Annual cycles of urinary reproductive steroid concentrations in wild and captive endangered Fijian ground frogs (Platymantis vitiana)

Annual cycles of reproductive steroid metabolites were measured in urine collected from free-living and captive tropical endangered Fijian ground frogs (Platymantis vitiana) a terrestrial breeding. Free-living frogs were sampled on Viwa Island, Fiji and captive frogs were maintained in an outdoor enclosure in Suva, Fiji. Urinary estrone, progesterone and testosterone metabolite concentrations increased in male and female frogs after hCG challenges, with clear peaks in steroid concentrations 2 or 3 days after the challenges. There were annual cycles of testosterone metabolites in wild and captive males, and of estrone and progesterone metabolites in wild and captive females. Peaks of steroid concentrations in the wet season corresponded with periods of mating and egg laying in females in December and January. Steroid concentrations declined in January and February when maximum egg sizes in females were also declining. Body weights of wild male and vitellogenic female frogs showed annual cycles. Body weights of non-vitellogenic female frogs varied significantly between months, although there was no clear pattern of annual changes. Body weights of the 3 captive male frogs and 4 captive female frogs were similar to those of the wild frogs. Estrone metabolites were 80% successful in identifying non-vitellogenic females from males. The results suggest that the Fijian ground frog is a seasonal breeder with an annual gonadal cycle, and this species is likely to be photoperiodic. Urinary steroid measurements can provide useful information on reproductive cycles in endangered amphibians.     

Plasmodium falciparum immunodetection in bone remains of members of the Renaissance Medici family (Florence, Italy, sixteenth century)

Medical accounts and ancient autopsy reports imply that tertian malarial fevers caused the death of four members of the Medici family of Florence: Eleonora of Toledo (1522-1562), Cardinal Giovanni (1543-1562), don Garzia (1547-1562) and Grand Duke Francesco I (1531-1587).All members of the Medici family hunted in the endemic malarial areas of Tuscany, such as the marshy areas surrounding their villas and along the swampy Maremma and were, therefore, highly exposed to the risk of being infected by Falciparum malaria. To determine if the original death certificates issued by the court physicians were correct, we carried out immunological investigations and then compared the biological results to the historical sources.Bone samples were examined for the presence of Plasmodium falciparum histidine-rich- protein-2 (PfHRP2) and P. falciparum lactate dehydrogenase (PfLDH) using two different qualitative double-antibody immunoassays.Our findings provide the first modern laboratory evidence of the presence of P. falciparum ancient proteins in the skeletal remains of four members of the Medici family. We confirm the clinical diagnosis of the court physicians, using modern methods.Finally, this study demonstrates that immunodetection can be successfully applied not only to mummified tissues but also to skeletal remains, thus opening new paths of investigation for large archaeological series.     

Fourth generation assays for HIV testing

Introduction: Laboratory diagnosis of HIV infection is essential for the prevention of infection and the identification of infected individuals who could benefit from highly active antiretroviral therapy. Since the release of the first assays for the detection of anti-HIV antibodies, the technology of immunoassays has improved.

Areas covered: Fourth generation assays – simultaneously detecting HIV p24 antigen and antibodies – have been developed and have been a major improvement in the detection of HIV infection, with a reduction of the diagnostic window. Studies have provided definite evidence for their clinical utility. Combination assays with separate results for anti-HIV antibodies and p24 antigen have been developed.

Expert Commentary: In conclusion, fourth generation assays are an effective tool for the laboratory diagnosis of HIV infection. The ADVIA Centaur HIV Ag/Ab Combo assay is in line with most recent fourth generation assays and its clinical utility has been assessed.     

Low light level in vitro monitoring of cellular and antigen-antibody reactions using a photon detection camera system — New perspectives for clinical diagnosis and research

Summary This article briefly describes the use of a photon counting system (ARGUS-100) in the detection of low levels of light. The ARGUS-100 was used in determining ATP in cell sections from tumor tissues and in measuring a luminescenceenhanced immunoluminometric assay, using ferritin as the analyte, based on the luminol-peroxide-4-iodophenol reaction with peroxidase as the enzyme. The aim is not so much the presentation of data, but rather to show the potentials of the photon counting camera in increasing our knowledge of the cellular and subcellular levels, as well as lowering the detection limits in already sensitive systems, such as immunoassays.Supported by the Bundesministerium für Forschung und Technologie (DFVLR grant No. 01Z08801)     

Comparison of five different immunoassays for the detection of Borrelia burgdorferi IgM and IgG antibodies

The performances of five commercially available enzyme immunoassays were compared for the detection of Borrelia burgdorferi IgM and IgG antibodies. Sensitivity was assessed with European serum samples collected from 45 patients with clinically defined Lyme disease in conjunction with a positive immunoblot (n = 44) or other serological test (n = 1). Sensitivities for the detection of IgM and IgG with each test were: Dako IgM 64%; Dako IgG 53%; Serion IgM 89%; and Serion IgG 88%. The Immunetics assay makes no distinction between IgM and IgG antibodies and had a sensitivity of 91%. Specificity was calculated by testing a control group comprising 40 patients with acute Epstein-Barr virus infection, cytomegalovirus infection, syphilis or rheumatoid factor positivity. The specificities achieved for each test were: Dako IgM 78%; Dako IgG 100%; Serion IgM 52%; Serion IgG 92%; and Immunetics 92%. The discriminatory power between control and patient samples appeared highest for the Immunetics assay. Between-run variation was comparable for the five tests and did not exceed 13%. When the Immunetics assay was used as an initial screening test, with low-titre positive results confirmed by an immunoblot, a sensitivity of 91% and a specificity of 100% were achieved. To attain maximal sensitivity, the Serion IgM and IgG tests were also performed on samples with negative Immunetics results. All positive Serion IgM and IgG results were also confirmed by immunoblot. In conclusion, the Immunetics assay, based on a synthetic C6 peptide, can be used reliably as an initial screening test for the serodiagnosis of Lyme disease.     

Analytical Comparability Assessments of 5 Recombinant CRM197 Proteins From Different Manufacturers and Expression Systems

Cross-reacting material 197 (CRM₁₉₇), a single amino acid mutant of diphtheria toxoid, is a commonly used carrier protein in commercial polysaccharide protein conjugate vaccines. In this study, CRM₁₉₇ proteins from 3 different expression systems and 5 different manufacturers were obtained for an analytical comparability assessment using a wide variety of physicochemical and in vitro antigenic binding assays. A comprehensive analysis of the 5 CRM₁₉₇ molecules demonstrate that recombinant CRM₁₉₇'s expressed in heterologous systems (Escherichia coli and Pseudomonas fluorescens) are overall highly similar (if not better in some cases) to those expressed in the traditional system (Corynebacterium diphtheriae) in terms of primary sequence/post-translational modifications, higher order structural integrity, apparent solubility, physical stability profile (vs. pH and temperature), and in vitro antigenicity. These results are an encouraging step to demonstrate that recombinant CRM₁₉₇ expressed in alternative sources have the potential to replace CRM₁₉₇ expressed in C diphtheriae as a source of immunogenic carrier protein for lower cost polysaccharide conjugate vaccines. The physicochemical assays established in this work to monitor the key structural attributes of CRM₁₉₇ should also prove useful as complementary characterization methods (to routine quality control assays) to support future process and formulation development of lower cost CRM₁₉₇ carrier proteins for use in various conjugate vaccines.     

Comparison of the performance of enzyme immunoassays for hepatitis B and C detection in dried blood spot

Dried blood spots (DBSs) could be an alternative to serum for hepatitis B virus (HBV) and hepatitis C virus (HCV) diagnosis. This study aims to evaluate two enzyme immunoassays (EIAs) for HBsAg and anti-HCV detection using DBS. Serum was tested using commercial EIA. DBS was tested using optimized EIA developed for serum and commercial EIA developed for DBS (Imunoscreen). Concordances between DBS and serum samples for both markers and EIAs were higher than 97%. Both EIAs demonstrated good performance for HBsAg and anti-HCV detection using DBS, and these methods could be used unchangeably increasing the access for HBV and HCV diagnosis.     

Evaluating the Performance of Lateral Flow Devices for Total Aflatoxins with Special Emphasis on Their Robustness under Sub-Saharan Conditions

As aflatoxins are a global risk for humans and animals, testing methods for rapid on-site screening are increasingly needed alongside the standard analytical laboratory tools. In the presented study, lateral flow devices (LFDs) for rapid total aflatoxin screening were thoroughly investigated with respect to their matrix effects, cross-reactivity, their performance under harsh conditions in Sub-Saharan Africa (SSA), and their stability, as well as when compared with liquid chromatography-tandem mass spectrometry (LC-MS/MS). To analyze the matrix effects, qualitative test kits offering a certain cutoff level were used to screen different nut samples. In addition, these tests were challenged on their cross-reactivity with 230 fungal toxins and metabolites. Furthermore, the resulting measurements performed under harsh tropical conditions (up to 38.4 °C and 91% relative humidity) in SSA, specifically Burkina Faso and Mozambique, were compared with the results from a well-established and validated LC-MS/MS-based reference method. The comparison of the on-site LFD results with the reference method showed a good agreement: 86.4% agreement, 11.8% non-agreement, and 1.8% invalid test results. To test the robustness of the cutoff tests, short- and long-term stability testing was carried out in Mozambique and Nigeria. For both experiments, no loss of test performance could be determined. Finally, a subset of African corn samples was shipped to Austria and analyzed under laboratory conditions using semiquantitative aflatoxin tests. A good correlation was found between the rapid strip tests and the LC-MS/MS reference method. Overall, the evaluated LFDs showed satisfying results regarding their cross-reactivity, matrix effects, stability, and robustness.