Stephan Ölschläger

Management of Accidental Exposure to Ebola Virus in the Biosafety Level 4 Laboratory, Hamburg, Germany

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 Freunds adjuvant. Neither an approved treatment nor a postexposure prophylaxis (PEP) exists for Ebola hemorrhagic fever. Following a risk-benefit assessment, it was recommended the exposed person take an experimental vaccine that had shown PEP efficacy in ZEBOV-infected nonhuman primates (NHPs) [12]. The vaccine, which had not been used previously in humans, was a live-attenuated recombinant vesicular stomatitis virus (recVSV) expressing the glycoprotein of ZEBOV. A single dose of 5 × 10(7) plaque-forming units was injected 48 hours after the accident. The vaccinee developed fever 12 hours later and recVSV viremia was detectable by polymerase chain reaction (PCR) for 2 days. Otherwise, the person remained healthy, and ZEBOV RNA, except for the glycoprotein gene expressed in the vaccine, was never detected in serum and peripheral blood mononuclear cells during the 3-week observation period.

Evaluation of Antiviral Efficacy of Ribavirin, Arbidol, and T-705 (Favipiravir) in a Mouse Model for Crimean-Congo Hemorrhagic Fever

Background Mice lacking the type I interferon receptor (IFNAR−/− mice) reproduce relevant aspects of Crimean-Congo hemorrhagic fever (CCHF) in humans, including liver damage. We aimed at characterizing the liver pathology in CCHF virus-infected IFNAR−/− mice by immunohistochemistry and employed the model to evaluate the antiviral efficacy of ribavirin, arbidol, and T-705 against CCHF virus. Methodology/Principal Findings CCHF virus-infected IFNAR−/− mice died 2–6 days post infection with elevated aminotransferase levels and high virus titers in blood and organs. Main pathological alteration was acute hepatitis with extensive bridging necrosis, reactive hepatocyte proliferation, and mild to moderate inflammatory response with monocyte/macrophage activation. Virus-infected and apoptotic hepatocytes clustered in the necrotic areas. Ribavirin, arbidol, and T-705 suppressed virus replication in vitro by ≥3 log units (IC50 0.6–2.8 µg/ml; IC90 1.2–4.7 µg/ml). Ribavirin [100 mg/(kg×d)] did not increase the survival rate of IFNAR−/− mice, but prolonged the time to death (p<0.001) and reduced the aminotransferase levels and the virus titers. Arbidol [150 mg/(kg×d)] had no efficacy in vivo. Animals treated with T-705 at 1 h [15, 30, and 300 mg/(kg×d)] or up to 2 days [300 mg/(kg×d)] post infection survived, showed no signs of disease, and had no virus in blood and organs. Co-administration of ribavirin and T-705 yielded beneficial rather than adverse effects. Conclusions/Significance Activated hepatic macrophages and monocyte-derived cells may play a role in the proinflammatory cytokine response in CCHF. Clustering of infected hepatocytes in necrotic areas without marked inflammation suggests viral cytopathic effects. T-705 is highly potent against CCHF virus in vitro and in vivo. Its in vivo efficacy exceeds that of the current standard drug for treatment of CCHF, ribavirin.

Molecular Diagnostics for Lassa Fever at Irrua Specialist Teaching Hospital, Nigeria: Lessons Learnt from Two Years of Laboratory Operation

Background Lassa fever is a viral hemorrhagic fever endemic in West Africa. However, none of the hospitals in the endemic areas of Nigeria has the capacity to perform Lassa virus diagnostics. Case identification and management solely relies on non-specific clinical criteria. The Irrua Specialist Teaching Hospital (ISTH) in the central senatorial district of Edo State struggled with this challenge for many years. Methodology/Principal Findings A laboratory for molecular diagnosis of Lassa fever, complying with basic standards of diagnostic PCR facilities, was established at ISTH in 2008. During 2009 through 2010, samples of 1,650 suspected cases were processed, of which 198 (12%) tested positive by Lassa virus RT-PCR. No remarkable demographic differences were observed between PCR-positive and negative patients. The case fatality rate for Lassa fever was 31%. Nearly two thirds of confirmed cases attended the emergency departments of ISTH. The time window for therapeutic intervention was extremely short, as 50% of the fatal cases died within 2 days of hospitalization—often before ribavirin treatment could be commenced. Fatal Lassa fever cases were older (p = 0.005), had lower body temperature (p<0.0001), and had higher creatinine (p<0.0001) and blood urea levels (p<0.0001) than survivors. Lassa fever incidence in the hospital followed a seasonal pattern with a peak between November and March. Lassa virus sequences obtained from the patients originating from Edo State formed—within lineage II—a separate clade that could be further subdivided into three clusters. Conclusions/Significance Lassa fever case management was improved at a tertiary health institution in Nigeria through establishment of a laboratory for routine diagnostics of Lassa virus. Data collected in two years of operation demonstrate that Lassa fever is a serious public health problem in Edo State and reveal new insights into the disease in hospitalized patients.

Improved Detection of Lassa Virus by Reverse Transcription-PCR Targeting the 5′ Region of S RNA

ABSTRACT The method of choice for the detection of Lassa virus is reverse transcription (RT)-PCR. However, the high degree of genetic variability of the virus poses a problem with the design of RT-PCR assays that will reliably detect all strains. Recently, we encountered difficulties in detecting some strains from Liberia and Nigeria in a commonly used glycoprotein precursor (GPC) gene-specific RT-PCR assay (A. H. Demby, J. Chamberlain, D. W. Brown, and C. S. Clegg, J. Clin. Microbiol. 32:2898-2903, 1994), which prompted us to revise the protocol. The design of the new assay, the GPC RT-PCR/2007 assay, took into account 62 S RNA sequences from all countries where Lassa fever is endemic, including 40 sequences generated from the strains in our collection. The analytical sensitivity of the new assay was determined with 11 strains from Sierra Leone, Liberia, Ivory Coast, and Nigeria by probit analysis; the viral loads detectable with a probability of 95% ranged from 342 to 2,560 S RNA copies/ml serum, which corresponds to 4 to 30 S RNA copies/assay. The GPC RT-PCR/2007 assay was validated with 77 serum samples and 1 cerebrospinal fluid sample from patients with laboratory-confirmed Lassa fever. The samples mainly originated from Liberia and Nigeria and included strains difficult to detect in the assay of 1994. The GPC RT-PCR/2007 assay detected virus in all clinical specimens (100% sensitivity). In conclusion, a new RT-PCR assay, based in part on the protocol developed by Demby et al. in 1994, for the detection of Lassa virus is described. Compared to the assay developed in 1994, the GPC RT-PCR/2007 assay offers improved sensitivity for the detection of Liberian and Nigerian Lassa virus strains.

Acute liver failure, multiorgan failure, cerebral oedema, and activation of proangiogenic and antiangiogenic factors in a case of Marburg haemorrhagic fever

A woman developed Marburg haemorrhagic fever in the Netherlands, most likely as a consequence of being exposed to virus-infected bats in the python cave in Maramagambo Forest during a visit to Uganda. The clinical syndrome was dominated by acute liver failure with secondary coagulopathy, followed by a severe systemic inflammatory response, multiorgan failure, and fatal cerebral oedema. A high blood viral load persisted during the course of the disease. The initial systemic inflammatory response coincided with peaks in interferon-γ and tumour necrosis factor-α concentrations in the blood. A terminal rise in interleukin-6, placental growth factor (PlGF), and soluble vascular endothelial growth factor receptor-1 (sVEGF-R1) seemed to suggest an advanced pathophysiological stage of Marburg haemorrhagic fever associated with vascular endothelial dysfunction and fatal cerebral oedema. The excess of circulating sVEGF-R1 and the high sVEGF-R1:PlGF ratio shortly before death resemble pathophysiological changes thought to play a causative part in pre-eclampsia. Aggressive critical-care treatment with renal replacement therapy and use of the molecular absorbent recirculation system appeared able to stabilise--at least temporarily--the patients condition.

Current Molecular Epidemiology of Lassa Virus in Nigeria

ABSTRACT Recent Lassa virus strains from Nigeria were completely or partially sequenced. Phylogenetic analysis revealed the predominance of lineage II and III strains, the existence of a previously undescribed (sub)lineage in Nigeria, and the directional spread of virus in the southern part of the country. The Bayesian analysis also provided estimates for divergence times within the Lassa virus clade.

Depletion of GTP pool is not the predominant mechanism by which ribavirin exerts its antiviral effect on Lassa virus

Ribavirin (1-β-d-ribofuranosyl-1,2,4-triazole-3-carboxamide) is the standard treatment for Lassa fever, though its mode of action is unknown. One possibility is depletion of the intracellular GTP pool via inhibition of the cellular enzyme inosine monophosphate dehydrogenase (IMPDH). This study compared the anti-arenaviral effect of ribavirin with that of two other IMPDH inhibitors, mycophenolic acid (MPA) and 5-ethynyl-1-β-d-ribofuranosylimidazole-4-carboxamide (EICAR). All three compounds were able to inhibit Lassa virus replication by ≥2 log units in cell culture. Restoring the intracellular GTP pool by exogenous addition of guanosine reversed the inhibitory effects of MPA and EICAR, while ribavirin remained fully active. Analogous experiments performed with Zaire Ebola virus showed that IMPDH inhibitors are also active against this virus, although to a lesser extent than against Lassa virus. In conclusion, the experiments with MPA and EICAR indicate that replication of Lassa and Ebola virus is sensitive to depletion of the GTP pool mediated via inhibition of IMPDH. However, this is not the predominant mechanism by which ribavirin exerts its in-vitro antiviral effect on Lassa virus.

Performance and clinical validation of the RealStar ® MERS-CoV Kit for detection of Middle East respiratory syndrome coronavirus RNA

Abstract Background A highly pathogenic human coronavirus causing respiratory disease emerged in the Middle East region in 2012. In-house molecular diagnostic methods for this virus termed Middle East respiratory syndrome coronavirus (MERS-CoV) allowed sensitive MERS-CoV RNA detection in patient samples. Fast diagnosis is important to manage human cases and trace possible contacts. Objectives The aim of this study was to improve the availability of existing nucleic acid amplification-based diagnostic methods for MERS-CoV infections by providing a real-time RT-PCR kit, including an internal control and two target regions recommended by the World Health Organization (WHO). And to validate this kit (RealStar® MERS-CoV RT-PCR kit 1.0, Altona Diagnostics GmbH, Hamburg, Germany) using clinical samples of one MERS-CoV case from Munich and respiratory samples of patients with other respiratory diseases. Study design An internal amplification control was included into the RT-PCR assays targeting the genomic region upstream of the Envelope gene (upE) and within open reading frame (ORF) 1A. Based on these assays, a ready-to-use real-time RT-PCR kit featuring both the upE and ORF1A assays was developed, validated and compared to the established in-house versions. Results The performance of both RT-PCR assays included in the kit is comparable to the in-house assays. They show high analytical sensitivity (upE: 5.3 copies/reaction; ORF1A: 9.3 copies/reaction), no cross-reactivity with other respiratory pathogens and detected MERS-CoV RNA in patient samples in almost the same manner as the in-house versions. Conclusion The kit is a valuable tool for assisting in the rapid diagnosis, patient management and epidemiology of suspected MERS-CoV cases.

Lassa Fever, Nigeria, 2005–2008

To the Editor: Lassa fever affects ≈100,000 persons per year in West Africa (1). The disease is caused by Lassa virus, an arenavirus, and is associated with bleeding and organ failure. The case-fatality rate in hospitalized patients is 10%–20%. The reservoir of the virus is multimammate mice (Mastomys natalensis). Investigations in the 1970s and 1980s pointed to the existence of 3 disease-endemic zones within Nigeria: the northeastern region around Lassa, the central region around Jos, and the southern region around Onitsha (2,3). The current epidemiologic situation is less clear because no surveillance system is in place. In 2003 and 2004, we conducted a hospital-based survey in Irrua, which demonstrated ongoing transmission of the virus in Edo State, Nigeria (4). Since then, laboratory capacity at the University of Lagos for diagnosing Lassa fever has been improved and used for small-scale passive surveillance in other parts of the country. Public health officials or hospital staff reported suspected cases. Blood samples were sent to Lagos, or staff from Lagos collected samples on site. Confirmatory testing, sequencing, and virus isolation were performed at the Bernhard Nocht Institute for Tropical Medicine in Hamburg, Germany. Primary testing was done by reverse transcription–PCR (RT-PCR) that targeted the glycoprotein (GP) gene (5,6). An RT-PCR that targeted the large (L) gene was used as a secondary test (7), and PCR products were sequenced. Serologic testing for Lassa virus–specific immunoglobulin (Ig) G and IgM was performed by immunofluorescent antibody test using Vero cells infected with Lassa virus. Virus isolation with Vero cells was conducted in the BioSafety Level 4 laboratory in Hamburg. From 2005 through 2008, 10 cases of Lassa fever were confirmed by virus detection (cases 3–10) or implicated by epidemiologic investigation and serologic testing (cases 1 and 2) (Appendix Table). Case-patients 1–4 were involved in a nosocomial outbreak that occurred in February 2005 at the Ebonyi State University Teaching Hospital (EBSUTH) in Abakaliki. Retrospective investigation suggests the following transmission chain. The presumed index case-patient was a male nurse living in Onitsha, who became ill on January 21, 2005, and traveled ≈200 km to EBSUTH for better medical treatment. The detection of Lassa virus–specific IgM during his convalescent phase indicates that he had Lassa fever. The second case-patient was a female nurse who had contact with the index case-patient on February 4. She was admitted on February 7 and died 6 days later. Her clinical features were compatible with Lassa fever, but laboratory confirmation is lacking because specimens were not collected. Two additional case-patients among hospital staff (case-patients 3 and 4) were seen on February 21; each had had contact with case-patient 2. Case-patient 3 took care of case-patient 2 and slept in the same room with her for 4 days. Lassa fever was confirmed in case-patients 3 and 4 by RT-PCR as well as by IgM and IgG seroconversion in the surviving patient (case-patient 3). Case-patient 4, a pregnant nurse, had a spontaneous abortion and died on day 9 of hospitalization. Sequencing the GP and L gene PCR fragments showed that case-patients 3 and 4 were infected with the same virus strain (100% identity). In March and April 2005, blood was collected from 50 hospital staff members (including those who had had contact with the case-patients) and screened for Lassa virus–specific IgM and IgG. No positive blood samples were found, which indicated that no additional staff members were involved in the outbreak. Case-patients 5 and 6 were admitted to EBSUTH in 2008 on January 17 and March 5, respectively. Both were medical doctors, one at a local hospital and the other at EBSUTH, and both died. Encephalopathy with generalized seizures and loss of consciousness preceded death in both cases. The source of infection is unknown, although it is likely that they became infected while they treated patients without knowing they had Lassa fever. In agreement with the epidemiology, the viruses from the 2 patients were similar, though not identical (89% and 87% identity in the GP and L genes, respectively). Cases 7 to 10 occurred in Abuja and Jos from December 2007 through March 2008. Healthcare workers appeared not to be involved, and no molecular epidemiologic evidence indicated that transmission occurred among the 3 case-patients from Jos (94–97% and 90–94% identity in the GP and L genes, respectively). In conjunction with our previous report (4), the cases presented here demonstrate current Lassa fever activity in the states of Edo, Ebonyi, Federal Capital Territory, and Plateau. These findings correspond to early reports on Lassa fever in southern and central parts of Nigeria. That healthcare workers are still at as high a risk of contracting and dying from the disease as they were 20 years ago (8) is alarming. A key to solving this problem would be the establishment of diagnostic facilities that can provide rapid molecular testing at referral centers in the disease-endemic zones. This testing would facilitate appropriate case and contact management, including early treatment and postexposure prophylaxis with ribavirin, and eventually raise awareness that Lassa fever should be considered in every severe febrile illness in these regions.

First International External Quality Assessment of Molecular Detection of Crimean-Congo Hemorrhagic Fever Virus

Crimean-Congo hemorrhagic fever (CCHF) is a zoonosis caused by a Nairovirus of the family Bunyaviridae. Infection is transmitted to humans mostly by Hyalomma ticks and also by direct contact with the blood or tissues of infected humans or viremic livestock. Clinical features usually include a rapid progression characterized by hemorrhage, myalgia and fever, with a lethality rate up to 30%. CCHF is one of the most widely distributed viral hemorrhagic fevers and has been reported in Africa, the Middle East and Asia, as well as parts of Europe. There is no approved vaccine or specific treatment against CCHF virus (CCHFV) infections. In this context, an accurate diagnosis as well as a reliable surveillance of CCHFV infections is essential. Diagnostic techniques include virus culture, serology and molecular methods, which are now increasingly used. The European Network for the Diagnostics of “Imported” Viral Diseases organized the first international external quality assessment of CCHVF molecular diagnostics in 2011 to assess the efficiency and accurateness of CCHFV molecular methods applied by expert laboratories. A proficiency test panel of 15 samples was distributed to the participants including 10 different CCHFV preparations generated from infected cell cultures, a preparation of plasmid cloned with the nucleoprotein of CCHFV, two CCHFV RNA preparations and two negative controls. Forty-four laboratories worldwide participated in the EQA study and 53 data sets were received. Twenty data sets (38%) met all criteria with optimal performance, 10 (19%) with acceptable performance, while 23 (43%) reported results showing a need for improvement. Differences in performance depended on the method used, the type of strain tested, the concentration of the sample tested and the laboratory performing the test. These results indicate that there is still a need for improving testing conditions and standardizing protocols for the molecular detection of Crimean-Congo hemorrhagic fever virus.