Sonja Linke

Highly sensitive real-time PCR for specific detection and quantification of Coxiella burnetii

BackgroundCoxiella burnetii, the bacterium causing Q fever, is an obligate intracellular biosafety level 3 agent. Detection and quantification of these bacteria with conventional methods is time consuming and dangerous. During the last years, several PCR based diagnostic assays were developed to detect C. burnetii DNA in cell cultures and clinical samples. We developed and evaluated TaqMan-based real-time PCR assays that targeted the singular icd (isocitrate dehydrogenase) gene and the transposase of the IS1111a element present in multiple copies in the C. burnetii genome.ResultsTo evaluate the precision of the icd and IS1111 real-time PCR assays, we performed different PCR runs with independent DNA dilutions of the C. burnetii Nine Mile RSA493 strain. The results showed very low variability, indicating efficient reproducibility of both assays. Using probit analysis, we determined that the minimal number of genome equivalents per reaction that could be detected with a 95% probability was 10 for the icd marker and 6.5 for the IS marker. Plasmid standards with cloned icd and IS1111 fragments were used to establish standard curves which were linear over a range from 10 to 107 starting plasmid copy numbers. We were able to quantify cell numbers of a diluted, heat-inactivated Coxiella isolate with a detection limit of 17 C. burnetii particles per reaction. Real-time PCR targeting both markers was performed with DNA of 75 different C. burnetii isolates originating from all over the world. Using this approach, the number of IS1111 elements in the genome of the Nine Mile strain was determined to be 23, close to 20, the number revealed by genome sequencing. In other isolates, the number of IS1111 elements varied widely (between seven and 110) and seemed to be very high in some isolates.ConclusionWe validated TaqMan-based real-time PCR assays targeting the icd and IS1111 markers of C. burnetii. The assays were shown to be specific, highly sensitive and efficiently reproducible. Cell numbers in dilutions of a C. burnetii isolate were reliably quantified. PCR quantification suggested a high variability of the number of IS1111 elements in different C. burnetii isolates, which may be useful for further phylogenetic studies.

First International Proficiency Study on West Nile Virus Molecular Detection

Abstract 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 ≥1.8 × 104 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.

West Nile virus monitoring in migrating and resident water birds in Iran: are common coots the main reservoirs of the virus in wetlands?

A molecular and serological study was carried out to determine the West Nile virus (WNV) status in different species of wild water birds. From 2003 to 2007, samples were collected from 519 birds representing 26 different species in Iran. Out of 519 serum samples tested for WNV antibodies, 78 (15%) were positive when tested using virus neutralization and immunofluorescence. Antibodies of WNV were detected in 71 out of 131 common coot (Fulica atra) samples. In comparison, only 7 out of 388 birds that were belonged to 25 other species of water birds revealed positive results. For most Anatidae species, no positive duck in serological tests was found. Further, no WNV viral RNA-positive samples were found in this study. Results of this investigation provide important information about the prevalence of WNV in wild water birds in Iran and indicate the potential role and importance of common coots in ecology of WNVs.

Second external quality assessment of the molecular diagnostic of West Nile virus: Are there improvements towards the detection of WNV?

BACKGROUND WNV epidemics occur worldwide, new WNV isolates were isolated in southern-east Europe belonging to WNV lineage 2. A first international proficiency study on WNV indicted that some laboratories were not able to detect WNV lineage 2 virus genome by their PCR diagnostic assays. Therefore an actual External Quality Assessment with both virus lineages was performed to monitor the improvements in molecular diagnostics. OBJECTIVES To asses the proficiency of laboratories to detect West Nile virus with molecular diagnostic tests. STUDY DESIGN A test panel of different WNV isolates and virus dilutions was given to 26 laboratories to test the samples with their routine diagnostic methods. RESULTS Twenty-one participating laboratories provided 28 data set results. WNV lineage 1 was detected with high overall efficiency of 92% (67.9-100%) but two different WNV lineage 2 strains were detected at lower rates (mean = 73%, 67.9-75%) by the different PCR assays. 93% of the laboratories were able to detect a WNV lineage 1 with a concentration of 1.2×10(4)copies/ml but the detection rate was decreased to 68% for 1.2×10(3)copies/ml. One laboratory generated false-positive result from the non-virus control samples and 29% of the datasets showed false-positive results for non-WNV flavivirus samples. CONCLUSIONS The WNV EQA showed an improved proficiency of laboratories as compared to the first EQA. However, the data suggest that problems in the detection of both lineages were still present since the first proficiency test was performed in 2006. Further proceedings versus the detection of both lineages are needed particularly for in-house assays.

Generation and characterisation of monoclonal antibodies against influenza virus A, subtype H5N1

Abstract The emergence of highly pathogenic avian influenza A virus (HPAIV) subtype H5N1 in 1997 has since resulted in large outbreaks in poultry and in transmission from poultry to humans, mostly in southeast Asia, but also in several European countries. Effective diagnosis and control measures are essential for the management of HPAIV infections. To develop a rapid diagnostic test, a panel of murine monoclonal antibodies (mAbs) against influenza virus A subtype H5 was generated. Eleven mAbs were produced and characterised according to their reactivity by indirect and sandwich ELISA and western blotting against different H5 subtypes representing past and viruses currently circulating. Ten out of 11 mAbs reacted strongly with the haemagglutinin (HA) protein of H5 viruses, whereas one mAb reacted with the M1 protein. Targeted HA protein epitopes seemed to be conformational. One hybridoma clone binds to a linear epitope of the M1 protein. One specific mAb reacts with HPAIV H5 in the immunofluorescence test, and two antibodies neutralised H5 viruses. On the basis of the results, the set of seven mAbs is appropriate for developing diagnostic tests. With the generated mAbs, a sandwich ELISA was developed recognising all H5N1 strains tested but no other influenza viruses. With this ELISA, as little as 0.005 HA units or 0.1ng/ml H5N1 was detected, surpassing other ELISA tests. The novel reagents have the potential to improve significantly available rapid antigen detection systems.

Lack of detectable West Nile virus RNA in brains and kidneys of dogs and cats with immunohistological precipitates using virus-specific antibodies

Five dogs and four cats from Germany suffering from encephalitis revealed positive immunoreactivity using two West Nile virus (WNV) specific monoclonal antibodies in brain and in kidney. However, WNV-infection could not be confirmed by additional PCR analyses. This study indicated that positive immunoreactivity for WNV in dogs and cats must be interpreted cautiously and should be confirmed by a second virus-specific technique.

Assessing the exposure of German and Austrian bird ringers to West Nile virus (Flavivirus) and evaluating their potential risk of infection

West Nile virus (WNV) is a mosquito-borne Flavivirus with birds as its natural reservoir. Mosquitoes are able to transmit WNV to humans. In Europe, WNV is most probably imported by migrating birds from WNV-endemic regions. In this study, the risk of WNV infection during bird-ringing activities in Germany and Austria was investigated. Several serological test systems were used to study WNV antibody prevalence among 137 bird ringers. Neutralising antibodies were detected in three of the bird ringers. This seropositivity could be explained by alternative factors such as traveling to endemic areas. The application of different serological methods showed that WNV IgG ELISAs were less specific than WNV IgG immunofluorescence tests (IFT). The neutralisation test (NT) appears to be the gold standard for Flavivirus differentiation. At present, we assume that bird-ringing activity in Germany and Austria harbours a very low risk of WNV infection.

Investigations on the aetiology of pinching off syndrome in four white-tailed sea eagles (Haliaeetus albicilla) from Germany

The purpose of this study was to investigate the aetiology of the pinching off syndrome (POS), a generalized feather abnormality affecting free-living nestling of the white-tailed sea eagle (Haliaeetus albicilla) in Europe. For the first time, extensive clinical, haematological, biochemical, virological, bacteriological, nutritional, histopathological, parasitological and electron microscopical examinations were performed on three females and one male suffering from POS. Early and increased cytokeratin formation at the base of regenerating feathers and their follicle was observed in affected birds. Ultrathin sections of the feather papillae revealed an extended stratum transitivum and a compact, thickened keratinized stratum corneum. The transitional cells in POS feathers contained vacuoles often associated with the nucleus. Lipofuscin accumulations in neurons, glial cells and islet cells of the pancreas were found in all examined birds. It was not clear whether there is an association between the occurrence of lipofuscin and POS. No evidence was found to suggest that infectious agents (parasites, bacteria, fungi or viruses), malnutrition or hormonal imbalances are involved in the aetiology of POS in white-tailed sea eagles. It remains unclear whether there is a genetic background of POS.