Response to Imported Case of Marburg Hemorrhagic Fever, the Netherlands

Adventure tourism may bring this disease to Western countries.

person left for Morocco 1 day before the end of the monitoring period, but he kept in touch with the Dutch authorities.
To anticipate possible needlestick accidents or gross breaches of isolation measures by healthcare workers, use of experimental vaccines were assessed in a teleconference with international experts. They favored the vaccine in which attenuated recombinant vesicular stomatitis virus vector expresses the Marburg virus (MARV) glycoprotein (1)(2)(3) and developed protocols for its use, including regulatory aspects and measures to contain environmental shedding of VSV.

Transportation and Processing of Samples
Transport of samples must be organized before sample collection to avoid bottlenecks.
We therefore arranged for certified couriers to link hospitals quarantine facilities to laboratories, including the nearest reference laboratory in Germany.
Protocols were designed to encompass essential laboratory testing of severely ill patients, including chemical and bacteriologic diagnostic techniques, biosafety considerations, and methods for decontamination of equipment. No existing preparedness protocols included these considerations. We decided that diagnostic work-ups would be limited to contacts in whom fever developed. In that case, essential equipment for blood chemistry analyses would be placed inside the Intensive Care isolation facility.

Laboratory Assessment of Febrile Contacts: Differential Diagnosis
Protocols were developed for diagnosis of the most probable causes of illness, given the seasonal patters, in which prodromal symptoms resemble those seen in patients with a filovirus infection. These include fever, myalgia, and diarrhoea. Data from physician-based studies of respiratory diseases and gastroenteritis were used as a reference (4,5). Contacts with such symptoms would be tested for a range of pathogens to provide an alternative diagnosis.
However, their removal from isolation would not be based solely on this testing because common pathogens are often detected in healthy controls.

Filovirus Evaluation in Contact Monitoring
Acute viremia develops in persons infected with Ebola virus, and viral antigens and RNA are detectable in serum, plasma, saliva, and occasionally other secreta (6,7). In early stages of infection, results of PCR-based assays have been positive 24-48 earlier than antigen-capture assays, making the PCR the method of choice. Although viral loads in severely ill patients are high, in the early course of illness, viral loads may be barely detectable (8). Therefore, proper evaluation of PCR-based methods, with particular emphasis on detection limits, is crucial for reliance on these diagnostics during monitoring. The filovirus diagnostics would therefore be conducted simultaneously in at least 2 laboratories. The Bernhard-Nocht-Institute for Tropical Medicine (BNI) in Hamburg, Germany, provided protocols for PCR-based detection of MARVs.
They had been validated in a joint study between P4 laboratories, using all MARV isolates available in these laboratories as reference material (8).
Sequence analysis of the patient's MARV strain showed it was most closely related to the first-identified Marburg virus isolate from Uganda, the Popp strain. Therefore, we assumed that detection limits reported for the Popp strain would apply to this strain as well. Reagent kits based on the Panning protocol were assembled at our request and kindly provided within a few days (Thomas Laue; QIAGEN, Hamburg, Germany). Evaluation of this kit, using extracts from patient serum and other possible sample types (throat swab, plasma, serum, feces), provided reliable results. Additionally, strain specific Taqman PCR was designed at the Department of Virology at the Erasmus University Hospital, with detection limits similar to those of the Panning protocol.

Laboratory Procedures Used in the Follow-up Survey
After inactivation and fixation on immunofluorescent antibody assay slides, the samples were stored at -20°C outside the high-containment laboratory, and further investigations using the inactivated virus were performed under BioSafety Level 2 conditions. Testing was performed observed. Therefore, all tested sera were considered negative for IgG and IgM antibodies to MARV.