Studies towards the standardization of lepromin*

Bull. Org. mond. Sante 1970, 42, 703-709Bull. Wid Hlth Org.J Studies Towards the Standardization of Lepromin* Progress and Prospects JOHN H. HANKS,1 M. ABE,' T. NAKAYAMA,' M. TUMA,3 L. M. BECHELLI 4 & V. MARTINEZ DOMfNGUEZ4 Because of the wide range of concentrations of Mycobacterium leprae in existing lepromins the authors studied methods ofproducing a standardizable lepromin containing 160 million bacilli/ml. The effects ofusing different dilutions oflepromin on the incidence of false-positive reactions were also studied. Progress reported includes a conyenient method for preparing large batches of non- sedimenting lepromin, which is directly suitable for microscopic counting of Myco. leprae cells; and a validation of current methods for microscopic enumeration of Myco. leprae. Skin tests with diluted lepromins have demonstrated that dilutions up to 1:16 increase progressively the ability to distinguish between lepromatous and tuberculoid leprosy. This work has provided further evidence that 20 million bacilli/ml (a 1:8 dilution of the initial lepromin) should produce adequate Mitsuda reactions in general populations, provided that 3-mm reactions are taken as the criterion for 1+ positivity. The net effect of these findings is equivalent to expanding the world supply of lepromin by 8 times. Recommendations for further research are proposed. The Mitsuda reaction to lepromin is of prime importance for determining prognosis in leprosy patients (particularly those of indeterminate and borderline forms) and for evaluating the relative resistance of contacts and populations. In order to obtain reliable data that can be compared in different countries, the standardization of lepromin is necessary. Recent research has been directed towards the production of an improved lepromin and the investigation of Mitsuda reactions to diluted lepro- mins. It is hoped to retain all significant reactions to the bacilli while decreasing false-positive 1 + reactions, which have been attributed to tissue com- ponents. Major defects inMHW (Mitsuda-Hayashi-Wade)- type lepromins have been the uncontrolled amounts * Report of investigators from research centres co- operating with WHO on the standardization of lepromin and WHO Leprosy unit staff, prepared in conjunction with consultations held in London, 14 September 1968. l Johns Hopkins-Leonard Wood Memorial Leprosy Research Laboratory, Baltimore, Md., USA. 2National Institute for Leprosy Research, Tokyo, Japan. 'Instituto de Leprologia, Rio de Janeiro, Brazil. 'Leprosy, World Health Organization, Geneva, Switzer- and. of tissue debris and leprosy bacilli, usually grouped in clumps and globi, so that a variable number of those tested was rejected. An improved lepromin should possess 3 useful properties: (1) bacilli that have been subjected to minimal, controlled mech- anical trauma, (2) a uniform range of bacterial clump sizes, and (3) freedom from visible, rapidly-settling tissue particles. These qualities would increase the reliability of bacterial counts as a primary means of standardization and ensure the injection of uniform bacterial dosages with minimal tissue component. A study of late (Mitsuda) reactions to diluted lepromins has revealed 2 interesting points: (1) that distinctions between Mitsuda positive and negative individuals increase with dilution and (2) that in patients and other people either the 1:4 or the 1:8 dilution and a 3-mm criterion for the 1+ category was equivalent to " standard " lepromin and a 5-mm criterion. Problems requiring further study include stabili- zation of the number of bacterial cells during the storage of lepromin, selection of the optimal dilution of lepromin and whether it is possible to reduce the number of false-positive reactions without increasing the rate of false-negative reactions. 2569 - 703- J. H. HANKS AND OTHERS PRODUCTION AND USE OF A STANDARD LEPROMIN The use ofa standard By 1961, Abe et al. (1961) had demonstrated that the average number of countable Mycobacterium leprae in Japanese and Philippine lepromins was 160x 106/ml and had shown the feasibility of repro- ducing this level. During the Eighth International Congress ofLeprology, Rio de Janeiro, 1963, a level of 160 million bacilli/ml was recommended for use as a standard. Lepromins containing this concentration of bacilli will be regarded as " standard " in the present report. Microscopic counts In 1963, Nakayama & Chatterjee (unpublished) found counts by the micro-pipette method of Nakayama and the pin-head method of Hanks, Chatterjee & Lechat (1964) to yield excellent agree- ment. Nakayama et al. (1965) later reported that the inherent errors in both methods are below 8%: they considered the pin-head method to be the more rapid and convenient. A table of conversion factors for the pin-head method has since been published (Hanks, 1968c) in order to eliminate calculations and minimize errors. Defects in Mitsuda-Hayashi-Wade-type lepromins The lepromas, the rough materials from which the lepromins are made, are very different from each other as far as the quantities of the bacilli, lipids, fibrous tissue, etc., are concerned. The bacilli are almost always grouped in clumps or globi and they can have granular or solid appearance. Different practices in the manual grinding of lepromas disrupt variable proportions of the bacilli. This is not com- patible with standardization on the basis of micro- scopic counts. Tuma observed Mitsuda reactions with Mickle-disintegrated lepromins. Abe et al. (1963a) demonstrated that Mitsuda reactions can be caused by fragments of sonically disrupted bacilli. Further problems are created by the coarse tissue particles that settle in syringes, and result in the injection of unequal doses of tissue and bacterial clumps. Although suggestions have been made on methods for minimizing this problem, it is evident that a new approach to the production of lepromin is necessary. "Blender type " lepromin Hanks (1968b) found that electric blenders liber- ate practically all the bacilli from 4 g of lepromas within 60 seconds. Since mechanical action on the bacilli is minimal and accurately controlled, there should be a fairly constant ratio between disrupted and intact, countable bacilli. The bacterial counts should predict clinical potency more reliably. Suspensions lacking sedimenting tissue compo- nents and containing a standard range of bacterial clump sizes were obtained by recovering successive supernates (free of visible particles) during gravity sedimentation. These supernates contained 75% of the total bacilli that could be recovered. Recovery of Myco. leprae from tissue residues Tissue residues remaining after the sedimentations mentioned above are nearly devoid of single bacilli and medium-sized clumps, but are rich in large clumps and globi. Hanks & Jacob (unpublished) recovered these bacilli from pooled tissue residues by using the minimal concentration of chloroform (7 %) that can be expected to declump them (Hanks, 1959), by immediately applying a vacuum to remove all the excess chloroform, and by rinsing the tissues nearly free of bacilli by flotation in phenol saline during gravity sedimentation. The procedures and recoveries are outlined in Annex 1 at the end of this paper. The data show that only 25% of the recover- able bacilli need be exposed to low concentrations ofchloroform. Tuma's method (1963) of recovering bacilli from tissue residues differs from the above in that a higher proportion of the bacilli are exposed to concentrated chloroform for longer periods of time. All chloroform treatments decrease the acid- fastness of the bacilli (Hanks, 1959). Studies with Dharmendra-type antigens have shown that the chloroform treatment accentuates the Fernandez phase of skin reactions and weakens the Mitsuda reaction. Since chloroform is the best reagent for declumping and suspending mycobacteria (Hanks, 1959), further consideration must be given to the conditions under which bacilli recovered from tissue residues can be combined with the non-treated bacilli in the original supernates: some indications of how to achieve this are described by Tuma (1963). Trial of blender-type lepromin Abe has compared the Fernandez and Mitsuda reactions produced by " standard" MHW-type lepromins and blender lepromins in tuberculoid and lepromatous patients. After 28 days, the blender type produced slightly stronger reactions in both 704 STUDIES TOWARDS THE STANDARDIZATION OF LEPROMIN tuberculoid and lepromatous patients. Further trials are desirable. Storage of lepromin Abe et al. (1961) observed that lyophilized lepro- mins retain their original potency and bacterial numbers for more than 3 years. In a further un- published communication to the World Health Orga- nization in 1968, Abe reported stability of these properties for 5 years. Refrigeration at 40C-10'C is less reliable, and after storage at these temperatures Abe found counts to decline within 3 years and skin-test potency to decline in 5 years. Hanks reported that microscopic counts decline progressively during refrigeration and rapidly during the shipment of lepromin in vials. It would be ideal, therefore, to dehydrate all lepromins as soon as the bacilli have been counted. Utilization ofMHW-type lepromins The inequality of serial doses while using existing stocks of lepromin can be minimized in one of two ways. Hanks (1968a) has suggested some precau- tions that are applicable during clinical use. Hanks & Jacob (Annex 2) have shown that it is feasible to pool such lepromins and recover homo- geneous suspensions by means of gravity sedimenta- tion. The laboratory method has the advantage that it permits standardization of bacterial counts. CONCENTRATION OF MYCO. LEPRAE CELLS REQUIRED FOR THE MITSUDA REACTION IN DIFFERENT POPULATIONS The usefulness of tuberculin and other skin-test reagents is enhanced by employing different con- centrations, adjusted to the probable immunological experience of the individual and the purpose of the trial. Proper dilution of lepromin should result in definite advantages: (1) a decrease in false-positive reactions; (2) fewer severe local reactions; (3) improved distinctions between lepromin reactors and non-reactors; and (4) great economy in the use of the available supplies of lepromin. False-positive reactions Histological studies in leprosy patients (Nolasco, 1940) and contacts (Bechelli et al., 1957) have shown that some 20% of 1+ (3 mm-5 mm) Mitsuda reactions to full-strength lepromin may be devoid of tuberculoid response and that clinical false- positive reactions are less frequent in 2+ reactors. The false-positive reactions are characterized by infiltrates or granulomas with bacilli and without tuberculoid structure. Abe et al. (1963b) have attributed these reactions to the tissue components, finding the liability to be greater in lepromatous than in tuberculoid patients. Yanagisawa et al. (1957) demonstrated that a 1:8 dilution of standard MHW lepromin reduced sharply the frequency of 1 + reactions in lepro- matous patients. Employing washed suspensions of Myco. leprae in comparison with standard MHW lepromin, Abe et al. (1965) more recently concluded that even the 1:16 dilution of partially purified lepromin may cause weak reactions in a small pro- portion of lepromatous patients. Since routine preparation of purified Myco. leprae is not yet feasible, dilution of lepromin remains the best means of decreasing false-positive reactions. The concentrations of Myco. leprae required Adequate responses to 1:8 dilutions of lepromin are not confined to leprosy patients. A study by Guinto & Wade (1955) in children aged 6-9 years permits comparisons between the frequency of 5-mm reactions to full strength lepromin and of 3-mm reactions to the 1:10 dilution. Although the 1: 10 dilution caused slight loss of sensitivity, calcula- tions show that a 1: 7.5 dilution and the 3-mm criterion would reproduce the results obtained with full-strength lepromin and the 5-mm criterion. During the past 4 years, Hanks (unpublished) has studied the pattern of reactions to the 1:8 dilution of standard lepromin obtained from some 50 students at the Johns Hopkins School of Hygiene. Using the 3-mm criterion, all but 2 individuals have given positive Mitsuda reactions: 3-mm-5-mm ulcers occurred in some persons. In the population studied (aged 19-61 years) only 70% of the reactions became positive within 28 days. The remaining reactions appeared between the sixth and the twelfth week. The 2 persons regarded as negatives produced 2-mm reactions, which were firm and persistent. Studies in leprosy patients provide additional guidelines. Employing 2 standard lepromins, Yana- gisawa et al. (1957) found the 1: 8 dilutions to reduce the average diameter of 1+ reactions by 44% (from 7 mm to 3.9 mm). If the criterion for 1 + reactions is set at 5 mm (WHO Expert Committee on Leprosy, 1960), the expected diameter of 1 + reactions to the 1: 8 dilution is 2.8 mm. 705 706 J. H. HANKS AND OTHERS TABLE 1 RELATION BETWEEN THE AVERAGE DIAMETERS OF THE MITSUDA REACTIONS OF TUBERCULOID AND LEPRO- MATOUS PATIENTS WITH DIFFERENT DILUTIONS OF LEPROMIN No. of Average diameters of Mitsuda Lepromin bacteria/skin reactions (mm) dilution site(x 106) T M Ratio T/L Full strength 16 9.47 1.44 6.6 1:1.3 12 8.73 0.88 9.9 1:2 8 8.30 0.82 10.1 1:4 4 7.30 0.35 20.8 1:16 1 5.70 0.18 31.7 Recent unpublished studies by Abe evaluated a series of dilutions. As noted in Table 1 the ratio between the sizes of the Mitsuda reactions of tuber- culoid and lepromatous patients was about 5 times greater with the 1:16 dilution of lepromin than with full-strength lepromin. Thus the 1:16 dilution was considerably more effective in distinguishing between lepromatous and tuberculoid leprosy. Tuma compared similar serial dilutions of lepromin in 4 groups of persons. The essential results are given in Table 2. It will be noted that in the contacts, the tubercu- loid patients and the indeterminate patients the criteria adopted above were satisfied more frequently by the 1: 4 dilution than by standard lepromin. This means that dilution greater than 1:4 and less than 1:16 will be required for equivalence with full- strength lepromin. Tuma carried out a similar comparative study with the lepromin recommended for use as a stan- dard (160 million bacilli/ml) and diluted antigens containing 120, 80, 40 and 10 million bacilli/ml and concluded that the 1:16 dilution lacks adequate potency. He also used the 1: 8 lepromin in the work of the Instituto de Leprologia for some years and found this concentration ideal for the work in any kind of individual tested.1 Fernandez reactions. General experience, even with lepromins containing 160 million bacilli/ml shows a poor correlation between Fernandez and Mitsuda reactions. Fernandez reactions occur in only a low proportion of persons who give 3-mm (or greater) Mitsuda reactions to the 1:4 and 1:8 dilutions of lepromin. It is suggested that 48-hour readings need not be made during further investiga- tions of cutaneous reactivity to the diluted lepromins. RECOMMENDATIONS AND FUTURE PROSPECTS (1) To reduce false-positive reactions, the present standards of lepromin production must be main- tained. The original stocks should be made from lepromas yielding 160 million bacilli/ml, when employing 4 g-5 g of leproma per 100 ml of final suspension. These basic stocks should be free of visible tissue particles and preferably preserved by lyophilization. (2) Clinical comparisons should be made be- tween 0.1 ml of such lepromins and lepromins diluted 1:4 (40 million bacilli/ml) and 1:8 (20 million bacilli/ml). It would be desirable to conduct these tests in at least 2 countries and to test the materials in at least 2 types of subjects: healthy persons and tuberculoid patients. 1 A comparative study of the suitability of lepromins containing 20 million (1:8) and 40 million (1:4) bacilli/ml is in progress. Preliminary results reported to WHO by Tuma indicated that the former antigen (1:8) can be safely sub- stituted for the latter (1:4) when an induration of 3 mm is considered as the lower limit of positivity. TABLE 2 PERCENTAGE OF POSITIVE REACTORS TO DIFFERENT DILUTIONS AMONG FOUR GROUPS OF PERSONS Lepri bi Minimum Groups of persons sizeof-dLutroion sicterasi Mitsuda Healthy Indeter-ilution s(1e reaction (non- Contacts Tuberculoid minate(mm) contacts) patients patients Full strength 16 5 100 95 86 70 1:4 4 3 100 100 95 75 1:16 1 3 93 60 76 50 STUDIES TOWARDS THE STANDARDIZATION OF LEPROMIN 707 (3) The Mitsuda reactions in leprosy patients and in contacts should be recorded at 4 weeks. Reactions in other people should be recorded at 4 or 5 weeks; for the purposes of an investigation, or if the first records are negative or doubtful, further records should be made at 7 and 9 weeks. (4) The size of each reaction should be recorded in millimetres denoting the average diameter of firm induration. The letter " u " should be added as exponent to designate ulceration. When it is necessary to group results, the following scale is suggested: 0, no reaction; ±, induration less than 3 mm; +, 3 mm-5 mm; 2+, 6 mm-10 mm; and 3+, >10 mm or with ulceration. (5) Renewed histological studies are required to investigate the frequency of false-positive reactions and to determine whether further refinements in lepromin production are necessary. (6) It is expected that the laboratories working in collaboration with WHO will produce lepromins for experimental investigation and co-operate in solving the problems related to the production of reliably standardized lepromins and that they will assist in the training ofpersonnel in other laboratories. RtSUME' tTUDES EN VUE DE LA STANDARDISATION DE LA LEPROMINE: RE-SULTATS DI-JA ACQUIS ET PERSPECTIVES Les centres travaillant en cooperation avec l'OMS a la standardisation de la l6promine ont effectu6 des recherches pour une mise au point d'un meilleur antigene et des etudes sur les reactions de Mitsuda suscitees par les lepromines diluees. L'objectif ultime est de mettre en evidence toutes les reactions specifiques dues aux bacilles tout en r6duisant le nombre des reactions faussement positives de valeur 1+, attribuees a la pr6sence d'el6ments tissulaires. Une 1lpromine renfermant 160 millions de bacilles par millilitre a ete proposee comme antig6ne ( etalon * et le 8e Congres international de Leprologie (Rio de Janeiro, 1963) a recommand6 cette preparation comme etalon international provisoire. Deux methodes de num6ration microscopique ont ete developpees aux fins de cette etude, l'utilisation de la micropipette et le procede dit ((de la tete d'epingle #, qui foumissent des resultats tres concordants. Grace a l'emploi de melangeurs electriques, on obtient une lepro- mine dans laquelle les bacilles n'ont ete soumis qu'a des effets mecaniques minimaux qui peuvent etre doses avec precision. Des techniques de laboratoire ont e mises au point qui permettent de recuperer Mycobacterium leprae a partir des alaments tissulaires des suspensions de lepromes. Les lepromines lyophilisees conservent leur pouvoir d'origine et leur teneur en bacilles pendant 5 ans, alors que la refrigeration a 4°C-10°C entraine une perte progressive d'activite. On a signale par ailleurs une diminution rapide du nombre des bacilles lorsque la lepromine est expediee en flacons. On a procede 'a une etude comparative de 1lpromines diluees contenant 160, 120, 80, 40 ou 10 millions de bacilles par millilitre. Les resultats semblent indiquer que si l'on prend comme critere de positivit6 I'apparition d'un nodule de 3 mm de diametre, la preparation contenant 40 millions d'organismes par millilitre permet d'effectuer valablement la reaction de Mitsuda chez les malades, les contacts et les personnes non expos&es a la contagion. D'apres les premieres donnees resultant d'un autre essai comparatif, il semble que la l6promine renfermant 20 millions de bacilles par millilitre puisse sans inconve- nient etre substituee ai la lepromine ayant une teneur double en bacilles, si l'on choisit comme limite inferieure de positivit6 un nodule de 3 mm de diametre. Les auteurs formulent les recommandations suivantes: creation de Centres OMS regionaux de ref6rence pour la preparation et la distribution de lepromine et pour la mise au point methodologique d'une lepromine standard; evaluation ulterieure des concentrations optimales en Myco. leprae pour l'utilisation generalis6e des epreuves cutanees; definition des limites dans lesquelles la lepro- mine contenant 20 millions de bacilles par millilitre reduit les reactions faussement positives chez les malades lepromateux; suggestions concemant les moments de lecture des reactions de Mitsuda ainsi que la notation des reactions 1+, 2+ et 3+. REFERENCES A te, M., Nakayama, T., Tachikawa, N. & Okamura, K. (1963a) La Lepro, 32, 137-140 A be, M., Nakayama, T., Yanagisawa, K., Maeda, M., Asami, N., Tachikawa, N. & Okamura, K. (1961) La Lepro, 30, 163-168 Abe, M. et al. (1963b) La Lepro, 32, 133-136 Abe, M. et al. (1965) La Lepro, 34, 341-347 Bechelli, et al. (1957) Rev. bras. Leprol., 25, 21-58. (Also in: Transactions ofthe Eighth International Congress of Leprosy, Tokyo, 1958 (1959), pp. 183-189) Guinto, R. S. & Wade, H. W. (1958) Int. J. Leprosy, 26, 328-345 Hanks, J. H. (1959) Int. J. Leprosy, 27, 134-140 708 J. H. HANKS AND OTHERS Hanks, J. H. (1968a) Int. J. Leprosy, 36, 64-65 Hanks, J. H. (1968b) Int. J. Leprosy, 36, 66-75 Hanks, J. H. (1968c) Int. J. Leprosy, 36, 76-77 Hanks, J. H. Chatterjee, B. R. & Lechat, M. F. (1964) Int. J. Leprosy, 32, 156-167 Nakayama, T., Ozawa, T., Otaka, S., Hayashi, K. & Ito, M. (1965) Research activities: special issue for the tenth anniversary, July 1965, Tokyo, National Institute for Leprosy Research, pp. 1-15 Nolasco, J. 0. (1940) Int. J. Leprosy, 8, 285-298 Tuma, M. (1963) Bol. Serv. Nac. Lepra (Rio), 22, 15-20 WHO Expert Committee on Leprosy (1960) Wid Hlth Org. techn. Rep. Ser., No. 189 Yanagisawa, K. et al. (1957) La Lepro, 26, 66-69 Annex I RECOVERY OF MYCO. LEPRAE FROM TISSUE RESIDUES Material Tissue residues from cycle IV, accumulated from processing 25 g of Estimated volumes: sediment, 55 ml; lepromas supemate, 30 ml Procedure (1) Pooling. Supernate from the above (30 ml) was used to transfer the sediments to the Waring blender. Additional NaCl (15 ml) was used to rinse the stock bottles.' Total volume: 100 ml (2) Chloroform declumping. 7 ml of chloroform (7 %/vol) was added. The blender was operated for 10 seconds after adding 7 ml of chloro- form. The suspension was promptly drained to a French square bottle: vacuum (550 mm Hg) was applied immediately while shaking the bottle. The temperature was raised to 50°C to facilitate the removal of all "free " chloroform. Total time: 30 min Volume Bacteria/ml Total no. (Ml) (x 1061) of bacteria(xlOG1) (3) Recovery of suspensions. Settling intervals were only 10 min as the chloroform-treated tissues settle more rapidly than normal tissues. Cycle I supernates . . . . . . . . . . . . . . . . . . . 60 ml 130 204 26 520 Cycle II supemates (after adding 80 ml NaCl)2 . . . . . . . 70 ml Volume of sediment: 50 ml Bacteria/ml (x 106): 250 ml Cycle III supernate (50 ml sediment + 100 ml NaCl)2 . . 90 ml 130 70 9100 Residue centrifuged for 1 min at 199 g - supernate . . . . 40 ml 1 7 Total no. of bacteria from residues of 25 g of lepromas ( x 10') . . 35 620 No. of bacteria recovered per gram of leproma . . . . . . . . . . 1 430 No. of bacteria Acid-fastness Evaluation recovered ( x 10) Comparative yields of bacteria per gram of leproma: (a) Supernates I-IV, usual method . . . . . . . . . . . . . . 3 620 (72%) + + + + (b) Supernates I-III from chloroform-treated tissue residues . . . 1 430 (28%) + ++ Unpublished method evolved by J. H. Hanks & C. V. Jacob. I NaCl = phenol saline containing 0.02% sodium lauryl sulfate. STUDIES TOWARDS THE STANDARDIZATION OF LEPROMIN Conclusions (1) The usual method had recovered about 75% of the bacilli obtainable. It would not pay to recover from the tissue residues of each batch of lepromin prepared. (2) By accumulating tissue residues, a further 25% of the obtainable bacilli could be collected periodically. The chloroform-treated bacilli are less acid-fast than the originals, but are clearly recognizable. (3) Since the bacilli from tissue residues are altered, the Mitsuda reactions will probably not develop at the normal rate. The chloroform-treated bacilli could be com- bined with the original bacilli if the ratios are maintained at 75% non-treated and 25 % chloroform-treated bacilli. nex 2 RECLAMATION OF SMOOTH SUSPENSIONS FROM MHW-TYPE LEPROMINS A " pool " was prepared by combining the remnants of MHW-type lepromins that had been submitted by various investigators during an earlier study. This sus- pension contained the usual rapidly-settling tissue par- ticles and all sizes of bacterial clumps. The basic pro- cedure for recovering a smooth suspension was that described by Hanks (1968b, pp. 68, 70). Three successive supernates were recovered each 20 min to yield supernate 1. After adding phenol saline to the original volume and shaking, 3 cycle II supemates were recovered and pooled with the above. This pro- cedure recovers between 87% and 95% of the total bacilli yielded by 4 collection cycles. Starting with 100 ml of pooled lepromins, the yield was 90 ml of stable lepromin containing 87 x 108 bac- teria/ml. The tissue residue, largely freed of single bacilli and medium-sized clumps but rich in large clumps and globi, was included with tissue residues fractionated as described in Annex 1. 709