and tear over time may compromise the HVAC system’s effectiveness to maintain adequate indoor air quality. Likewise, economic issues may curtail the completion of necessary renovations to increase its effectiveness. Therefore, when exposure to airborne infectious pathogens is a risk, the use of an in-room air cleaner to reduce the concentration of airborne pathogens and prevent the spread of airborne infectious diseases has been proposed as an alternative to renovating a HVAC system.
Airborne transmission is the spread of infectious pathogens over large distances through the air. Infectious pathogens, which may include fungi, bacteria, and viruses, vary in size and can be dispersed into the air in drops of moisture after coughing or sneezing. Small drops of moisture carrying infectious pathogens are called droplet nuclei. Droplet nuclei are about 1 to 5μm in diameter. This small size in part allows them to remain suspended in the air for several hours and be carried by air currents over considerable distances. Large drops of moisture carrying infectious pathogens are called droplets. Droplets being larger than droplet nuclei, travel shorter distances (about 1 metre) before rapidly falling out of the air to the ground. Because droplet nuclei remain airborne for longer periods than do droplets, they are more amenable to engineering infection control methods than are droplets.
Droplet nuclei are responsible for the airborne transmission of infectious diseases such as tuberculosis, chicken pox (varicella), measles (rubeola), and dessiminated herpes zoster, whereas close contact is required for the direct transmission of infectious diseases transmitted by droplets, such as influenza (the flu) and SARS.
The Technology
In-room air cleaners are supplied as portable or fixed devices. Fixed devices can be attached to either a wall or ceiling and are preferred over portable units because they have a greater degree of reliability (if installed properly) for achieving adequate room air mixing and airflow patterns, which are important for optimal effectiveness.
Through a method of air recirculation, an in-room air cleaner can be used to increase room ventilation rates and if used to exhaust air out of the room it can create a negative-pressure room for airborne infection isolation (AII) when the building’s HVAC system cannot do so. A negative-pressure room is one where clean air flows into the room but contaminated air does not flow out of it. Contaminated room air is pulled into the in-room air cleaner and cleaned by passing through a series of filters, which remove the airborne infectious pathogens. The cleaned air is either recirculated into the room or exhausted outside the building. By filtering contaminated room air and then recirculating the cleaned air into the room, an in-room air cleaner can improve the room’s ventilation. By exhausting the filtered air to the outside the unit can create a negative-pressure room. There are many types of in-room air cleaners. They vary widely in the airflow rates through the unit, the type of air cleaning technology used, and the technical design.
Crucial to maximizing the efficiency of any in-room air cleaner is its strategic placement and set-up within a room, which should be done in consultation with ventilation engineers, infection control experts, and/or industrial hygienists. A poorly positioned air cleaner may disrupt airflow patterns within the room and through the air cleaner, thereby compromising its air cleaning efficiency.
The effectiveness of an in-room air cleaner to remove airborne pathogens from room air depends on several factors, including the airflow rate through the unit’s filter and the airflow patterns in the room. Tested under a variety of conditions, in-room air cleaners, including portable or ceiling mounted units with either a HEPA or a non-HEPA filter, portable units with UVGI lights only, or ceiling mounted units with combined HEPA filtration and UVGI lights, have been estimated to be between 30% and 90%, 99% and 12% and 80% effective, respectively. However, and although their effectiveness is variable, the United States Centers for Disease Control and Prevention has acknowledged in-room air cleaners as alternative technology for increasing room ventilation when this cannot be achieved by the building’s HVAC system with preference given to fixed recirculating systems over portable ones.
Importantly, the use of an in-room air cleaner does not preclude either the need for health care workers and visitors to use personal protective equipment (N95 mask or equivalent) when entering AII rooms or health care facilities from meeting current regulatory requirements for airflow rates (ventilation rates) in buildings and airflow differentials for effective negative-pressure rooms.
The Plasmacluster ion technology, developed in 2000, is an air purification technology. Its manufacturer, Sharp Electronics Corporation, says that it can disable airborne microorganisms through the generation of both positive and negative ions. (1) The functional unit is the hydroxyl, which is a molecule comprised of one oxygen molecule and one hydrogen atom.
Plasmacluster ion air purifier uses a multilayer filter system composed of a prefilter, a carbon filter, an antibacterial filter, and a HEPA filter, combined with an ion generator to purify the air. The ion generator uses an alternating plasma discharge to split water molecules into positively and negatively charged ions. When these ions are emitted into the air, they are surrounded by water molecules and form cluster ions which are attracted to airborne particles. The cluster ion surrounds the airborne particle, and the positive and negative ions react to form hydroxyls. These hydroxyls steal the airborne particle’s hydrogen atom, which creates a hole in the particle’s outer protein membrane, thereby rendering it inactive.
Because influenza is primarily acquired by large droplets and direct and indirect contact with an infectious person, any in-room air cleaner will have little benefit in controlling and preventing its spread. Therefore, there is no role for the Plasmacluster ion air purifier or any other in-room air cleaner in the control of the spread of influenza. Accordingly, for purposes of this review, the Medical Advisory Secretariat presents no further analysis of the Plasmacluster.
Review Strategy
The objective of the systematic review was to determine the effectiveness of in-room air cleaners with built in UVGI lights and HEPA filtration compared with those using HEPA filtration only.
The Medical Advisory Secretariat searched the databases of MEDLINE, EMBASE, Cochrane Database of Systematic Reviews, INAHATA (International Network of Agencies for Health Technology Assessment), Biosis Previews, Bacteriology Abstracts, Web of Science, Dissertation Abstracts, and NIOSHTIC 2.
A meta-analysis was conducted if adequate data was available from 2 or more studies and where statistical and clinical heterogeneity among studies was not an issue. Otherwise, a qualitative review was completed. The GRADE system was used to summarize the quality of the body of evidence comprised of 1 or more studies.
Summary of Findings
There were no existing health technology assessments on air cleaning technology located during the literature review. The literature search yielded 59 citations of which none were retained. One study was retrieved from a reference list of a guidance document from the United States Centers for Disease Control and Prevention, which evaluated an in-room air cleaner with combined UVGI lights and HEPA filtration under 2 conditions: UVGI lights on and UVGI lights off. Experiments were performed using different ventilation rates and using an aerosolized pathogen comprised of Mycobaterium parafortuitum, a surrogate for the bacterium that causes tuberculosis. Effectiveness was measured as equivalent air changes per hour (eACH). This single study formed the body of evidence for our systematic review research question.
Experimental Results
The eACH rate for the HEPA-UVGI in-room air cleaner was statistically significantly greater when the UV lights were on compared with when the UV lights were off. (P < .05). However, subsequent experiments could not attribute this to the UVGI. Consequently, the results are inconclusive and an estimate of effect (benefit) is uncertain.
The study was reviewed by a scientific expert and rated moderate for quality. Further analysis determined that there was some uncertainty in the directness of the outcome measure (eACH); thus, the GRADE level for the quality of the evidence was low indicating that an estimate of effect is very uncertain.
There is uncertainty in the benefits of using in-room air cleaners with combined UVGI lights and HEPA filtration over systems that use HEPA filtration alone. However, there are no known risks to using systems with combined UVGI and HEPA technology compared with those with HEPA alone. There is an increase in the burden of cost including capital costs (cost of the device), operating costs (electricity usage), and maintenance costs (cleaning and replacement of UVGI lights) to using an in-room air cleaner with combined UVGI and HEPA technology compared with those with HEPA alone. Given the uncertainty of the estimate of benefits, an in-room air cleaner with HEPA technology only may be an equally reasonable alternative to using one with combined UVGI and HEPA technology
Conclusions
In-room air cleaners may be used to protect health care staff from air borne infectious pathogens such as tuberculosis, chicken pox, measles, and dessiminated herpes zoster. In addition, and although in-room air cleaners are not effective at protecting staff and preventing the spread of droplet-transmitted diseases such as influenza and SARS, they may be deployed in situations with a novel/emerging infectious agent whose epidemiology is not yet defined and where airborne transmission is suspected.
It is preferable that in-room air cleaners be used with a fixed and permanent room placement when ventilation requirements must be improved and the HVAC system cannot be used. However, for acute (temporary) situations where a novel/emerging infectious agent presents whose epidemiology is not yet defined and where airborne transmission is suspected it may be prudent to use the in room air cleaner as a portable device until mode of transmission is confirmed. To maximize effectiveness, consultation with an environmental engineer and infection control expert should be undertaken before using an in-room air cleaner and protocols for maintenance and monitoring of these devices should be in place.
If properly installed and maintained, in room air cleaners with HEPA or combined HEPA and UVGI air cleaning technology are effective in removing airborne pathogens. However, there is only weak evidence available at this time regarding the benefit of using an in-room air cleaner with combined HEPA and UVGI air cleaner technology instead of those with HEPA filter technology only.
PMCID: PMC3382390  PMID: 23074468
17.  Biosafety Guidelines for Handling Microorganisms in the Teaching Laboratory: Development and Rationale† 
The safe handling of microorganisms in the teaching laboratory is a top priority. However, in the absence of a standard set of biosafety guidelines tailored to the teaching laboratory, individual educators and institutions have been left to develop their own plans. This has resulted in a lack of consistency, and differing levels of biosafety practices across institutions. Influenced by the lack of clear guidelines and a recent outbreak of Salmonella infections that was traced back to teaching laboratory exposures, the Education Board of the American Society for Microbiology charged a task force to develop a uniform set of biosafety guidelines for working with microorganisms in the teaching laboratory. These guidelines represent best practices for safely handling microbes, based on the safety requirements found in the Centers for Disease Control and Prevention’s (CDC’s) Biosafety in Microbiological and Biomedical Laboratories (BMBL). Guidelines for safely handling microbes at both biosafety level 1 (BSL1) and biosafety level 2 (BSL2) were developed. The guidelines are brief by design for ease of use and are accompanied by an extensive appendix containing explanatory notes, sample documents, and additional resources. These guidelines provide educators with a clear and consistent way to safely work with microorganisms in the teaching laboratory.
doi:10.1128/jmbe.v14i1.531
PMCID: PMC3706168  PMID: 23858356
18.  Construction and Organization of a BSL-3 Cryo-Electron Microscopy Laboratory at UTMB 
Journal of structural biology  2012;181(3):223-233.
A unique cryo-electron microscopy facility has been designed and constructed at the University of Texas Medical Branch (UTMB) to study the three-dimensional organization of viruses and bacteria classified as select agents at biological safety level (BSL)-3, and their interactions with host cells. A 200 keV high-end cryo-electron microscope was installed inside a BSL-3 containment laboratory and standard operating procedures were developed and implemented to ensure its safe and efficient operation. We also developed a new microscope decontamination protocol based on chlorine dioxide gas with a continuous flow system, which allowed us to expand the facility capabilities to study bacterial agents including spore-forming species. The new unified protocol does not require agent-specific treatment in contrast to the previously used heat decontamination. To optimize the use of the cryo-electron microscope and to improve safety conditions, it can be remotely controlled from a room outside of containment, or through a computer network world-wide. Automated data collection is provided by using JADAS (single particle imaging) and SerialEM (tomography). The facility has successfully operated for more than a year without an incident and was certified as a select agent facility by the Centers for Disease Control.
doi:10.1016/j.jsb.2012.12.007
PMCID: PMC3593667  PMID: 23274136
cryo-electron microscopy; single particle imaging; electron tomography; biological safety containment
19.  A Unique BSL-3 Cryo-Electron Microscopy Laboratory at UTMB 
This article describes a unique cryo-electron microscopy (CryoEM) facility to study the three-dimensional organization of viruses at biological safety level 3 (BSL-3). This facility, the W. M. Keck Center for Virus Imaging, has successfully operated for more than a year without incident and was cleared for select agent studies by the Centers for Disease Control and Prevention (CDC). Standard operating procedures for the laboratory were developed and implemented to ensure its safe and efficient operation. This facility at the University of Texas Medical Branch (Galveston, TX) is the only such BSL-3 CryoEM facility approved for select agent research.
PMCID: PMC3156611  PMID: 21852942
20.  Developing a Behavioral Health Screening Program for BSL-4 Laboratory Workers at the National Institutes of Health 
To ensure that biomedical research is performed in the safest and most responsible manner possible, the NIH established the Biological Surety Program (BSP) in 2006 to reflect the unique human and security factors associated with biosafety level 4 (BSL-4) laboratories. The program sets forth control measures to ensure there is a trained, responsible, and reliable workforce, with rigorous procedures to protect employee health and maintain a safe work environment.
The events and aftermath of September 11, 2001, accelerated a search for personnel reliability test measures to identify individuals who could pose a threat to our nation's security and safety. The creation and administration of a behavioral health screen for BSL-4 laboratory workers at the National Institutes of Health represents a pioneering effort to proactively build a BSL-4 safety culture promoting worker cohesiveness, trust, respect, and reliability with a balance of worker privacy and public safety.
doi:10.1089/bsp.2010.0048
PMCID: PMC3063697  PMID: 21361798
21.  The importance of implementing safe sharps practices in the laboratory setting in Europe 
Biochemia Medica  2014;24(1):45-56.
Healthcare workers are at risk of sharps injuries and subsequent infection from more than 40 bloodborne pathogens or species. Hepatitis B Virus (HBV), Hepatitis C Virus (HCV) and Human Immunodeficiency Virus (HIV) together account for the vast majority of cases. The Directive 2010/32/EU “Prevention from sharp injuries in the hospital and healthcare sector”, issued to protect workers from these risks, requires an integrated approach to prevention including awareness-raising, education, training, elimination of unnecessary needles, safe procedures for sharps use and disposal, banning of recapping, vaccination, use of personal protective equipment, provision of safety-engineered devices, and appropriate surveillance, monitoring, response and follow-up.
As laboratories represent a high-risk setting both in the preanalytical and analytical phase, we reviewed accidents and prevention in this setting in the light of the new legislation.
Phlebotomy is the procedure carrying the highest risk of exposure and infection, involved in 30–50% of HIV and HCV cases detected in nationwide systems following accidental blood exposures implemented since the 1990s in Italy and France. In laboratories, problems in the management of sharps containers, recapping, needle disassembly by hand and blood transfer from syringes into tubes were observed and accounted for two-thirds of injuries. These accidents could be reduced through education and monitoring of behaviours, and introduction of medical devices incorporating safety-engineered protection mechanisms with appropriate training. Laboratory staff should be immunized against HBV, and know policies and procedures for the post-exposure management and prophylaxis. The management commitment to safety is crucial to ensure the necessary support to these changes.
doi:10.11613/BM.2014.007
PMCID: PMC3936965  PMID: 24627714
occupational exposure; needlestick injuries; bloodborne pathogens; accident prevention; laboratories
22.  HIV safety guidelines and laboratory training. NIOSH. 
Public Health Reports  1991;106(6):727-732.
At the Centers for Disease Control (CDC), educational activities concerning acquired immunodeficiency syndrome (AIDS) are directed to many target audiences; important among these are health care and public safety workers. Several CDC programs are designed to address the specific education and training needs of these groups. The National Institute for Occupational Safety and Health (NIOSH) has developed a set of occupational safety guidelines directed to fire service personnel, emergency medical technicians, paramedics, and law enforcement and correctional facility personnel. These guidelines provide information on modes of transmission of human immunodeficiency virus (HIV) in the workplace, the risk of transmission, the control of risk, and specific risk-control recommendations. NIOSH also has developed a model curriculum, based on the principles and practices discussed in the guidelines, for use in training workers. The Hospital Infections Program (HIP) at CDC's National Center for Infectious Diseases is responsible for assessing the risk of HIV infection for both health care workers and patients. As part of this effort, HIP has developed guidelines to prevent transmission of HIV and other bloodborne pathogens in health care settings, as well as statements regarding management of occupational exposure to HIV. The Public Health Practice Program Office provides laboratory training to health care workers who are performing HIV- and AIDS-related testing. This training is delivered through the National Laboratory Training Network and through courses given at CDC headquarters in Atlanta. The delivery of laboratory training is supported by the development of training materials and by performance evaluation programs.
PMCID: PMC1580331  PMID: 1659723
23.  Influence of laboratory animal hosts on the life cycle of Hyalomma marginatum and implications for an in vivo transmission model for Crimean-Congo hemorrhagic fever virus 
Crimean-Congo hemorrhagic fever virus (CCHFV) is one of the most geographically widespread arboviruses and causes a severe hemorrhagic syndrome in humans. The virus circulates in nature in a vertebrate-tick cycle and ticks of the genus Hyalomma are the main vectors and reservoirs. Although the tick vector plays a central role in the maintenance and transmission of CCHFV in nature, comparatively little is known of CCHFV-tick interactions. This is mostly due to the fact that establishing tick colonies is laborious, and working with CCHFV requires a biosafety level 4 laboratory (BSL4) in many countries. Nonetheless, an in vivo transmission model is essential to understand the epidemiology of the transmission cycle of CCHFV. In addition, important parameters such as vectorial capacity of tick species, levels of infection in the host necessary to infect the tick, and aspects of virus transmission by tick bite including the influence of tick saliva, cannot be investigated any other way. Here, we evaluate the influence of different laboratory animal species as hosts supporting the life cycle of Hyalomma marginatum, a two-host tick. Rabbits were considered the host of choice for the maintenance of the uninfected colonies due to high larval attachment rates, shorter larval-nymphal feeding times, higher nymphal molting rates, high egg hatching rates, and higher conversion efficiency index (CEI). Furthermore, we describe the successful establishment of an in vivo transmission model for CCHFV in a BSL4 biocontainment setting using interferon knockout mice. This will give us a new tool to study the transmission and interaction of CCHFV with its tick vector.
doi:10.3389/fcimb.2013.00039
PMCID: PMC3747357  PMID: 23971007
Crimean-Congo hemorrhagic fever; Hyalomma marginatum; BSL4; transmission; Crimean-Congo hemorrhagic fever virus; bunyavirus; tick; tick-borne virus
24.  Trauma care — a participant observer study of trauma centers at Delhi, Lucknow and Mumbai 
The Indian Journal of Surgery  2009;71(3):133-141.
Background
Trained doctors and para-medical personnel in accident and emergency services are scant in India. Teaching and training in trauma and emergency medical system (EMS) as a specialty accredited by the Medical Council of India is yet to be started as a postgraduate medical education program. The MI and CMO (casualty medical officer) rooms at military and civilian hospitals in India that practice triage, first-aid, medico-legal formalities, reference and organize transport to respective departments leads to undue delays and lack multidisciplinary approach. Comprehensive trauma and emergency infrastructure were created only at a few cities and none in the rural areas of India in last few years.
Aim
To study the infrastructure, human resource allocation, working, future plans and vision of the established trauma centers at the 3 capital cities of India — Delhi (2 centres), Lucknow and Mumbai.
Setting and design
Participant observer structured open ended qualitative research by 7 days direct observation of the facilities and working of above trauma centers.
Material and methods
Information on, 1. Infrastructure; space and building, operating, ventilator, and diagnostic and blood bank facilities, finance and costs and pre-hospital care infrastructure, 2. Human resource; consultant and resident doctors, para-medical staff and specialists and 3. Work style; first responder, type of patients undertaken, burn management, surgical management and referral system, follow up patient management, social support, bereavement and postmortem services were recorded on a pre-structured open ended instrument interviewing the officials, staff and by direct observation. Data were compressed, peer-analyzed as for qualitative research and presented in explicit tables.
Results
Union and state governments of Delhi, Maharashtra and Uttar Pradesh have spent heavily to create trauma and emergency infrastructure in their capital cities. Mostly general and orthopedics surgeons with their resident staff were managing the facilities. Comprehensively trained accident and emergency (AandE) personnel were not available at any of the centers. Expert management of cardiac peri-arrest arrhythmias, peripheral and microvascular repair were occasionally available. Maxillo-facial, dental and prosthodontic facilities, evenomation grading and treatment of poisoning — anti venom were not integrated. Ventilators, anesthetist, neuro and plastic surgeons were available on call for emergency care at all the 4 centers. Emergency diagnostic radiology (X-ray, CT scan, and ultrasound) and pathology were available at all the 4 centers. On the spot blood bank and component blood therapy was available only at the Delhi centers. Pre-hospital care, though envisioned by the officials, was lacking. Comprehensively trained senior A and E personnel as first responders were unavailable. Double barrier nursing for burn victims was not witnessed. Laparoscopic and fibreoptic endoscopic emergency procedures were also available only at Delhi. Delay in treatment on account of incomplete medico-legal formalities was not seen. Social and legal assistance, bereavement service and cold room for dead body were universally absent. Free treatment at Delhi and partial financial support at Lucknow were available for poor and destitute.
Conclusion
Though a late start, evolution of trauma services was observed and huge infrastructure for trauma have come up at Delhi and Lucknow. Postgraduate accreditation in Trauma and EMS and creation of National Injury Control Program must be mandated to improve trauma care in India. Integration of medical, non traumatic surgical and pediatric emergency along with pre-hospital care is recommended.
doi:10.1007/s12262-009-0037-0
PMCID: PMC3452474  PMID: 23133136
Trauma Care; India; Trauma Centers; Participant observer
25.  Serological Assays Based on Recombinant Viral Proteins for the Diagnosis of Arenavirus Hemorrhagic Fevers  
Viruses  2012;4(10):2097-2114.
The family Arenaviridae, genus Arenavirus, consists of two phylogenetically independent groups: Old World (OW) and New World (NW) complexes. The Lassa and Lujo viruses in the OW complex and the Guanarito, Junin, Machupo, Sabia, and Chapare viruses in the NW complex cause viral hemorrhagic fever (VHF) in humans, leading to serious public health concerns. These viruses are also considered potential bioterrorism agents. Therefore, it is of great importance to detect these pathogens rapidly and specifically in order to minimize the risk and scale of arenavirus outbreaks. However, these arenaviruses are classified as BSL-4 pathogens, thus making it difficult to develop diagnostic techniques for these virus infections in institutes without BSL-4 facilities. To overcome these difficulties, antibody detection systems in the form of an enzyme-linked immunosorbent assay (ELISA) and an indirect immunofluorescence assay were developed using recombinant nucleoproteins (rNPs) derived from these viruses. Furthermore, several antigen-detection assays were developed. For example, novel monoclonal antibodies (mAbs) to the rNPs of Lassa and Junin viruses were generated. Sandwich antigen-capture (Ag-capture) ELISAs using these mAbs as capture antibodies were developed and confirmed to be sensitive and specific for detecting the respective arenavirus NPs. These rNP-based assays were proposed to be useful not only for an etiological diagnosis of VHFs, but also for seroepidemiological studies on VHFs. We recently developed arenavirus neutralization assays using vesicular stomatitis virus (VSV)-based pseudotypes bearing arenavirus recombinant glycoproteins. The goal of this article is to review the recent advances in developing laboratory diagnostic assays based on recombinant viral proteins for the diagnosis of VHFs and epidemiological studies on the VHFs caused by arenaviruses.
doi:10.3390/v4102097
PMCID: PMC3497043  PMID: 23202455
arenavirus; viral hemorrhagic fever; diagnosis; recombinant protein

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