Christian Drosten

Newly discovered coronavirus as the primary cause of severe acute respiratory syndrome

n Summaryn n Backgroundn The worldwide outbreak of severe acute respiratory syndrome (SARS) is associated with a newly discovered coronavirus, SARS-associated coronavirus (SARSCoV). We did clinical and experimental studies to assess the role of this virus in the cause of SARS.n n n Methodsn We tested clinical and postmortem samples from 436 SARS patients in six countries for infection with SARSCoV, human metapneumovirus, and other respiratory pathogens. We infected four cynomolgus macaques (Macaca fascicularis) with SARS-CoV in an attempt to replicate SARS and did necropsies on day 6 after infection.n n n Findingsn SARS-CoV infection was diagnosed in 329 (75%) of 436 patients fitting the case definition of SARS; human metapneumovirus was diagnosed in 41 (12%) of 335, and other respiratory pathogens were diagnosed only sporadically. SARS-CoV was, therefore, the most likely causal agent of SARS. The four SARS-CoV-infected macaques excreted SARS-CoV from nose, mouth, and pharynx from 2 days after infection. Three of four macaques developed diffuse alveolar damage, similar to that in SARS patients, and characterised by epithelial necrosis, serosanguineous exudate, formation of hyaline membranes, type 2 pneumocyte hyperplasia, and the presence of syncytia. SARS-CoV was detected in pneumonic areas by virus isolation and RT-PCR, and was localised to alveolar epithelial cells and syncytia by immunohistochemistry and transmission electron microscopy.n n n Interpretationn Replication in SARS-CoV-infected macaques of pneumonia similar to that in human beings with SARS, combined with the high prevalence of SARS-CoV infection in SARS patients, fulfill the criteria required to prove that SARS-CoV is the primary cause of SARS.n Published online July 22, 2003 http://image.thelancet.com/extras/03art6318web.pdfn n

Chikungunya Fever in Travelers: Clinical Presentation and Course

BACKGROUNDnAn outbreak of chikungunya virus infection emerged in the southwest Indian Ocean islands in 2005, spread out to India, and resulted in an ongoing outbreak that has involved >1.5 million patients, including travelers who have visited these areas.nnnMETHODSnOur study investigated 69 travelers who developed signs and symptoms compatible with chikungunya fever after returning home from countries involved in the epidemic. Twenty cases of infection that were confirmed by serological analysis, polymerase chain reaction, and/or cell culture were investigated.nnnRESULTSnAll patients experienced flulike symptoms with fever and joint pain. No serious complications were observed, but 69% of the patients had persistent arthralgia for >2 months, and 13% had it for >6 months. Viral RNA could be detected in blood samples using reverse-transcriptase polymerase chain reaction in 4 of 4 patients who presented to a health care facility during their first week of illness, and the virus was successfully isolated from blood samples obtained from 2 of these patients. Chikungunya virus-specific immunoglobulin M and/or immunoglobulin G antibodies were detected in all patients. However, initial testing of serum samples yielded negative results for 3 of 5 patients during the first week.nnnCONCLUSIONSnChikungunya fever must be considered in travelers who develop fever and arthritis after traveling to areas affected by an ongoing epidemic. Related arthritis mainly affects smaller joints and often persists for extended periods. Serological testing may have negative results during the first week of the disease; diagnosis using polymerase chain reaction appears to be more reliable during this time. Travelers to areas of epidemicity should be informed of the risk of infection and of adequate preventive measures, such as protection against mosquitos.

Detection of Ebola Virus in Oral Fluid Specimens during Outbreaks of Ebola Virus Hemorrhagic Fever in the Republic of Congo

BACKGROUNDnPatients who have refused to provide blood samples has meant that there have been significant delays in confirming outbreaks of Ebola virus hemorrhagic fever (EVHF). During the 2 EVHF outbreaks in the Republic of Congo in 2003, we assessed the use of oral fluid specimens versus serum samples for laboratory confirmation of cases of EVHF.nnnMETHODSnSerum and oral fluid specimens were obtained from 24 patients with suspected Ebola and 10 healthy control subjects. Specimens were analyzed for immunoglobulin G antibodies by enzyme-linked immunosorbent assay (ELISA) and for Ebola virus by antigen detection ELISA and reverse-transcriptase polymerase chain reaction (RT-PCR). Oral fluid specimens were collected with a commercially available collection device.nnnRESULTSnWe failed to detect antibodies against Ebola in the oral fluid specimens obtained from patients whose serum samples were seropositive. All patients with positive serum RT-PCR results also had positive results for their oral fluid specimens.nnnCONCLUSIONSnThis study demonstrates the usefulness of oral fluid samples for the investigation of Ebola outbreaks, but further development in antibodies and antigen detection in oral fluid specimens is needed before these samples are used for filovirus surveillance activities in Africa.

Diagnosing Schistosomiasis by Detection of Cell-Free Parasite DNA in Human Plasma

Introduction Schistosomiasis (bilharzia), one of the most relevant parasitoses of humans, is confirmed by microscopic detection of eggs in stool, urine, or organ biopsies. The sensitivity of these procedures is variable due to fluctuation of egg shedding. Serological tests on the other hand do not distinguish between active and past disease. In patients with acute disease (Katayama syndrome), both serology and direct detection may produce false negative results. To overcome these obstacles, we developed a novel diagnostic strategy, following the rationale that Schistosoma DNA may be liberated as a result of parasite turnover and reach the blood. Cell-free parasite DNA (CFPD) was detected in plasma by PCR. Methodology/Principal Findings Real-time PCR with internal control was developed and optimized for detection of CFPD in human plasma. Distribution was studied in a mouse model for Schistosoma replication and elimination, as well as in human patients seen before and after treatment. CFPD was detectable in mouse plasma, and its concentration correlated with the course of anti-Schistosoma treatment. Humans with chronic disease and eggs in stool or urine (nu200a=u200a14) showed a 100% rate of CFPD detection. CFPD was also detected in all (nu200a=u200a8) patients with Katayama syndrome. Patients in whom no viable eggs could be detected and who had been treated for schistomiasis in the past (nu200a=u200a30) showed lower detection rates (33.3%) and significantly lower CFPD concentrations. The duration from treatment to total elimination of CFPD from plasma was projected to exceed one year. Conclusions/Significance PCR for detection of CFPD in human plasma may provide a new laboratory tool for diagnosing schistosomiasis in all phases of clinical disease, including the capacity to rule out Katayama syndrome and active disease. Further studies are needed to confirm the clinical usefulness of CFPD quantification in therapy monitoring.

Monitoring of clinical and laboratory data in two cases of imported Lassa fever

During 2000, four cases of fatal Lassa fever were imported from Africa to Europe. In two patients, consecutive serum samples were available for monitoring of virus load and cytokine levels in addition to standard laboratory data. Both patients had non-specific early clinical symptoms including high fever. Patient 1 developed multi-organ failure and died of hemorrhagic shock on day 15 of illness, while patient 2 died of respiratory failure due to aspiration without hemorrhage on day 16. Ribavirin was administered to both patients beginning only on day 11. High serum aspartate aminotransferase and lactate dehydrogenase (LDH) levels were remarkable in both patients. Patient 1 had an initial virus load of 10(6) S RNA copies/ml as measured by real-time RT-PCR. Viremia increased steadily and reached a plateau of approximately 10(8)-10(9) copies/ml 4 days before death, while IFN-gamma and TNF-alpha rose to extremely high levels only shortly before death. In contrast, in patient 2 the virus load decreased from 10(7) to 10(6) copies/ml during the late stage of illness which was paralleled by a decrease in the IFN-gamma and TNF-alpha levels. The IL-10 level increased when specific IgM and IgG appeared. These data suggest that a high virus load and high levels of pro-inflammatory cytokines in the late stage of Lassa fever play an important role in the pathogenesis of hemorrhage, multi-organ failure, and shock in Lassa fever.

Sequence and Phylogenetic Analysis of Hepatitis B Virus Genotype G Isolated in Germany

Genotype G of hepatitis B virus (HBV) has only recently been discovered. This report describes the full-length sequence of genotype G HBV (designated 235/01) isolated in Germany from a chronic HBV carrier. The patient was hepatitis B e antigen-positive, had high HBV DNA levels of about 1010 copies/ml serum, lacked a measurable anti-HBc response, and was coinfected with human immunodeficiency virus type 1. Genome 235/01 showed characteristic genotype G-specific features: stop codons at codon 2 and 28 of the pre-C region and insertion of 36 nucleotides at the 5′ end of the C gene. It was nearly identical (≥99.7% identity) to both genotype G genomes (B1-89 and FR1 from France) described so far, suggesting either close epidemiological link among European genotype G isolates or high genetic stability of genotype G.

Management and Outcomes after Multiple Corneal and Solid Organ Transplantations from a Donor Infected with Rabies Virus

BACKGROUNDnThis article describes multiple transmissions of rabies via transplanted solid organ from a single infected donor. The empirical Milwaukee treatment regimen was used in the recipients.nnnMETHODSnSymptomatic patients were treated by deep sedation (ketamine, midazolam, and phenobarbital), ribavirin, interferon, and active and passive vaccination. Viral loads and antibodies were continuously monitored.nnnRESULTSnRecipients of both cornea and liver transplants developed no symptoms. The recipient of the liver transplant had been vaccinated approximately 20 years before transplantation. Two recipients of kidney and lung transplants developed rabies and died within days of symptomatic disease. Another kidney recipient was treated 7 weeks before he died. The cerebrospinal fluid viral load remained at constant low levels (<10,000 copies/mL) for approximately 5 weeks; it increased suddenly by almost 5 orders of magnitude thereafter. After death, no virus was found in peripheral compartments (nerve tissue, heart, liver, or the small intestine) in this patient, in contrast to in patients in the same cohort who died early.nnnCONCLUSIONSnOur report includes, to our knowledge, the longest documented treatment course of symptomatic rabies and the first time that the virus concentration was measured over time and in different body compartments. The postmortem virus concentration in the periphery was low, but there was no evidence of a reduction of virus in the brain.

Severe acute respiratory syndrome: identification of the etiological agent.

n Abstractn n The severe acute respiratory syndrome (SARS) emerged in late 2002 in southern China and rapidly spread to countries around the globe. Three research groups within a World Health Organization (WHO)-coordinated network have independently and simultaneously shown that a novel coronavirus is linked to SARS. A fourth group has completed the Kochs postulates by infecting monkeys with the agent. Sequencing of the complete genome was achieved only weeks after the first isolate of the virus became available.n n

Evaluation of the first commercial chikungunya virus indirect immunofluorescence test

The chikungunya virus (CHIKV), an arbovirus of the genus Alphavirus, family Togaviridae, is mainly transmitted by Aedes mosquitoes. It causes an acute infection, characterized by high fever, polyarthralgia and rash and was responsible for a major outbreak which started in 2005 and spread over many islands of the south western Indian Ocean before it hit the Indian subcontinent. As nucleic acid amplification can be used only during the viremic period, serological tests are most widely used for the diagnosis of CHIKV infections. CHIKV IgM and IgG antibodies can be detected as soon as 3-6 days after clinical onset, respectively. Presently only in-house ELISA and immunofluorescence tests exist for analysing the CHIKV specific immune response. The first commercial indirect immunofluorescence test (IIFT) (EUROIMMUN AG, Lüebeck, Germany) was evaluated using two sera panels of patients from La Reunion and travellers returning with CHIKV infections from the Indian Ocean region. The IgM IIFT shows a specificity of 98.3% and a sensitivity of 96.9%. The specificity and sensitivity for the IgG IIFT are 100.0% and 95.4%, respectively. This commercial IIFT is a valuable tool for the diagnosis of CHIKV infections and antibody seroprevalence studies.

Diagnostic Reverse-Transcription Polymerase Chain Reaction Kit for Filoviruses Based on the Strain Collections of all European Biosafety Level 4 Laboratories

Abstract A network of European biosafety level 4 laboratories has designed the first industry-standard molecular assay for all filoviruses species, based on the strain collections of all participants. It uses 5 optimized L gene primers and 3 probes, as well as an internal control with a separate detection probe. Detection limits (probit analysis, 95% detection chance) were as follows: Zaire ebolavirus, 487 copies/mL of plasma; Sudan ebolavirus Maleo, 586 copies/mL; Sudan ebolavirus Gulu, 1128 copies/mL; Cote dIvoire ebolavirus, 537 copies/mL; Reston ebolavirus, 4546 copies/mL; Lake Victoria marburgvirus Musoke, 860 copies/mL; and Lake Victoria marburgvirus Ravn, 1551 copies/mL. The assay facilitates reliable detection or exclusion screening of filovirus infections.