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Biomedical subjects

Christian Drosten

Publications and source records attributed to Christian Drosten.

At least 19 recordsLinked to original sources
PubMed2007

Generic detection of coronaviruses and differentiation at the prototype strain level by reverse transcription-PCR and nonfluorescent low-density microarray.

A nonfluorescent low-cost, low-density oligonucleotide array was designed for detecting the whole coronavirus genus after reverse transcription (RT)-PCR. The limit of detection was 15.7 copies/reaction. The clinical detection limit in patients with severe acute respiratory syndrome was 100 copies/sample. In 39 children suffering from coronavirus 229E, NL63, OC43, or HKU1, the sensitivity was equal to that of individual real-time RT-PCRs.

Bacterial Typing Techniques↗
PubMed2006

High throughput screening for spores and vegetative forms of pathogenic B. anthracis by an internally controlled real-time PCR assay with automated DNA preparation.

Human infections with Bacillus anthracis have become rare but in cases of intentional release, masses of samples would have to be expected. Current PCR assays for anthrax are appropriate for use in single cases, but they have not been formulated for high throughput screening. This article describes a high throughput real-time PCR for anthrax, including automated sample preparation without the need for pre-culturing of samples. The assay detects single copies of target gene. An internal control monitors the whole assay including sample preparation. The limit of detection in blood was 1,066 (95%CI, 741-1,739) copies/ml, corresponding to 4.4-32.3 organisms/ml. Using spore preparations, 20 colony-forming units (CFU) per sample could be detected reliably (0.8 CFU per PCR). The extraction procedures depleted viable spores from solution by factors of 10,000 (automated procedure) and >100,000 (conventional column procedure). One hundred and ten clinical and environmental specimens were retested, 50 of them sampled during a period of heightened anthrax awareness in 2001. A widely used assay yielded two false positive results (cross-reaction with B. cereus), while the new assay tested all samples negative. The internal control operated stable in all clinical samples. The assay is capable of testing for anthrax in the high throughput mode.

Automation↗
PubMed2006

Reverse ELISA for IgG and IgM antibodies to detect Lassa virus infections in Africa.

BACKGROUND: Anti-Lassa antibodies are detected by indirect immunofluorescence assay (IFA) or by enzyme-immunoassay (ELISA). Both methods have problems to detect low amounts of specific antibodies. OBJECTIVES: We report here highly sensitive and specific reverse ELISAs to detect Lassa virus IgG and IgM antibodies. Due to the reverse techniques, serum samples could be applied at dilutions of 1:10 without increasing non-specific background reactions. STUDY DESIGN: For IgM antibody detection microtiter plates were coated with anti-IgM antibodies and for IgG antibody detection with rheumatoid factor (RF) (Sachers M, Emmerich P, Mohr H, Schmitz H. Simple detection of antibodies to different viruses using rheumatoid factor and enzyme-labelled antigen (ELA). J Virol Methods 1985;10:99-110). In both assays a tissue culture antigen was used in combination with a labeled anti-Lassa monoclonal antibody (Hufert FT, Ludke W, Schmitz H. Epitope mapping of the Lassa virus nucleoprotein using monoclonal anti-nucleocapsid antibodies. Arch Virol 1989;106(3-4):201-12). RESULTS: The reverse ELISA turned out to detect virus-specific IgG and IgM antibody in all 20 samples of West African patients collected 2-8 weeks after onset of Lassa fever. Moreover, both IFA and reverse ELISA found IgG antibodies in 53 out of 643 samples of healthy West Africans (sensitivity of 100%). Six of the 643 samples were positive by reverse IgG ELISA only. Thus, the specificity compared to IIF was 99.0%, but it may be even higher, because compared to IFA the IgG ELISA was clearly more sensitive in detecting low antibody titers. CONCLUSIONS: In Ghana 3% seropositives were found by IFA, but 4% by the reverse ELISA. The reverse ELISAs can be performed with high sensitivity and specificity under field conditions in Africa.

Africa↗
PubMed2006

First international proficiency study on West Nile virus molecular detection.

BACKGROUND: West Nile virus (WNV) molecular detection is being conducted by a growing number of laboratories, but the degree of proficiency may vary between them. External quality control is needed. METHODS: We have conducted an international quality assurance study on WNV molecular detection. Participating laboratories tested noninfectious samples inactivated by heat and gamma irradiation. Participants received 7 coded lyophilized samples containing WNV of genetic lineages 1a, 1b, and 2 at 2600 to 18 000 000 RNA copies/mL, 3 samples containing heterologous flaviviruses, and 2 negative samples. RESULTS: Thirty laboratories participated. The average laboratory achieved 50% detection probability from 7762 copies/mL onward (probit analysis; 95% CI = 1174-24547 copies/mL). Lineages 1a and 1b were detected with equal efficiencies, but the lineage 2 strain (Ug37) was detected at significantly lower rates. Only 27% of participants were able to detect the 6 samples containing > or =1.8 x 10(4) copies/mL. Three laboratories generated false-positive results in negative samples. Six of 30 laboratories reported correct strain identification in 3 samples containing non-WNV flaviviruses. We observed a significant positive correlation between the capability of detecting non-WNV flaviviruses and detecting WNV lineage 2. CONCLUSIONS: Most participants showed good performance in detecting lineage 1 WNV, the predominant virus in the Northern Hemisphere. The inability of some laboratories to detect even highly concentrated lineage 2 WNV downgraded the overall outcome. The lineage 2 material received through this study will provide laboratories with the necessary template for improving their assays. Such material is otherwise hard to obtain.

Animals↗
PubMed2006

Detection of Ebola virus in oral fluid specimens during outbreaks of Ebola virus hemorrhagic fever in the Republic of Congo.

BACKGROUND: Patients 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. METHODS: Serum 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. RESULTS: We 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. CONCLUSIONS: This 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.

Adult↗
PubMed2006

Ultrasensitive monitoring of HIV-1 viral load by a low-cost real-time reverse transcription-PCR assay with internal control for the 5' long terminal repeat domain.

BACKGROUND: Current HIV-1 viral-load assays are too expensive for resource-limited settings. In some countries, monitoring of antiretroviral therapy is now more expensive than treatment itself. In addition, some commercial assays have shown shortcomings in quantifying rare genotypes. METHODS: We evaluated real-time reverse transcription-PCR with internal control targeting the conserved long terminal repeat (LTR) domain of HIV-1 on reference panels and patient samples from Brazil (n = 1186), South Africa (n = 130), India (n = 44), and Germany (n = 127). RESULTS: The detection limit was 31.9 IU of HIV-1 RNA/mL of plasma (> 95% probability of detection, Probit analysis). The internal control showed inhibition in 3.7% of samples (95% confidence interval, 2.32%-5.9%; n = 454; 40 different runs). Comparative qualitative testing yielded the following: Roche Amplicor vs LTR assay (n = 431 samples), 51.7% vs 65% positives; Amplicor Ultrasensitive vs LTR (n = 133), 81.2% vs 82.7%; BioMerieux NucliSens HIV-1 QT (n = 453), 60.5% vs 65.1%; Bayer Versant 3.0 (n = 433), 57.7% vs 55.4%; total (n = 1450), 59.0% vs 63.8% positives. Intra-/interassay variability at medium and near-negative concentrations was 18%-51%. The quantification range was 50-10,000,000 IU/mL. Viral loads for subtypes A-D, F-J, AE, and AG yielded mean differences of 0.31 log(10) compared with Amplicor in the 10(3)-10(4) IU/mL range. HIV-1 N and O were not detected by Amplicor, but yielded up to 180 180.00 IU/mL in the LTR assay. Viral loads in stored samples from all countries, compared with Amplicor, NucliSens, or Versant, yielded regression line slopes (SD) of 0.9 (0.13) (P < 0.001 for all). CONCLUSIONS: This method offers all features of commercial assays and covers all relevant genotypes. It could allow general monitoring of antiretroviral therapy in resource-limited settings.

Brazil↗
PubMed2006

Follow-up on diagnostic proficiency of laboratories equipped to perform orthopoxvirus detection and quantification by PCR: the second international external quality assurance study.

Two years after the first external quality assurance study on bioterrorism-relevant viruses, we have conducted a follow-up study on orthopoxvirus detection by PCR. Thirty-three laboratories (27 European, 4 Austral-Asian, and 2 American) participated. Samples contained 0 to 40,000,000 DNA copies of lyophilized monkeypox, cowpox, and vaccinia virus per ml. Laboratories achieved a >80% detection chance above 56,234 copies per ml. Global sensitivity was not significantly improved over that of the first study. Twenty-seven and 9 participants, respectively, were able to genotype and quantify virus. Four of 27 genotyping results were incorrect. Quantification accuracy was significantly better for vaccinia virus than for the other viruses. False-positive results occurred in 22 (11.8%) of all 186 tests on negative samples, but 18 of these were contributed by only five laboratories. Fifty-five percent of laboratories could appropriately detect PCR inhibition. The use of either real-time PCR or commercial diagnostic kits had significant positive influence on laboratory performance.

Clinical Laboratory Techniques↗
PubMed2006

MassTag polymerase chain reaction for differential diagnosis of viral hemorrhagic fever.

Viral hemorrhagic fevers are associated with high rates of illness and death. Although therapeutic options are limited, early differential diagnosis has implications for containment and may aid in clinical management. We describe a diagnostic system for rapid, multiplex polymerase chain reaction identification of 10 different causes of viral hemorrhagic fevers.

Diagnosis, Differential↗
PubMed2005

SARS transmission pattern in Singapore reassessed by viral sequence variation analysis.

BACKGROUND: Epidemiological investigations of infectious disease are mainly dependent on indirect contact information and only occasionally assisted by characterization of pathogen sequence variation from clinical isolates. Direct sequence analysis of the pathogen, particularly at a population level, is generally thought to be too cumbersome, technically difficult, and expensive. We present here a novel application of mass spectrometry (MS)-based technology in characterizing viral sequence variations that overcomes these problems, and we apply it retrospectively to the severe acute respiratory syndrome (SARS) outbreak in Singapore. METHODS AND FINDINGS: The success rate of the MS-based analysis for detecting SARS coronavirus (SARS-CoV) sequence variations was determined to be 95% with 75 copies of viral RNA per reaction, which is sufficient to directly analyze both clinical and cultured samples. Analysis of 13 SARS-CoV isolates from the different stages of the Singapore outbreak identified nine sequence variations that could define the molecular relationship between them and pointed to a new, previously unidentified, primary route of introduction of SARS-CoV into the Singapore population. Our direct determination of viral sequence variation from a clinical sample also clarified an unresolved epidemiological link regarding the acquisition of SARS in a German patient. We were also able to detect heterogeneous viral sequences in primary lung tissues, suggesting a possible coevolution of quasispecies of virus within a single host. CONCLUSION: This study has further demonstrated the importance of improving clinical and epidemiological studies of pathogen transmission through the use of genetic analysis and has revealed the MS-based analysis to be a sensitive and accurate method for characterizing SARS-CoV genetic variations in clinical samples. We suggest that this approach should be used routinely during outbreaks of a wide variety of agents, in order to allow the most effective control.

DNA, Viral↗
PubMed2005

Analysis of two imported cases of yellow fever infection from Ivory Coast and The Gambia to Germany and Belgium.

BACKGROUND: Yellow fever remains one of the great burdens for public health in the endemic regions in Africa and South America. The under reporting of yellow fever cases in the respective regions and lack of international interest leads to an underestimation of the constant danger in these areas. Non-vaccinated travelers take a high risk without the effective protection of YFV 17D vaccination. OBJECTIVES: Two YF cases were imported to Europe in the last 4 years. We characterized two yellow fever virus (YFV) isolates from severely infected patients coming back from Africa, Ivory Coast and The Gambia, by genome sequencing and phylogenetic analysis. STUDY DESIGN: The virus infections in different organs were analyzed with pathological, immunohistological, electronmicroscopical and quantitative real-time PCR methods. RESULTS AND CONCLUSION: High virus loads in spleen and liver (2.4 x 10 (6) to 3 x 10 (7)GE/mL) demonstrated by real time PCR show massive virus replication leading to extraordinary progression of the disease in these patients. Immunohistological and electronmicroscopical analysis confirms virus particles in liver tissue. In all other organs no virus could be detected. A fast, specific and sensitive virus PCR detection is recommended for diagnostic of acute infections. The further sequence alignments show that the new isolates belong to the type II West African strain with great homology to over 40-year old YF isolates from Senegal and Ghana. The divergence observed was on average 3.3%, ranging from 0.0% to 5.0% in the coding region of Gambia 2001 strain and 2.9 %, ranging from 0.0% to 4.3% in the coding region of the Ivory C 1999 strain. Most mutations (5.0%/4.3%, respectively) occurred in the envelope protein.

Base Sequence↗
PubMed2005

Establishment of conventional and fluorescence resonance energy transfer-based real-time PCR assays for detection of pathogenic New World arenaviruses.

BACKGROUND: Five of the known arenaviruses cause viral hemorrhagic fever in humans and are classified as biosafety level 4 pathogens. Four of the viruses, namely Junin, Guanarito, Machupo, and Sabia, belong to clade B of New World arenaviruses that also comprises the nonpathogenic viruses Tacaribe, Cupixi, and Amapari. OBJECTIVES: To establish real-time reverse transcription (RT)-PCR assays for Junin and Guanarito virus based on fluorescence resonance energy transfer (FRET) probes, and a universal RT-PCR assay for all known clade B viruses with conventional read-out. RESULTS: Conserved sequences in the nucleoprotein gene were chosen as target sites for primers and FRET probes. A common set of primers was designed for all three assays. The assays were based on one-step RT-PCR reagents and were optimised with respect to analytical sensitivity using synthetic RNA templates. The real-time PCR assays detected about 0.5 and 5TCID(50) of cell culture-derived Junin and Guanarito virus, respectively. The universal clade B PCR amplified cell culture-derived RNA of Junin, Guanarito, Machupo, and Sabia virus (5-500TCID(50) per reaction), as well as RNA of Tacaribe, Cupixi, and Amapari virus. CONCLUSIONS: The PCR assays may be used as complementary diagnostic tests for pathogenic New World arenaviruses. The universal PCR assay could also be suitable for the detection of novel clade B arenaviruses in patients as well as in animal reservoirs.

Animals↗
PubMed2005

Topographic and quantitative display of integrated human immunodeficiency virus-1 provirus DNA in human lymph nodes by real-time polymerase chain reaction.

In situ polymerase chain reaction (isPCR) has been applied in many fields that require detection of a genomic marker in combination with its topographic localization in tissue. We describe here a novel approach that circumvents the major drawbacks of in situ PCR, ie, low sensitivity, leakage of DNA from cells, and inability to quantify the DNA input. Frozen sections of a lymph node from a human immunodeficiency virus (HIV)-1-infected patient were fixed on glass microscope slides, and the glass was scored into square fragments of 0.5-mm edge length using a diamond cutting device. Slides were then attached to adhesive, elastic plastic foil and finally broken, and the foil was extended to allow sorting of fragments into PCR microtiter plates. The material was tested for HIV-1 proviral DNA by a sensitive real-time PCR protocol. Subjacent sections were stained for follicular dendritic cells to identify follicles. The fragmentation process prevented leakage of amplified DNA to neighboring areas as often experienced with in situ PCR. Provirus was clearly associated with follicular areas, in which provirus-carrying cells represented an average of 0.8% of the total cell population (peak density, 3.1% of all follicular cells). The results of this method suggest that the high density of provirus-containing cells in follicles may be important for the persistence of proviral DNA in infected persons.

Adult↗
PubMed2005

Cinanserin is an inhibitor of the 3C-like proteinase of severe acute respiratory syndrome coronavirus and strongly reduces virus replication in vitro.

The 3C-like proteinase (3CLpro) of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is one of the most promising targets for anti-SARS-CoV drugs due to its crucial role in the viral life cycle. In this study, a database containing structural information of more than 8,000 existing drugs was virtually screened by a docking approach to identify potential binding molecules of SARS-CoV 3CLpro. As a target for screening, both a homology model and the crystallographic structure of the binding pocket of the enzyme were used. Cinanserin (SQ 10,643), a well-characterized serotonin antagonist that has undergone preliminary clinical testing in humans in the 1960s, showed a high score in the screening and was chosen for further experimental evaluation. Binding of both cinanserin and its hydrochloride to bacterially expressed 3CLpro of SARS-CoV and the related human coronavirus 229E (HCoV-229E) was demonstrated by surface plasmon resonance technology. The catalytic activity of both enzymes was inhibited with 50% inhibitory concentration (IC50) values of 5 microM, as tested with a fluorogenic substrate. The antiviral activity of cinanserin was further evaluated in tissue culture assays, namely, a replicon system based on HCoV-229E and quantitative test assays with infectious SARS-CoV and HCoV-229E. All assays revealed a strong inhibition of coronavirus replication at nontoxic drug concentrations. The level of virus RNA and infectious particles was reduced by up to 4 log units, with IC50 values ranging from 19 to 34 microM. These findings demonstrate that the old drug cinanserin is an inhibitor of SARS-CoV replication, acting most likely via inhibition of the 3CL proteinase.

Animals↗
PubMed2004

Rapid detection and differentiation of human pathogenic orthopox viruses by a fluorescence resonance energy transfer real-time PCR assay.

BACKGROUND: The orthopox viruses that are pathogenic for humans include variola major virus (VAR), monkeypox virus (MPV), cowpox virus (CPV), and to a lesser extent, camelpox virus (CML) and vaccinia virus (VAC). PCR is a powerful tool to detect and differentiate orthopox viruses, and real-time PCR has the further advantages of rapid turnaround time, low risk of contamination, capability of strain differentiation, and use of multiplexed probes. METHODS: We used real-time PCR with fluorescence resonance energy transfer technology to simultaneously detect and differentiate VAR, MPV, CPV/VAC, and CML. An internal control generated by cloning and mutating the PCR target gene facilitated monitoring of PCR inhibition in each individual test reaction. RESULTS: Strain differentiation results showed little interassay variability (CV, 0.4-0.6%), and the test was 100-fold more sensitive than virus culture on Vero cells. Low copy numbers of DNA could be detected with > or =95% probability (235-849 genome copies/mL of plasma). CONCLUSIONS: The real-time PCR assay can detect and differentiate human pathogenic orthopox viruses. The use of an internal control qualifies the assay for high sample throughput, as is likely to be needed in situations of suspected acts of biological terrorism, e.g., use of VAR.

Animals↗
PubMed2004

Application of real-time PCR for testing antiviral compounds against Lassa virus, SARS coronavirus and Ebola virus in vitro.

This report describes the application of real-time PCR for testing antivirals against highly pathogenic viruses such as Lassa virus, SARS coronavirus and Ebola virus. The test combines classical cell culture with a quantitative real-time PCR read-out. The assay for Lassa virus was validated with ribavirin, which showed an IC(50) of 9 micrograms/ml. Small-scale screening identified a class of imidazole nucleoside/nucleotide analogues with antiviral activity against Lassa virus. The analogues contained either dinitrile or diester groups at the imidazole 4,5-positions, and many of which possessed an acyclic sugar or sugar phosphonate moiety at the imidazole 1-position. The IC(50) values of the most active compounds ranged from 5 to 21 micrograms/ml. The compounds also inhibited replication of SARS coronavirus and Ebola virus in analogous assays, although to a lesser extent than Lassa virus.

Antiviral Agents↗