Efficiency of CMV serodiagnosis during pregnancy in daily laboratory routine.

BACKGROUND: Maternal acute primary cytomegalovirus (CMV) infection during the first trimester may cause severe long-term sequelae in newborns. For risk assessment, serological screening is routinely performed in pregnant women based on IgM, IgG and avidity tests using whole-virus antigen. A recent study evaluated the diagnostic value of recombinant protein-based ELISAs as second-line tests in pregnancy CMV screening, including anti-p52 IgM and anti-gB IgG as markers defining the early and late phase of infection, respectively. In the present study, these recombinant ELISAs were used as first-line screening tests in daily laboratory routine and compared to lysate-based assays with respect to [i] the number of conclusive results obtained with the initial sample and [ii] the underlying workload. METHODS: 553 unselected routine serum samples from pregnant women were tested for anti-CMV IgM and IgG antibodies using lysate-based ELISAs and avidity testing. Anti-CMV IgM antibodies against recombinant p52 and anti-CMV IgG antibodies against recombinant glycoprotein B (gB) were also determined by ELISA. All assays were performed and interpreted according to the manufacturer's instructions. RESULTS: For lysate-based IgM, IgG and avidity testing, 84.6 % of samples yielded conclusive results in a total of 1156 tests, while 15.4 % needed follow-up testing of a consecutive sample. Anti-p52 CMV IgM and anti-gB CMV IgG testing produced conclusive results for 92.8 % of samples in a total of 1026 tests, while 7.2 % samples required follow-up testing. CONCLUSIONS: The first-line use of ELISAs measuring anti-p52 CMV IgM and anti-gB CMV IgG antibodies to test for maternal CMV infection increases the number of conclusive results derived from an initial serum sample while requiring a considerably lower number of tests compared to the lysate-based approach. For day-to-day routines in a diagnostic laboratory, this high efficiency of the recombinant testing approach has significant practical relevance.

A new ELISA and western blot technique based on recombinant TES antigen and/or larval antigen for the detection of toxocariasis in humans.

Serological antibody detection by enzyme-linked immunosorbent assay (ELISA)- and immunoblot-based methods constitutes the best indicator of human Toxocara infection. Nevertheless, the availability of serological tests, particularly western blots (WB), evaluated for sensitivity and specificity is limited. Therefore, an Anti-Toxocara-ELISA immunoglobulin g (IgG) prototype (Proto-ELISA) and an Anti-Toxocara-Westernblot (IgG) prototype (Proto-WB) were evaluated by testing 541 human sera pre-determined for Toxocara infection by an established in-house Anti-Toxocara-ELISA (IH-ELISA). To evaluate sensitivity and specificity of the newly developed ELISA and WB prototypes, results were compared to IH-ELISA and a commercial WB (Com-WB). Compared to the IH-ELISA, a sensitivity of 93.1% (229/246) and a specificity of 94.6% (279/295) of the Proto-ELISA with a Cohen's κ of 0.88 were obtained. The sensitivity of the Proto-WB was 76.7% (240/313) and specificity was 99.6% (227/228) with a Cohen's κ of 0.73 compared to those of Com-WB. A comparison to the IH-ELISA revealed 91.5% (225/246) sensitivity and 94.6% (279/295) specificity of the Proto-WB with a Cohen's κ of 0.86. Cross-reactivity was observed for some samples positive for Ascaris and Trichinella spp. in the Proto-ELISA, Proto-WB and Com-WB. Overall, the evaluated ELISA and WB prototypes showed high sensitivity and specificity, indicating high reliability of these newly developed tests.

Added value of IgA antibodies against Zika virus non-structural protein 1 in the diagnosis of acute Zika virus infections.

Zika virus (ZIKV) is a mosquito-borne flavivirus posing a public health threat due to its association with neurological complications in newborns and adults. In flavivirus-endemic areas, coming mosquito seasons will require the differentiation of primary versus secondary and acute versus past ZIKV/flavivirus infections. This is complicated by two major difficulties: [i] secondary infections often present with low or undetectable titres of specific IgM and with early-positive IgG, [ii] previous flavivirus infection(s) or vaccinations cause elevated cross-reactivities. Here, we analysed the anti-ZIKV IgA, IgG, and IgM responses at different stages of infection in an endemic setting, scrutinising the diagnostic relevance of specific IgA. Anti-ZIKV antibodies were measured by ELISA based on ZIKV non-structural protein 1 (NS1) in paired sera from 31 patients with suspected primary or (flavivirus-primed) secondary ZIKV infection. The control panel comprised samples from 136 DENV-infected patients. Among ZIKV samples collected 8-16 days after symptom onset, ELISA sensitivities for detecting anti-ZIKV NS1 IgA, IgG, and IgM were 93.5%, 100%, and 48.4%, respectively. The proportion of cases with negative IgM but positive IgA was higher in suspected secondary (61.9%) than in primary (30.0%) ZIKV infections. Combined IgA/IgM detection yielded a sensitivity of 100% at a specificity of 97.1%. In conclusion, at time points after PCR can detect the virus, the determination of anti-ZIKV NS1 IgA may improve the accuracy in diagnosing acute ZIKV infection in flavivirus-endemic regions in the context of both primary and secondary infection, especially when IgM is undetectable.

Serodiagnosis of Zika virus (ZIKV) infections by a novel NS1-based ELISA devoid of cross-reactivity with dengue virus antibodies: a multicohort study of assay performance, 2015 to 2016.

Serological diagnosis of Zika virus (ZIKV) infections is challenging due to high cross-reactivity between flaviviruses. We evaluated the diagnostic performance of a novel anti-ZIKV ELISA based on recombinant ZIKV non-structural protein 1 (NS1). Assay sensitivity was examined using sera from 27 patients with reverse transcription (RT)-PCR-confirmed and 85 with suspected ZIKV infection. Specificity was analysed using sera from 1,015 healthy individuals. Samples from 252 patients with dengue virus (n = 93), West Nile virus (n = 34), Japanese encephalitis virus (n = 25), chikungunya virus (n = 19) or Plasmodium spp. (n = 69) infections and from 12 yellow fever-vaccinated individuals were also examined. In confirmed ZIKV specimens collected ≥ 6 days after symptom onset, ELISA sensitivity was 58.8% (95% confidence interval (CI): 36.0-78.4) for IgM, 88.2% (95% CI: 64.4-98.0) for IgG, and 100% (95% CI: 78.4-100) for IgM/IgG, at 99.8% (95% CI: 99.2-100) specificity. Cross-reactivity with high-level dengue virus antibodies was not detected. Among patients with potentially cross-reactive antibodies anti-ZIKV positive rates were 0.8% (95% CI: 0-3.0) and 0.4% (95% CI: 0-2.4) for IgM and IgG, respectively. Providing high specificity and low cross-reactivity, the NS1-based ELISA has the potential to aid in counselling patients, pregnant women and travellers after returning from ZIKV-endemic areas.

Inclusion of targeted therapies in the standard of care for metastatic colorectal cancer patients in a German cancer center: the more the better?!

PURPOSE: Significant prolongation of overall survival (OS) has been reached in metastatic colorectal cancer (mCRC) treatment within the last 5-10 years. Our study was conducted in order to evaluate and compare OS of different standard of care treatment options in a university-based outpatient clinic. METHODS: One hundred and three mCRC patients were identified by retrospective analysis and treated according to available guidelines. OS was analyzed according to the different combinations of first- and second-line treatments. RESULTS: mCRC patients revealed an mOS of 34.4 months. Patients receiving anti-vascular endothelial growth factor (VEGF) blockade in at least one treatment line showed a significantly longer survival time (p = 0.0056) versus patients without any bevacizumab. No OS differences were detected comparing the different first- and second-line chemotherapy (CTX) strategies in the unselected population. However, wild-type (wt) Kras patients treated with anti-epidermal growth factor receptor (EGFR) therapy plus CTX in first-line therapy showed significantly longer OS compared to those receiving only additional VEGF inhibition or no targeted therapy (p = 0.0056; mOS 46.8 vs. 20.4 months, respectively). wt Kras patients profited in trend (p = 0.076) from CTX combinations of first-line anti-EGFR followed by second-line anti-VEGF compared to first-line anti-VEGF followed by second-line anti-EGFR (mOS 46.8 vs. 19.2 months, respectively). CONCLUSIONS: Our results indicate successful allocation of the current mCRC treatment according to the Kras status. Differences in OS of wt Kras patients indicated the further need for randomized trials to define the potential benefit of sequential therapy with EGFR inhibition in first-line therapy followed by VEGFR inhibition vice versa.

A robust methodology to study urine microRNA as tumor marker: microRNA-126 and microRNA-182 are related to urinary bladder cancer.

OBJECTIVES: MicroRNAs have been shown to be related to specific types of malignant cell growth. In case of urothelial bladder cancer (BCa), novel noninvasive diagnosis is particularly required and it is attractive to consider, as urine is an easily available source for molecular markers including RNA. In this context, we aimed to develop a clinically applicable and sensitive protocol for the preparation and molecular analysis of low molecular weight RNA from urine samples obtained from bladder cancer patients or healthy volunteers. MATERIALS AND METHODS: First, a method was developed for the preparation of low molecular weight RNA from a set of urine samples from different donor groups: (1) patients with low-grade BCa, (2) patients with high-grade BCa, (3) patients with urinary tract infections, (4) healthy donors; each n = 9. The RNA extracts were then used to monitor a number of 157 microRNA species by quantitative reverse transcriptase-polymerase chain reaction. Subsequently, those microRNAs that showed a higher abundance in urine samples from BCa patients were detected in an independent set of urine samples (n = 47). RESULTS: The significance and diagnostic usefulness of this methodology is reflected by the finding that the RNA ratio of microRNA-126:microRNA-152 enabled the detection of BCa from urine at a specificity of 82% and a sensitivity of 72%, with an area under the curve of 0.768 (95% confidence interval, 0.605-0.931). CONCLUSIONS: This study describes a novel, robust, and useful technology platform that is suitable to analyze small RNAs, including novel RNA-based tumor markers, in urine samples. A detailed technical analysis of this methodology provides new insights into the characteristics of urine microRNA such as composition and the donor-dependent variability.

Detailed technical analysis of urine RNA-based tumor diagnostics reveals ETS2/urokinase plasminogen activator to be a novel marker for bladder cancer.

BACKGROUND: The noninvasive detection of RNA tumor markers in body fluids represents an attractive diagnostic option, but diagnostic performance of tissue-derived markers is often poorer when measured in body fluids rather than in tumors. We aimed to develop a procedure for measurement of tumor RNA in urine that would minimize donor-dependent influences on the results. METHODS: RNA isolated from urinary cell pellet, cell-depleted fraction, and whole urine was quantified by reverse transcription quantitative-PCR. The donor-dependent influence of urine background on individual steps of the standardized procedure was analyzed using an external RNA standard. Using a test set of samples from 61 patients with bladder cancer and 37 healthy donors, we compared 4 putative RNA tumor markers identified in whole urine with 5 established, tissue-derived RNA tumor markers for the detection of bladder cancer. RESULTS: Of the markers analyzed by this system, the RNA ratio of v-ets erythroblastosis virus E26 oncogene homolog 2 (avian; ETS2) to urokinase plasminogen activator (uPA) enabled the most specific (100%) and sensitive (75.4%) detection of bladder cancer from whole urine, with an area under the curve of 0.929 (95% CI 0.882-0.976). CONCLUSIONS: The described methodology for RNA marker detection in urine appears to be clinically applicable. The ratio of ETS2 mRNA to uPA mRNA in urine is a potential marker for bladder cancer.

Investigation of the origin of extracellular RNA in human cell culture.

We have used the human ECV 304 cell line to study the origin and fate of extracellular RNA (exRNA) in cell culture. Quantification of different extracellular RNA species using reverse transcription followed by quantitative PCR revealed a prevalent fraction of ribosomal RNAs. Comparison of intracellular and extracellular ribosomal RNA copy numbers allowed the calculation of the number of destroyed cells that would result in the corresponding number of extracellular rRNAs. Interestingly, this number was comparable to the amount of destroyed cells as determined by the measurement of extracellular lactate dehydrogenase activity.

Improved conditions for isolation and quantification of RNA in urine specimens.

There is clear evidence that the occurrence of specific mRNAs in plasma and serum is associated with cancer, while the usefulness of other body fluids for nucleic acid-based cancer detection remains to be elucidated. Nevertheless, due to the principal advantages of urine (large sample quantities, easy to acquire), several attempts were made to use quantitative reverse transcriptase polymerase chain reaction (RT-PCR)-based detection of putative RNA tumor markers from urine as a tool for noninvasive tumor detection. Because most of the commercially available RNA isolation systems do not accommodate larger sample volumes, the majority of experiments were performed using urine pellets. During the centrifugation step, putative extracellular nucleic acids of low molecular weight as well as complexes containing nucleic acids with low density are lost. Furthermore, cells may be destroyed during this procedure, and the subsequently released nucleic acids will quickly be degraded by nucleases in the urine, which may give rise to inconsistent results. Therefore, we established an improved protocol for the isolation of RNA from urine and subsequent quantification steps. The isolation procedure was tested using a quantitative RT-PCR specific for Ki-67 RNA as well as a radioactive-based reverse transcription approach.