Accurate detection of Zika virus IgG using a novel immune complex binding ELISA.

OBJECTIVES: Accurate serological assays are urgently needed to support public health responses to Zika virus (ZIKV) infection with its potential to cause foetal damage during pregnancy. Current flavivirus serology for ZIKV infections lacks specificity due to cross-reacting antibodies from closely related other flaviviruses. In this study, we evaluated novel serological tests for accurate ZIKV IgG detection. METHODS: Our ELISAs are based on immune complex binding. The high specificity is achieved by the simultaneous incubation of labelled ZIKV antigen and unlabelled flavivirus homolog protein competitors. Two assays were validated with a panel of 406 human samples from PCR-confirmed ZIKV patients collected in Brazil (n = 154), healthy blood donors and other infections from Brazil, Europe, Canada and Colombia (n = 252). RESULTS: The highest specificity (100% [252/252, 95% confidence interval (CI) 98.5-100.0]) was shown by the ZIKV ED3 ICB ELISA using the ED3 antigen of the ZIKV envelope. A similar test using the NS1 antigen (ZIKV NS1 ICB ELISA) was slightly less specific (92.1% [232/252, 95% CI 88.0-95.1]). The commercial Euroimmun ZIKV ELISA had a specificity of only 82.1% (207/252, 95% CI 76.8-86.7). Sensitivity was high (93-100%) from day 12 after onset of symptoms in all three tests. Seroprevalence of ZIKV IgG was analysed in 87 samples from Laos (Asia) confirming that the ED3 ELISA showed specific reactions in other populations. CONCLUSIONS: The novel ED3 ICB ELISA will be useful for ZIKV-specific IgG detection for seroepidemiological studies and serological diagnosis for case management in travellers and in countries where other flavivirus infections are co-circulating.

Toscana virus encephalitis in Southwest Germany: a retrospective study.

BACKGROUND: Toscana virus (TOSV) is an arthropod-borne virus transmitted by phlebotomine sandflies (Phlebotomus sp.) widespread throughout the Mediterranean having the potential to cause meningoencephalitis in humans. In Germany, the vectors of TOSV are introduced recently and become endemic especially in Southwestern Germany. As TOSV is not investigated regularly in patients with meningoencephalitis, cases of TOSV-neuroinvasive disease may remain mostly undetected. METHODS: We conducted a retrospective cohort study on patients with meningoencephalitis without identification of a causal pathogen from 2006 to 2016. Serologic assessment for anti-TOSV-IgG and IgM was performed on serum and CSF. Demographic, clinical and CSF data from TOSV-positive patients were compared to a cohort of patients with meningoencephalitis due to enterovirus. Informed consent was obtained from all included patients. RESULTS: We found 138 patients with meningoencephalitis without identified causal pathogen. From 98 of these patients CSF and serum was available for further testing. Additionally, we included 27 patients with meningoencephalitis due to enterovirus. We identified two patients with serological confirmed TOSV-neuroinvasive disease (TOSV-IgM and IgG positive, 2%) and two patients with possible TOSV-neuroinvasive disease (isolated TOSV-IgM positive, 2%). Overall, TOSV-neuroinvasive was detected in 4% of our cases with suspected viral meningoencephalitis. None of them had a history of recent travel to an endemic area. CONCLUSIONS: We found cases of TOSV-neuroinvasive disease in our German cohort of patients with meningoencephalitis. As no recent history of travel to an endemic area was reported, it remains probable that these cases resemble autochthonous infections, albeit we cannot draw conclusions regarding the origin of the respective vectors. TOSV could be considered in patients with meningoencephalitis in Germany.

Autochthonous dengue virus infection in Japan imported into Germany, September 2013.

In September 2013, dengue virus (DENV) infection was diagnosed in a German traveller returning from Japan. DENV-specific IgM and IgG and DENV NS1 antigen were detected in the patient's blood, as were DENV serotype 2-specific antibodies. Public health authorities should be aware that autochthonous transmission of this emerging virus may occur in Japan. Our findings also highlight the importance of taking a full travel history, even from travellers not returning from tropical countries, to assess potential infection risks of patients.

Fatal dengue hemorrhagic fever imported into Germany.

Dengue virus (DENV) is an arthropod-borne virus (family Flaviviridae) causing dengue fever or dengue hemorrhagic fever. Here, we report the first fatal DENV infection imported into Germany. A female traveler was hospitalized with fever and abdominal pain after returning from Ecuador. Due to a suspected acute acalculous cholecystitis, cholecystectomy was performed. After cholecystectomy, severe spontaneous bleeding from the abdominal wound occurred and the patient died. Postmortem analysis of transudate and tissue demonstrated a DENV secondary infection of the patient and a gallbladder wall thickening (GBWT) due to an extensive edema.

Detection of Usutu virus infection in a healthy blood donor from south-west Germany, 2012.

From September 2011 until November 2012, 31 serum samples from German patients with clinically suspected acute Usutu virus (USUV) infections were tested for USUV-specific antibodies. All samples tested negative. In addition, 4,200 serum samples from healthy blood donors from south-west Germany were collected in January 2012 and also analysed for the presence of specific antibodies. One sample tested positive for USUV-IgG and -IgM. Thus, the seroprevalence of USUV antibodies in healthy blood donors from south-west Germany was low in January 2012.

Imported tropical virus infections in Germany.

Our routine tests for tropical viruses document that several hundreds of Dengue fever cases are imported into Germany every year. In contrast, hemorrhagic fever cases are rarely diagnosed in Germany. Our investigations suggest that this low number is due to the different living conditions of the local population in the tropics compared with that of travellers from Europe or North America. Improved methods for detecting Dengue virus infections, e.g. three different antibody tests and the reverse transcriptase-polymerase chain reaction (RT-PCR) for detection of viral RNA, have been developed.

Simple detection of antibodies to different viruses using rheumatoid factor and enzyme-labelled antigen (ELA).

A new one-step method was developed to detect IgG antibodies to different virus antigens. Human serum specimens were mixed with different enzyme-labelled antigen (ELA) preparations in microtitre plates coated with rheumatoid factor (RF). During the incubation in the wells of microtitre plates immune complexes form which are specifically bound to the RF on the solid phase. The test detects antibody to cytomegalovirus (CMV) and West-Nile virus with a high sensitivity and specificity.

International diagnostic accuracy study for the serological detection of chikungunya virus infection.

External quality assurance for serological detection of chikungunya virus infection was performed to assess the diagnostic quality of expert laboratories. Of 30 participants, only six correctly analysed all reference samples with their respective tests. Thirteen laboratories gave at least 85% correct results, and 11 laboratories 75% or less. IgM antibodies were detected less frequently than IgG antibodies (p <0.001). The study provides information on the quality of different serological tests and indicates that most of the participants need to improve the sensitivity of their assays, in particular to detect IgM antibodies more reliably and be able to detect acute infections adequately.

Detection of specific immunoglobulin M antibody to different flaviviruses by use of enzyme-labeled antigens.

An enzyme immunoassay was developed for the detection of human immunoglobulin M (IgM) antibody to different flavivirus antigens. The IgM antibody of human sera was selectively bound to anti-IgM antibody-coated solid-phase plates. Flavivirus IgM antibodies were then detected by use of various enzyme-labeled antigens. The flavivirus antigens (dengue type 2 virus, West Nile virus, and tick-borne encephalitis virus) were produced in suckling mice. The antigens were labeled with horseradish peroxidase by adding the activated enzyme at alkaline pH to sucrose-acetone-treated antigens. Addition of unlabeled mouse brain suspension of uninfected animals to the diluted enzyme-labeled antigens effectively reduced nonspecific binding to the solid phase. In patients with acute flavivirus infections, viral IgM antibody could be demonstrated with high sensitivity. Furthermore, the enzyme-labeled antigen-IgM test showed greater specificity than the hemagglutination inhibition test.

Chimeric human and mouse spheroids.

We investigated structures resembling embryoid bodies (EBs), grown intraperitoneally in nude mice after the injection of xenografted human teratocarcinoma cells. Following in situ hybridization of paraffin sections containing these EB-like structures with either human or mouse total genomic DNA, two species-specific types of cell nuclei were localized. Tumor cells of human origin were found centrally but flattened normal mouse cells formed an outer coat. Thus these spheric structures are of bispecies origin and do not meet the definition of EBs. For a clear distinction from EBs and spheroids, we termed these structures chimeric spheroids.

[Tonometer sterilization. The inactivation of herpes simplex virus type 1 (HSV-1) and adenovirus (type 2) by ultraviolet radiation]. / Tonometersterilisation. Inaktivierung von Herpes simplex Virus Typ 1 (HSV-1) und Adenovirus (Typ 2) durch ultraviolette Bestrahlung.

Thirty seconds of ultraviolet radiation was sufficient to completely sterilize the probes of hand applanation tonometers highly contaminated with herpes simplex virus type I and adenovirus type 2. Application of the method and clinical consequences are discussed.

Preliminary approach to elucidate the role of pigment as a binding site for drugs and chemicals in anagen hairs: pigments as carriers for 3H-haloperidol in HaCaT/Sk-Mel-1 co-cultures.

In view of the melanin-binding characteristics of haloperidol and its differential uptake by pigment- and non-pigment-producing cells, a co-culture of HaCaT with Sk-Mel-1 cell lines was performed to investigate whether melanosomes act as carriers for drug molecules associated with the pigments. Initially, HaCaT and Sk-Mel-1 cells were separately cultivated in the presence of 3H-haloperidol (400 pmol/ml medium ) for 28 days followed by subsequent co-cultivation in the absence of 3H-haloperidol for 5 days. The transfer of pigments into the keratinocytes during co-culture was confirmed by transmission electron microscopy. After the co-culture experiments a striking increase (> or = 50%) of 3H-haloperidol was observed in the pigmented HaCaT cells compared to the unpigmented keratinocytes. The present study proved the role of pigments as carriers for melanin-associated drug molecules. The results supported the hypothesis that hair pigment might be a factor affecting the outcome of hair assays for particular categories of commonly used licit and illicit substances. The chosen cell lines and the developed co-culture system may represent suitable in vitro models to study differential drug uptake into cell populations present in the skin or in the growing hair follicle as well as to elucidate drug uptake due to melanocyte-keratinocyte interactions.

Preliminary approach to elucidate the role of pigment as a binding site for drugs and chemicals in anagen hair: differential uptake of 3H-haloperidol by pigment-producing compared to non-pigment-producing cell lines.

A striking difference was observed for cellular-bound drug in HaCaT and Sk-Mel-1 cells for a fixed drug exposure time of 72 h and varying 3H-haloperidol concentrations in the culture media. Drug uptake was dependent on drug concentration and linearly correlated for both the non-pigment- and the pigment-producing cells which however was different in magnitude. In an additional investigation the time course of drug uptake during 3H-haloperidol exposure (400 pmol/ml; 28 days) revealed increasing drug concentrations in the Sk-Mel-1 population, whereas drug concentrations in the keratinocytes reached a plateau within a short time period. In contrast to the HaCaT cells no tendency to saturation was observed for the pigment-producing cell line. At the end of the experiments 3H-haloperidol concentrations in Sk-Mel-1 were found to be approximately tenfold higher than in HaCaT.

[Hantaan virus infections as a cause of acute kidney failure. 3 cases in West Germany]. / Hantavirusinfektionen als Ursache von akutem Nierenversagen. Drei Fallbeobachtungen in der Bundesrepulik Deutschland.

Three cases of Hantaan virus infection (Korean haemorrhagic fever) leading to acute renal failure are described. All three had mild haemorrhagic fever with a renal syndrome. It had started with acute fever followed by oliguria, proteinuria and microhaematuria (in two patients) in the further course of the disease, as well as urea and creatinine retention. One patient needed to be dialysed twice. Hantaan virus-specific IgG antibodies were demonstrated in all three patients; one also had IgM antibodies.

Detection of dengue virus RNA in patients after primary or secondary dengue infection by using the TaqMan automated amplification system.

In consecutive serum samples from 25 tourists with acute dengue fever, virus-specific RNA was detected by using fully automated TaqMan reverse transcriptase PCR. For this amplification technique new primers and special fluorochrome-labeled probes had to be synthesized. During amplification the increasing amount of viral DNA could simultaneously be measured in the tightly sealed tubes. Dengue virus RNA was found in almost all patients (17 of 18), if the samples had been taken soon after the onset of symptoms and before anti-dengue virus antibody had been produced. RNA was detectable in only one of five persons who had anti-dengue virus immunoglobulin M (IgM) antibodies but not yet IgG antibodies. In 30 late samples with both IgG and IgM antibodies viral RNA was no longer demonstrable. In two early samples from two frequent travelers obtained 1 and 2 days after the onset of symptoms significant IgG antibody titers were present but there were no anti-dengue virus IgM antibodies. In these samples a viral load of >5 x 10(6) dengue virus RNA copies (dengue types 1 and 2) was detectable. These findings of a high viral load in the presence of anti-dengue virus IgG antibody are suggestive of a secondary dengue virus infection. In the 20 tourists (17 plus 1 plus 2) in whom viral RNA was found, the dengue virus serotype could be related to the area where the infection had taken place. Most of our patients came from southeast Asia and most frequently had dengue virus type 1 infections (8 of 20).

Assessment of IgG antibodies against yellow fever virus after vaccination with 17D by different assays: neutralization test, haemagglutination inhibition test, immunofluorescence assay and ELISA.

We analysed serum samples of 209 subjects immunized with yellow fever vaccine 17D by different assays: neutralization test, immunofluorescence assay, haemagglutination inhibition test and ELISA, for presence of 17D-specific antibodies. Serum samples were taken from a few weeks up to 35 years after vaccination. The neutralization test had the highest sensitivity. There was no correlation of results between the serological assays. Considering NT titres > 1:10 as indicating protection, we found that about 75% of subjects remained immune even 10 years after vaccination, with a median NT titre of 1:40 in reactive sera.

Supernumerary chromosome 1 in interphase nuclei of atypical germ cells in paraffin-embedded human seminiferous tubules.

The so-called atypical germ cells or cells of carcinoma in situ morphologically resemble neoplastic cells in seminoma. Since seminomas show numerical aberrations of chromosome 1 we have used a DNA probe specific for chromosome region 1q12 to determine whether such aberrations can be detected in atypical germ cell nuclei in paraffin-embedded seminiferous tubules as well. One-third of intratubular nuclei, containing atypical germ cells, consistently showed three hybridization signals in contrast to two signals regularly observed in normal intestine and in spermatogonia. We thus show that cytogenetic studies of precancerous cells can be performed directly on the tissue where these cells originate.