Friday, April 11, 2014

Bio-Safety: What Happens when Viral Hemorrhagic Fever Escapes in the Laboratory?

"In 1967, 31 workers at a laboratory in Marburg, Germany began suffering from an array of horrifying symptoms: fever, diarrhea, vomiting, and massive bleeding from a variety of internal organs. Seven of the workers would eventually succumb to their illnesses.After an extensive investigation, scientists identified the source of the outbreak, a pair of grivet monkeys imported from Uganda for polio research. The primates were carrying a shocking, never-before-seen virus, which later was named Marburg for the city in which it was discovered." 

See: " 

"The virus was isolated, and found to exhibit a unique morphology, leading to the designation of a new group: the Filoviridae. In the Marburg outbreak the disease presented with a 32% mortality rate (7 deaths out of 31 infections). Between 1987 and 1998, reported cases of Marburg were due solely to laboratory accidents both in the former Soviet Union. While there have been several naturally occurring outbreaks what happens when it is laboratory acquired? " Marburg hemorrhagic fever (MHF) in human and non-human primates, characterized by person to person transmission and high case fatality rates (2). To date, 34 filovirus hemorrhagic fever (FHF) outbreaks and laboratory -acquired infections are known to have occurred in humans. (23 EHF and 11 MHF), all in or originating from sub-Saharan Africa and yielding approximately 2800 laboratory confirmed, suspected, or putative cases (3-7). Epidemiological studies were done in parallel with the microbiological studies. It became apparent very early that all the patients in Marburg were employees of Behringwerke, a producer of sera and vaccines, and that the patients in Frankfurt were employees of the Paul Ehrlich Institute, a control institute for sera and vaccines. The primary case patient in Belgrade, a veterinarian, was employed at Institute Torlak. A major activity of these institutions was the production and safety testing of live poliomyelitis vaccine. All patients with primary infections at the 3 locations had direct contact with blood, organs, and cell cultures from Cercopithecus aethiops monkeys. These animals were imported from Uganda and were used mainly for the production of kidney cell cultures, which were needed for the propagation of vaccine strains. .For complete repport on German Marburg outbreak" see:

While this outbreak is certainly ranked among the worst laboratory acquired, highly pathogenic infectious diseases, it is more concerning still that it happened in a German laboratory, given, at that time, what would have been relatively high standards.  Unfortunately, Germany has since witnessed other laboratory acquired diseases or LAD's. Recalling a sharps and sticks accident that happened as recently as 2009 in a Hamburg laboratory, the Journal of Infectious Diseases published a study entitled: "Management of Accidental Exposure to Ebola Virus in the Bio-Safety Level 4 Laboratory Hamburg, Germany", the report details this accident stating that "A needlestick injury occurred during an animal experiment in the biosafety level 4 laboratory in Hamburg, Germany in March 2009 The syringe contained Zaire ebolavirus (ZEBOV) mixed with Freund's adjuvant. Following a risk-benefit assessment, it was recommended the exposed person take an experimental vaccine. The syringe contained Zaire ebolavirus (ZEBOV) mixed with Freund's adjuvant. Despite high standards of protection in these laboratories, laboratory workers are still at risk of contracting Ebola hemorrhagic fever, in particular during animal experimentation. Three laboratory accidents iwth Ebola virus are documented in the literature: 1 case was fatal (4), 1 case was symptomatic and survived(5), and 1 case, there was no evidence that the accident resulted in infection (6). Here, we report on the management of laboratory accidents with Ebola virus that occurred in the BSL-4 facility at the Bernhard Nocht Insitute for Tropical Medicine in Hamburg, Germany." See full report:

Scientist separates cells in order to test for the Ebola virus at the European Mobile Laboratory Gueckedou, Guinea. Photo: Reuters

The Case Study, detailed in the Journal of Infectious Diseases Report ( included the following very detailed account. It gives us insights into what actually happens when a viral hemorrhagic fever such as Ebola or Marburg escape or in this terrible case, when there is exposure through sharps and sticks. The following is quite comprehensive account, but worth reading.

"A virologist working in the BSL-4 laboratory pricked herself in the finger during a mouse experiment on 12 March 2009. The syringe contained ZEBOV from culture supernatant that had been concentrated by ultracentrifugation and mixed 1:1 with incomplete Freund’s adjuvant for immunization of mice. The material was injected into the animal before the accident happened. When the laboratory worker tried to recap the needle, it penetrated the cap laterally and subsequently all 3 gloves. The puncture site on the skin was visible, but it did not bleed. The wound was disinfected after leaving the laboratory. Overnight, reverse-transcription polymerase chain reaction (RT-PCR) analysis revealed that the ultracentrifuged material, before mixing with Freund’s adjuvant, contained 2.6 × 1010 copies/mL ZEBOV. Traces of material in the syringe (about 2 μL) were recovered and tested as well: it contained 1.4 × 108copies/mL. The effect of incomplete Freund’s adjuvant on Ebola virus was retrospectively tested by immunofocus assay. Mixing cell culture supernatant with adjuvant reduced the virus titer 4.4-fold. Thus, Ebola virus mixed with incomplete Freund’s adjuvant essentially retains its infectivity.
An infectious disease specialist at the University Medical Center Hamburg saw the patient immediately after the accident. It was decided to consult colleagues from Canada and the United States to explore possibilities for PEP and treatment. The first teleconference was held in the evening of 12 March; a second one on 13 March. Filovirus experts from the Laboratory of Virology, National Institutes of Health, Hamilton, Montana; the Boston University School of Medicine; the Public Health Agency of Canada, Winnipeg, Canada; the Special Pathogens Branch, Centers for Disease Control and Prevention, Atlanta, Georgia; the US Army Medical Research Institute of Infectious Diseases in Fort Detrick, Maryland, and the University of Texas Medical Branch, Galveston, Texas, participated in the consultations. Various possibilities for PEP and treatment, which had previously been tested in nonhuman primates (NHPs), were discussed, including recombinant nematode anticoagulant protein c2 (rNAPc2) [7], recombinant human activated protein C [8], siRNA (unpublished at the time of accident) [9], interferon (L. Hensley, unpublished data), immune therapy with neutralizing antibodies [1011], and experimental vaccines [1217]. The expert panel eventually recommended postexposure vaccination with live-attenuated recombinant vesicular stomatitis virus (recVSV) expressing the glycoprotein of ZEBOV (VSVΔG/ZEBOVGP) for the following reasons: (1) VSVΔG/ZEBOVGP has shown PEP efficacy in NHP [12]; (2) it is well tolerated in immunocompromized NHPs [13]; (3) a similar vaccine shows PEP efficacy against Marburg virus [15] and SEBOV [18]; (4) at the time of the accident, unpublished data indicated PEP effect (33% survival) of the recVSV-based Marburg virus vaccine, even 48 hours postinfection [14]; and (5) recVSV vectors show good safety profile in NHPs as long as the vector is not inoculated directly into the central nervous system [19]. Immediately following the first teleconference, the VSVΔG/ZEBOVGP vaccine was shipped from Winnipeg, Canada, to Hamburg. An emergency clearance from customs was obtained in advance. The package arrived in Hamburg in the morning of 14 March. Relevant facts available on the vaccine by 14 March are listed in Table 1. While the vaccine was in transit, a risk–benefit assessment was made on whether the vaccine should be given or not based on the following considerations:
  • 1. a visible puncture site, but not definitive evidence that the skin was fully penetrated due to the lack of bleeding;
  • 2. the syringe looked empty when the accident happened, but material was probably in the lumen of the needle;
  • 3. a high concentration of ZEBOV in the syringe at the start of the manipulation, but was unknown whether mixing with adjuvant affected virus infectivity (retrospectively, we found Ebola virus does retain infectivity);
  • 4. a good safety profile of the vaccine in NHPs, but no safety data in humans, as experimental VSV vaccines have never been administered to human volunteers;
  • 5. an unknown mechanism of PEP effect in NHPs (not clear if effect is reproducible in humans);
  • 6. a diminished PEP efficacy of the related recVSV-based Marburg virus vaccine in NHPs when given 48 hours after inoculation compared with earlier administration [14] (but the infectious dose in our case was probably much lower than in the animal experiments, which expectedly could increase the efficacy of PEP);
  • 7. laboratory stock rather than Good Manufacturing Practice (GMP)–made vaccine;
  • 8. ZEBOV infection is associated with 80–90% case fatality rate [20];
  • 9. 1 of 3 documented needlestick injuries with Ebola virus had a fatal outcome [46].
View this table:
Table 1.
Facts on the recVSV Vaccine as Summarized in the PEP Protocol Signed by the Patient
The risk of ZEBOV infection and fatal outcome due to the accidental exposure was considered to be low, but real. A hypothetical risk of life-threatening adverse effects due to the experimental vaccine was considered acceptable in view of the anticipated benefit and the risk of ZEBOV infection. In addition, overwhelming recVSV replication was expected to be amenable to treatment with ribavirin and type I interferon, both of which strongly inhibit VSV replication in vitro and in vivo [2125]. Members of the Institutional Ethics Committee of the University Medical Center, who were available for evaluation of the planned interventions, also shared these views. As a result, the patient was recommended to take the experimental vaccine. A PEP and treatment protocol with informed consent was drafted and signed by the patient.
The patient voluntarily agreed on being hospitalized on 13 March. The responsible public health authorities, infectious disease specialists, and virologists considered the risk of virus transmission during the incubation period extremely low, as available epidemiological evidence indicates that Ebola virus is spread by ill or deceased patients through direct contact with infectious body fluids [13]. It was agreed that surveillance on a regular infectious disease ward in a single room with anteroom and negative pressure is appropriate. Standard barrier nursing precautions were implemented (gown, gloves, N95 mask, eye protection). It was also taken into account that this level of isolation is less stressful to the patient and the hospital staff. A maximum incubation period of 21 days was assumed and, therefore, surveillance was foreseen for this period of time. Daily monitoring during this period included (1) body temperature; (2) D-dimer level, which is an early marker of Ebola hemorrhagic fever in NHPs [7] and is increased in humans with Ebola hemorrhagic fever [26]; (3) hematology and blood chemistry; and (4) Ebola virus in plasma and peripheral blood mononuclear cells (PBMCs) using real-time RT-PCR [27,28]. PBMCs were tested because the virus was detected in these cells in asymptomatic Ebola virus infections [29]. Fever, a rise in the D-dimer level, or the detection of Ebola virus RNA by RT-PCR were defined as independent indications of Ebola virus infection. If any of these showed a positive indication, the patient would have to be transferred to the Biocontainment Patient Care Unit (BPCU) of the University Medical Center (Figure 1). Isolation in the BPCU was required twice during the observation period for the reasons given below. The BPCU was located in a separate building and included a single patient room with 2 airlocks. It had an independent ventilation system and was maintained under negative pressure. Exhaust air underwent high efficiency particulate air (HEPA) filtration. Staff was specifically trained and dressed in biosafety suits equipped with HEPA filters. A disinfectant shower enabled transfer of specimens out of the unit for laboratory analysis. All waste was treated in an autoclave. Personal protective equipment was decontaminated in the disinfectant shower before leaving the unit. Routine laboratory investigations were performed by the Clinical Chemistry Department without special precautions, as soon as a negative PCR result for a parallel sample had been communicated. If PCR results were not available in a timely manner, point of care diagnostics was performed within the BPCU.
Figure 1.
Biocontainment Patient Care Unit at the University Medical Center Hamburg used for isolation of the patient. The unit consists of 3 rooms: airlock 1 (a), airlock 2 (b), and the main room with a bed for a patient (c), which are separated by airtight ”doors.” It is maintained under negative pressure with an increasing pressure gradient from airlock 1 to the patient room. Equipment for patient care is placed outside the unit and cables or flexible tubes for artificial ventilation or suction are passed through the wall of the unit (d). Exhaust air is filtered through HEPA filters (e). Staff are wearing full-face respirators with a HEPA filter in combination with biohazard protective suits (f). The unit is entered via airlocks 1 and 2. Before exiting the unit, the suit is decontaminated in a chemical shower in airlock 2. The shower applies foam on the surface of the suit consisting of peracetic acid and alcapur that is mixed outside the unit (g).Exposure time is 12 minutes. Thereafter, the person leaves the unit through airlock 1 and undresses. The remaining decontamination fluid is pumped out of airlock 2 and stored in a barrel outside the unit (h) until the peracetic acid is decomposed and can be fed into the wastewater system.
If an Ebola virus infection would have been diagnosed in the patient, the expert panel recommended treatment with rNAPc2, an inhibitor of factor VIIa/tissue factor that has shown some therapeutic potential in NHPs infected with Ebola virus [7] and already undergone phase II studies for other medical conditions [30]. ARCA Biopharma generously released a batch of rNAPc2 on 13 March, which arrived in Hamburg on 15 March. In addition, Ebola virus–specific small interfering RNA (siRNA) preparations were provided by Tekmira Pharamaceuticals and arrived in Hamburg on 14 March. This siRNA had shown PEP effects in NHPs (unpublished at the time of accident) [9]." See: 
The recent outbreak of Ebola in a non-laboratory setting, serves to highlight the need for research and development of a vaccine candidate.

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