The quality control of tuberculin PPD products from seven Latin American laboratories.

The Pan American Health Organization, through its Pan American Zoonoses Center (CEPANZO), distributes the concentrated stock solution of PPD batch RT23 (1 mg ml-1 = 50,000 TU ml-1) to national central laboratories. In the laboratories the stock solution is diluted to 2 TU/0.1 ml and dispensed with the addition of Tween 80 and either chinosol or phenol as preservatives in rubber-stoppered vials for use in the Mantoux test. The expiry date is usually fixed at six months after filling. Samples of nine lots of PPD 2 TU from seven countries (Argentina, Brazil, Colombia, Dominican Republic, Ecuador, Paraguay and Uruguay) were received by CEPANZO for quality control. The biological activity and phenol concentrations of these samples were assayed periodically after storage at 4, 25 or 37 degrees C either unopened or opened and re-used in a manner simulating extreme field conditions of use and storage. The object of the study was to determine the stability of the biological activity as well as the maintenance of sterility of these conditions with a view to assessing the need for stricter requirements for the labelling of tuberculin. The results obtained showed that PPD 2 TU with 0.4-0.5% (w/v) phenol added, and dispensed in vials, was stable in its biological activity and maintained its sterility. Therefore, it seems unnecessary to issue stricter requirements than those currently applied.(ABSTRACT TRUNCATED AT 250 WORDS)

The production of mycobacterial antigens by homogeneous culture in a fermentor.

Mycobacterium bovis strain BCG, substrain 1173P2, has been grown in homogeneous culture in classical synthetic Sauton medium without supplementary ingredients. The culture conditions are described. The protein release in the culture medium and the tuberculin yield after 2% trichloroacetic acid precipitation were significantly improved. The antigenicity of the tuberculin has been successfully assayed on specifically sensitized guinea-pigs. It is concluded that homogeneous mycobacterium culture in a fermentor using synthetic medium is a suitable method for the large scale production of antigen.

An evaluation of the polyacrylamide gel electrophoresis fractionation method for the production of Mycobacterium tuberculosis skin test preparations. I. Production, physiochemical characterization and serological analyses.

As a part of a cooperative inter-laboratory WHO supported project raw tuberculins were produced and purified protein derivative (PPD, 18.7 g protein) was prepared. Employing a multistage preparative polyacrylamide gel electrophoresis (PAGE) method the PPD was separated into four fractions corresponding to 15, 7, 4.75 and 3.5% gel concentrations. The PAGE procedure resulted in three lots of material--each representing 11 electrophoretic runs. Immunodiffusion analyses showed that the largest number of precipitinogens was found in the 15% fractions and that some precipitinogens cross-reacted with preparations of Mycobacterium bovis BCG, M. intracellulare, M. kansasii, M. smegmatis and M. vaccae.

An evaluation of the polyacrylamide gel electrophoresis fractionation method for the production of Mycobacterium tuberculosis skin test preparations. II. Comparative skin testing on guinea-pigs.

As part of a cooperative inter-laboratory WHO supported project for the fractionation of Mycobacterium tuberculosis skin test preparations, four fractions (designated 15, 7, 4.75 and 3.5%) were evaluated by comparative skin tests on sensitized guinea-pigs. The 7% fraction was the most potent in both homologously and heterologously sensitized animals, and the 4.75% and 3.5% gel fractions showed the lowest activity. Significant levels of cross-reactivity in guinea-pigs immunized with M. bovis BCG, M. kansasii, M. avium and M. intracellulare were demonstrated for all fractions examined, thus reflecting the antigenic relationships among these mycobacteria. These four fractions may qualify as starting material for further studies aiming at a reduction of skin test cross-reactivity.

Comparison in sensitized and unsensitized guinea-pigs of tuberculin PPDs RT 23 and PPD-M by skin tests and lymphocyte stimulation tests. Effect of immunization time.

The biological activities of tuberculin PPD RT 23 and the International Standard for Purified Protein Derivative of Mammalian Tuberculin (PPD-M) were compared in sensitized and unsensitized guinea-pigs by skin tests and lymphocyte stimulation (LS) tests. Estimates of relative potency (RP) from skin test results were dependent on the dose level, on the immunogen used, and, in guinea-pigs immunized with killed tubercle bacilli in oil, also on the immunization time. Relative potency estimates from LS results were dependent on the source of the lymphocytes and were different from estimates obtained from skin tests. Lymphocyte stimulation dose-response curves for the tuberculins were qualitatively different. In contrast to RT 23, PPD-M gave rise to non-specific skin reaction in unsensitized guinea-pigs. Both tuberculins were mitogenic to lymph node lymphocytes isolated from unsensitized guinea-pigs, PPD-M being the more mitogenic of the two tuberculins. The present results confirm that qualitatively different tuberculins cannot be unambiguously calibrated in identical terms and thus emphasize that the uncritical use of (international) standards should be avoided in tuberculin calibration.

Disposable unit-dose PPD.

The new type of disposable unit-dose PPD has some advantages in that the injection can be performed anytime, anywhere when needed, if the sterile tuberculin syringe or the disposable tuberculin syringe is available, immediately after reconstitution of PPD without any decrease of potency. There is no risk of contamination such as hepatitis infection. The potency of the new type PPD was compared with the multi-dose PPD and the stability of potency in the storage of this PPD was observed.

The need to redefine the unit of the international standard for tuberculin purified protein derivative (PPD-S).

It is well known that the results of the relative potency tests between tuberculin preparations and the standard can be affected by many variables. The authors emphasize that the single most important variable to control, to ensure that the potency tests are reproducible, is to prevent the depletion by adsorption of the tuberculin from the preparation. Evidence is presented to show the importance that adsorption, to glass and plastic surfaces, could have on the loss of tuberculin PPD during the preparation of dilute tuberculin solutions. It is suggested that a) an anti-adsorption agent be added to all diluents used to prepare tuberculin solutions, and b) the assignment of a new value to the International Unit for tuberculin PPD be considered.

Potency testing of bovine tuberculins.

The diagnosis of bovine tuberculosis is based on the intradermal tuberculin test. Therefore in eradication campaigns HCSM or PPD tuberculins with high potency and maximal specificity per cattle dose are needed. In quality control and standardization assays the PPD tuberculins have the advantage that the protein content can serve as a basis for calculating the degree of dilution to be used for batches of the concentrated tuberculin, but this chemical estimation does not measure the biological activity of the tuberculo-proteins. Potency control of tuberculins necessitates a bio-assay method, which according to WHO report 384 should be performed under the conditions and in the animal species in which the tuberculin will be used in practice. This potency testing is performed in M. bovis sensitized cattle and guinea pigs, but the bio-assays in both species encounter several problems which must be overcome in order to obtain reliable and consistent results. At the C.V.I. numerous bio-assays have been carried out with various tuberculins and it is the aim of this report to demonstrate that so far the best correlation was obtained between tuberculous cattle and guinea pigs infected with a minimal dose of live M. bovis bacilli, whilst the results with heat killed M. bovis or BCG sensitized guinea pigs were evidently less reliable.

Multiple puncture tuberculin tests: relationship of false negative responses to variations in dried tuberculin coating.

Multiple puncture dried tuberculin tests compare favourably with the standard Mantoux test provided the tips of the metal prongs are thinly and uniformly coated with tuberculin. Unfortunately, surface tension, varying viscosity of the acacia solutions, and deeper dipping depths lead to increased quantities of tuberculin materials adhering to the metal prongs which results in thickening and retraction of the materials on the metal. Solutions of tuberculin in buffered phosphate produce thin and uniform coatings provided adequate cleaning of the metal--especially degreasing--is carried out. In the absence of this the coating layer may fragment. However, provided the distal 0.6-0.8 mm is covered, sufficient tuberculin will be deposited intradermally.

Comparative testing of the potency of different PPD preparations.

The potency of different PPD preparations, obtained from M. tuberculosis and M. bovis was investigated in the guinea-pig test. The groups of animals were immunized with dried mycobacterium powder (obtained from M. tuberculosis, M. bovis and M. tuberculosis + M. bovis), suspended in sterile liquid paraffin oil and with BCG vaccine. The potency tests were carried out according to the WHO requirements. Independently of the immunization method, a significantly greater potency of homologous versus heterologous PPD could not be demonstrated. The mixed PPD preparation (obtained from M. tuberculosis + M. bovis) was fractionated on SEPHAROSE 6 B and SEPHACRYL S 200 column. The potency of different fractions compared to the non-fractionated material was determined in the guinea-pig tests. The animals were immunized by dried mycobacterium powder (obtained from M. tuberculosis + M. bovis), suspended in sterile liquid paraffin oil. The IU content of fractions was calculated according to their protein N content, compared to the non-fractionated material. In these experimental conditions PPD fractions with significantly greater potency than the non-fractionated material could not be found. As a final conclusion we suggest that the desired improvement of tuberculosis diagnostics can be achieved not only by using PPDs derived from different mycobacteria but by further immunochemical purification of the tuberculin preparations and extensive testing of the purified materials in human beings as well.

Evaluation of difficulties in the standardization of human and bovine tuberculins.

The tuberculins are made of mixtures of antigens. Certain elements are common to all tuberculins, others are particular to each one. Bovine and human tuberculins are widely different. This is the reason why the activities of a bovine tuberculin using a human tuberculin standard will be in a ratio of 1 to 5 according to whether the guinea-pigs were sensitized with human or bovine mycobacteria. Also, the activities measured for a given tuberculin might be different if the mycobacteria used in sensitization were inactivated or not, depending on the amount of Antigen L in the tuberculin or in the standard. Because BCG is well defined and used worldwide we propose that a new standard be prepared from BCG cultures, and that the animals be sensitized by live BCG. Two new units are proposed: Bovine Clinical Unit (B.C.U.) and Human Clinical Unit (H.C.U.).

Laboratory assay methods for bovine tuberculin PPD (report of the Expert Group on Veterinary Sera and Vaccines of the European Pharmacopoeia Commission).

It had been found that preparations of bovine tuberculin PPD that appeared to be similar in potency when assayed in guinea-pigs sensitized with killed tubercle bacilli could differ markedly in potency when assayed in cattle. In an attempt to define a laboratory assay method that would reflect accurately the performance of bovine tuberculin PPD in cattle, the European Pharmacopoeia Group of Experts on Veterinary Sera and Vaccines carried out a collaborative study of various methods of sensitization. Seven laboratories in 6 countries participated. The only method of sensitization that gave results corresponding to those obtained in cattle was that using living, virulent, bovine, tubercle bacilli. The Group concluded that it was essential to use this method in laboratory assays of bovine tuberculin PPD.

Field trials to determine a suitable injection dose of bovine PPD tuberculin for the diagnosis of bovine tuberculosis in naturally infected cattle.

Biological assays carried out in the Republic of Ireland in naturally sensitized (M. bovis infected) tuberculous cattle have shown that Rotterdam and Weybridge bovine PPD tuberculins issued for routine use in 1981 and 1982 were equipotent with an estimated potency of about 24,000 Community Tuberculin Units (C.T.U.) per mg of PPD. However, previously it was shown that Weybridge bovine PPD produced in 1978 had only a potency of about 4,000 C.T.U./mg in M. bovis infected cattle. The clinical significance of the latter finding is seen in the results of field trials carried out in the Republic of Ireland in 1978 to determine the relative diagnostic abilities of Weybridge and Rotterdam, routine issue bovine PPD tuberculins. These trials involved the tuberculin testing and slaughter of naturally sensitized cattle. Post-mortem and laboratory examination confirmed that 68 of these animals were infected with M. bovis. It was concluded that the injection dose for the diagnosis of bovine tuberculosis in cattle should not contain less than 2,000 C.T.U. and that the use of an injection of double this dosage was desirable in known M. bovis infected herds.

The comparative tuberculin test in guinea pigs using PPD extracts prepared from mycobacteria killed with phenol.

Purified-protein-derivative (PPD) extracts were prepared from Mycobacterium tuberculosis (PPD-S), M. bovis (PPD-BS), M. avium (PPD-A) and M. kansasii (PPD-K), after killing the cultures with phenol. The reactions were assessed in guinea pigs sensitised to a range of mycobacteria, with a view to selecting a suitable pairing of the extracts to distinguish sensitivity due to M. tuberculosis or M. bovis from that due to other mycobacteria. Sensitisation induced by M. bovis was best distinguished from others using PPD-S and PPD-A; for sensitisation by atypical mycobacteria, PPD-BS was better than PPD-S, used with either PPD-K or PPD-A. PPD-BS was more specific than PPD-S. Using PPD-BS, PPD-A and PPD-K in a comparative test, 3 groups were recognised: those sensitive to M. bovis or M. tuberculosis (greatest reaction was to PPD-BS); those sensitive to M. avium, M. intracellulare and M. scrofulaceum (greatest reaction was to PPD-A); those sensitive to M. kansasii, M. marium, M. gordonae and M. fortuitum (greatest reaction was to PPD-K).