World Health Organization International Standard To Harmonize Assays for Detection of Mycoplasma DNA.

Nucleic acid amplification technique (NAT)-based assays (referred to here as NAT assays) are increasingly used as an alternative to culture-based approaches for the detection of mycoplasma contamination of cell cultures. Assay features, like the limit of detection or quantification, vary widely between different mycoplasma NAT assays. Biological reference materials may be useful for harmonization of mycoplasma NAT assays. An international feasibility study included lyophilized preparations of four distantly related mycoplasma species (Acholeplasma laidlawii, Mycoplasma fermentans, M. orale, M. pneumoniae) at different concentrations which were analyzed by 21 laboratories using 26 NAT assays with a qualitative, semiquantitative, or quantitative design. An M. fermentans preparation was shown to decrease the interassay variation when used as a common reference material. The preparation was remanufactured and characterized in a comparability study, and its potency (in NAT-detectable units) across different NATs was determined. The World Health Organization (WHO) Expert Committee on Biological Standardization (ECBS) established this preparation to be the "1st World Health Organization international standard for mycoplasma DNA for nucleic acid amplification technique-based assays designed for generic mycoplasma detection" (WHO Tech Rep Ser 987:42, 2014) with a potency of 200,000 IU/ml. This WHO international standard is now available as a reference preparation for characterization of NAT assays, e.g., for determination of analytic sensitivity, for calibration of quantitative assays in a common unitage, and for defining regulatory requirements in the field of mycoplasma testing.

International validation of novel pyrogen tests based on human monocytoid cells.

It is a requirement that parenteral medicines be tested for pyrogens (fever causing agents) using one of two animal-based tests: the rabbit pyrogen test and the bacterial endotoxin test. Understanding the human fever reaction has led to novel non-animal alternative tests based on in vitro activation of human monocytoid cells in response to pyrogens. Using 13 prototypic drugs, clean or contaminated with pyrogens, we have validated blindly six novel pyrogen tests in ten laboratories. Compared with the rabbit test, the new tests have a lower limit of detection and are more accurate as well as cost and time efficient. In contrast to the bacterial endotoxin test, all tests are able to detect Gram-positive pyrogens. The validation process showed that at least four of the tests meet quality criteria for pyrogen detection. These validated in vitro pyrogen tests overcome several shortcomings of animal-based pyrogen tests. Our data suggest that animal testing could be completely replaced by these evidence-based pyrogen tests and highlight their potential to further improve drug safety.

[Evaluation and further development of a pyrogenicity assay based on human whole blood]

When cells of the immune system, i.e. primarily blood monocytes and macrophages, come into contact with pyrogens (fever inducing contaminations) they release mediators transmitting the fever reaction within the organism. A new pyrogen test exploits this reaction for the detection of pyrogens: human whole blood taken from healthy volunteers is incubated in the presence of the test sample. In case of pyrogen contamination, the formation of interleukin-1 is induced, which is determined by ELISA. According to the various pharmacopoeia, the rabbit pyrogen test determines the fever reaction following injection of a test sample. In comparison, the new whole blood assay is more sensitive, less expensive and determines the reaction of the targeted species. In contrast to the well established in vitro alternative, i.e. the limulus amebocyte lysate assay (LAL), the blood assay is not restricted to endotoxins of Gram-negative bacteria and is not to the same extent disturbed by endotoxin-binding blood proteins. Here, interim results of the ongoing optimisation and prevalidation are demonstrated. Preliminary data of the evaluation for biological and pharmaceutical drugs are presented.

[First Results in the prevaluation of the human whole blood assay for pyrogens in biological pharmaceuticals]

When establishing the whole blood assay for pyrogens in our laboratory we did several investigations on the standard pyrogen to be used. Because of the hydrophobicity of the lipopolysaccharid (LPS) it is difficult to get a homogeneous solution. Therefore we used ultrasonic and biphenyl hydroxyl amine chloride for a better dispergation. Surprisingly the reactivity of the LPS in the whole blood assay for pyrogens dropped the more the LPS was dispergated. That's right inverse to the situation in the LAL. There are first experiences in the whole blood assay for pyrogens with biological pharmaceuticals. This includes immunoglobulins, vaccines and coagulation factors. Except of the vaccines and albumin the rabbit assay is the only way to test these pharmaceuticals for pyrogens. The albumins show a little amplification of the reactivity of LPS in the whole blood assay for pyrogens. The testing of albumin in the whole blood assay for pyrogens showed highly reproproducible results and a detection limit under 50 pg/ml LPS. The immunoglobulins show an amplifying effect as well. Immunoglobulins spiked with LPS were detected as pyrogenic in all cases. Some vaccines show a slight pyrogenity in the whole blood assay which gets visible when diluting the sample. Undiluted vaccines may cover a LPS contamination in the whole blood assay for pyrogens.

[Development and evaluation of a pyrogen test based on human whole blood]

When cells of the immune system, especially blood monocytes and macrophages, come into contact with pyrogenic (fever-inducing) contaminations, they secrete messenger molecules which initiate an hyperthermic reaction in the organism. Of this group of endogenous pyrogens, most is known about interleukin-1 (IL-1). A new pyrogen test makes use of this reaction as a system for detection: The substances which are to be screened are incubated with a small volume of blood from a healthy donor. Any pyrogens present induce the production of IL-1 which can be detected by ELISA. This test has a higher sensitivity and is more economical than the conventional pyrogen test in rabbits and furthermore reflects the reaction of the relevant species. In contrast to the customary alternative method, the Limulus amoebocyte lysate test (LAL), this test is not restricted to endotoxins from Gram-negative bacteria and is also not hindered by substances which bind endotoxins, such as blood proteins, to the same extent. Consequently, more than 50 non-endotoxin pyrogens have already been traced by this test. The whole blood test is even superior to the LAL in regard to the detection of endotoxins: in a comparison of about 60 endotoxins, there was a correlation of the potency of the individual endotoxins between the whole blood test and the pyrogen test in rabbits, but neither test correlated with the LAL test. In some cases, endotoxins with equal effects in the LAL test differed in potency in the human blood model by a factor of 10 000. A method has been developed by which cryopreserved blood can be put to use in the test. In this way, blood donations from a donor can be pre-tested so that uniform material may be employed in the test. This test opens up entirely new perspectives on pyrogen testing for Gram-positive or fungal pyrogens as well as in medicinal products. In addition, it could fill the dangerous security gap which might result from the limitations of testing medications and blood products with LAL only. The project was supported by ZEBET, D-Berlin, BMBF, D-Bonn, and set, D-Mainz.

[Characteristics of Toxoplasma-gondii-tachyzoites from different culture systems]

Toxoplasma gondii is an intracellular protozoan parasite of worldwide distribution. Parasites that habour a complete antigenic profile, that is necessary for the serological diagnosis of human Toxoplasma infections, are provided by in vivo culture methods only. It seems that the host immune pressure is responsible for the expression of a total antigen pattern. Thus, in most laboratories the asexual proliferative stage of the parasite, the tachyzoite, is maintained by successive intraperitoneal passages in highly susceptible animals such as mice. We would like to develop an in vitro method to provide sufficient amounts of high quality parasite antigen suitable for diagnosis of Toxoplasmosis. Using the RAPD-PCR (random amplified polymorphic DNA-PCR) technique, we were able to show that during different culture conditions (in vivo and in vitro culture) the parasites undergo no clonal selection. That means the entire parasite population changes the protein expression pattern due to the in vitro culture conditions. Based on the result described previously the work could be continued as follows. One strategy could be to simulate the host immune pressure during the in vitro cultivation of the tachyzoites. A more convinced approach may be the production of recombinant parasite antigens useful for diagnosis of a Toxoplasma infection in human adults and newborns.