DISTR. : LIMITED WORLD HEALTH ORGANIZATION DISTR -.: L1MITEE
ORGANISATION MONDIALE DE LA SANTE WHO/CDS/LEP/86.4 page i
ENGLISH ONLY
LABORATORY TECHNIQUES FOR LEPROSY
Table of Contents
PREFACE ···················· . 1
INTRODUCTION :...... 2
CHAPTER 1 - SAFETY WITH HYCOBACTERIUH LEPRAE 5
5
6
I·1 6
6
7
Accidental Inoculation...... 11
Cleaning and Sterilization...... 12
CLEANING...... 12
Cleaning Glassware...... 12
Cleaning Working Surfaces...... 13
STERILIZATION...... 13
Autoc1aving...... 13
Hot-Air Sterilization 13
Ethylene Oxide Sterilization · · 14
Irradiation...... 14
Other Methods...... 14
CHAPTER 2 - MICROSCOPY...... 15
The Microscope...... 15
Illumination...... 15
Micrometry...... 17
DIAMETER OF AREA ON SLIDE...... 17
DIAMETER OF MICROSCOPE FIELD ·.················· 18
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Care of the Microscope...... 19
CHAPTER 3 - THE SMEAR...... 21
Sites for Smear 21
MULTIBACILLARY LEPROSY...... 21
PAUCIBACILLARY LEPROSY...... 22
Smear Techniques...... 22
SMEARS OF SKIN SCRAPINGS...... 22
SMEARS OF.NASAL SECRETIONS ("NOSE BLOWS")...... 23
TRANSPORT OF SMEARS...... 23
Preparation of the Smear for Examination...... 23
FIXATION OF SMEARS ...... 23 ACID" FAST STAINING...... 24
PERIODATE OXIDATION...... 24
The Bacteriological Index...... 25
The Morphological Index and the Solid Ratio...... 25
MEASURING THE SOLID RATIO...... 29
CHAPTER 4 - THE BIOPSY...... 30
Sites for Biopsy...... 30
Biopsy Techniques...... 30
SKIN BIOPSY...... 30
Incision Method...... 30
Punch Method...... 31
NERVE BIOPSy...... 31 [. Procedure...... 31
DARTOS MUSCLE BIOPSY...... 33
Procedure...... 33
Preparation of the Specimen for Examination...... 34
STORAGE AND SHIPMENT OF SPECIMENS...... 34
Biopsy Specimens for Animal Inoculation...... 34
Biopsy Specimens for Histopathological Examination...... 34 WHO/CDS/LEP/86.4 page iii
CHAPTER 5 - HISTOPATHOLOGICAL EXAMINATION AND CLASSIFICATION...... 36
Fixation, Processing, Sectioning and Staining Tissue Specimens...... 36
FIXATION...... 36
PROCESSING...... 36
SECTIONING...... 37
STAINING...... 37
Haematoxylin and Eosin 37
Acid-Fast Stain 37
Histopathological Examination . 37
PATHOGENESIS OF THE SKIN LESION . 38
Host Cells of H. leprae . 38
"Activity" and "Regression" . 38
Macrophages . 38
Epithelioid cells . 39
Evolution of the Lesion . 39
Cellular Infiltrate . 39
Nerve Involvement . 40
EXAMINATION AND INTERPRETATION OF A SKIN SECTION . 40
Granu10ma and infiltrate . 40
Nerve bundles . 40
Hair-follicles and sweat- glands . 41
Epidermis and subepidermal zone . 41
Blood vessels . 41
Oedema . 41
Connective tissue . 42
Organisms . 42
Activity of the Process ·. 42
DIAGNOSIS . 43
Early Leprosy . 43
Tuberculoid Leprosy . 43 WHO/CDS/LEP/86.4 page iv
THE RIDLEY - JOPLING CLASSIFICATION...... 44
TT (Full Tuberculoid)...... 44
BT (Borderline Tuberculoid)...... 44
BB (Mid-Borderline)...... 44
BL (Borderline-Lepromatous)...... 44
LL (Full Lepromatous)...... 45
LLs (sub-polar lepromatous)...... 45
LLp (polar lepromatous)...... 45
Indeterminate (I) 46
Classification of Regressing Lesions...... 46
TT. BT...... 46
BB...... 46
BL...... 46
RELAPSE...... 46
REACTIONS...... 46
ENL. 46
Lepra (Borderline, Reversal or Upgrading) Reactions...... 46
Measurement of the Solid Ratio in Tissue Sections...... 47
CHAPTER 6 - WORK WITH EXPERIMENTAL ANIMALS · 48
""Immune-Competent Rodents...... 48
GROWTH OF H. LEPRAE IN THE IMMUNE-COMPETENT RODENT...... 48
Immune-Deficient Rodents...... 49
GROWTH OF H. LEPRAE IN IMMUNE-DEFICIENT RODENTS...... 50
Thymectomized-Irradiated Mice...... 50
Nude Mi.ce...... 52
Neonatally Thymectomized Rat...... 53
Congenitally Athymic Rat...... 53
Production of Thymectomized-Irradiated Mice ····· 54
T900R MICE...... 54
T200X5R MICE...... 54 WHO/CDS/LEP/86.4 page v
PROCEDURES...... 55
Mice...... 55
Anaesthesia...... 55
Thymectomy...... •...... 55
Irradiation...... 56
Bone-Marrow Replacement...... •...... 57
Other Experimental Animals...... 57
. ARMADILLO...... 57
OTHER SPECIES...... 57
Animal Husbandry and Breeding ·· ······· 57
HUSBANDRY OF IMMUNE-COMPETENT MICE ··· ········· 58
Housing...... 58
Nutrition...... 58
Animal Care...... 60
BREEDING...... 60
HUSBANDRY OF IMMUNE-DEFICIENT RODENTS ··· .. ······ 61
T900R and T200X5R Mice; Neonatally Thymectomized and Congenitally Athymic Rats...... 61
Nude Mice...... 62
Mouse Foot-Pad Techniques...... 63
SHEPARD'S TECHNIQUE · .. ················ 63
Recovery of H. leprae from Human Biopsy Specimens...... 63
Preparation of Smears and Counting AFB ··.·········· 63
Inoculation into Mice...... 64
Harvesting H. leprae from Mouse Footpads ··········· 64
REES' TECHNIQUE ··.···················· 65
Recovery of H. leprae from Human Biopsy Specimens ···· 65
Preparation of Smears and Counting AFB · .. ·········· 65
Inoculation into Mice...... 66
Harvesting H. leprae from Mouse Footpads ·············· 66 WHO/CDS/LEP/86.4 page vi
PREPARATION OF INOCULA FOR NUDE MICE . 67
Applications of Animal Inoculation . 67
CLINICAL APPLICATIONS . 67
Detection of Viable M. leprae . 67
Drug- Susceptibility Testing . 69
Procedure . 69
Preparation of dapsone-containing mouse diets . 71
Preparation of mouse diets containing other drugs . 72
Assaying dapsone in mouse diets . 73
Assay of mouse diets for other drugs . 73
Detection of Persisting M. leprae . 73
EXPERIMENTAL APPLICATIONS . 78
Drug Screening . 78
Continuous Technique . 78
Kinetic Technique . 79
Proportional Bactericide Technique . 81
Experimental Chemotherapy . 82
Calculations and Statistics . 83
RESULTS OF MULTIPLE HARVESTS OF M. LEPRAE . 83
FISHER'S EXACT PROBABILITY CALCULATION . 84
RESULTS OF THE PROPORTIONAL BACTERICIDE TECHNIqUE . 86
Calculation of the MPN . 86
Median Infectious Dose (IDsO) . 101
CHAPTER 7 - IDENTIFICATION OF MYCOBACTERIUM LEPRAE . 107
Morphology . 107
Multiplication of an Organism ( . 108
Non-Cultivability of H. leprae . 110
Growth of H. leprae in the Mouse Footpad . 110
Susceptibility of H. leprae to Dapsone . 110
"Lepromin" - Testing . 111 WHO/CDS/LEP/86.4 page vii
Oxidation of Dihydroxyphenylalanine . III
PROCEDURES . 111
Spectrophotometric Procedure . 111
Radioisotopic Procedure . 112
Spot Test . 112
Pyridine Extraction . 112
PROCEDURE . 112
Characterization and Identification of Bacteria by Study of their Genome . 113
GENOME SIZE . 113
BASE COMPOSITION OF THE BACTERIAL GENOME . 113
Chromatography of DNA . 113
Buoyant Density . 113
Thermal Denaturation . 113
HOMOLOGIES BETWEEN BACTERIAL GENOMES . 114
Hybridization on a Membrane Filter . 114
Hybridization in Solution . 114
Spectrophotometric Method . 115
Cell-Wall Composition . 115
Antigenic Constitution . 117
Identification of an Isolate as H. leprae . 117
CHAPTER 8 - IMMUNOLOGICAL STUDIES . 119
Skin-Testing . 119
APPLYING THE SKIN-TEST . 119
Equipment . 119
Administering the Test . 119
Reading the Reaction · . 120
LEPROMIN - TESTING . 121
Testing with Lepromin . 121
COMPARING SKIN-TEST ANTIGENS . 121
Range - Finding . 121 WHO/CDS/LEP/86.4 page viii
SKIN-TESTING WITH SOLUBLE ANTIGENS FROM H. LEPRAE 122
Serodiagnosis...... 123
SERODIAGNOSIS BY INDIRECT IMMUNOFLUORESCENCE 123
Reagents...... 123
Suspension of H. leprae 123
Suspension of BCG...... 123
Suspension of H. vaccae 124
Fluorescent antibody...... 124
The Test...... 124
Smear of H. leprae 124
Pretreatment of the smear...... 124
Absorption and dilution of test serum 124
Primary reaction...... 124
Secondary reaction...... 124
Washing and mounting...... 124
Reading...... 125
DETECTION OF ANTIBODIES AGAINST THE PHENOLIC GLYCOLIPID OF H. LEPRAE ..... 125
Procedure...... 125
Preparation of antigen " 125
Coating the plates...... 126
Blocking of non-specific binding 126
Incubation with test sera ·· 126
Enzyme-conjugated secondary antibody 126
Colour development...... 127
Calculation of Results...... 127
Use in Serodiagnosis...... 127
CHAPTER 9 - SURVEILLANCE OF DRUG-INTAKE ·· .. ·········· 128
Storage and Shipment Of Urine Specimens ····· 128
Monitoring Compliance With Dapsone 128
DAPSONE/CREATININE RATIO...... 128 WHO/CDS/LEP/86.4 page ix
Colorimetric Determination of Dapsone 129
Colorimetric Determination of Creatinine ···· 129
"ELISA" METHOD · ··············· 129
"SPOT" TEST · ················ 129
Monitoring Compliance with Other Drugs ····· 130
RIFAMPICIN...... 130
CLOFAZIMINE...... 130
ETHIONAMIDE AND PROTIONAMIDE ··.·.········· 130
APPENDIX 1 - PRODUCTION OF M. LEPRAE IN THE ARMADILLO ····· 132
APPENDIX 2 - PURIFICATION OF H. LEPRAE 134
Solutions and Chemicals...... 134
Homogenization Medium...... 134
Washing Buffer...... 134
DNAase Buffer ··· .. ·...... 134
Buffered Tween...... 134
Percol1R...... 134
Aqueous Two-Phase System 134
DNAase...... 134
Procedure...... 134
Comments...... 135
APPENDIX 3 - PREPARATION OF LEPROMIN ···.················ 137
APPENDIX 4 - PREPARATION OF REAGENTS ··.················ 139
Acid Alcohol...... 139
Ammonium Su1famate...... 139
Bouin's Fluid 139
Carbolfuchsin...... 139
Cardiolipin-Lecithin Solution...... 139
Celestine Blue...... 139
Citrate Buffer, pH 5· 139
Creatinine...... 139 WHO/CDS/LEP/86.4 page x
Dapsone...... 140
Diluent A (for Abe's FLA-ABS test) 140
Diluent B (for Abe's FLA-ABS test) 140
Dimethylaminobenzaldehyde-Oxalate Reagent (for spot-test for dapsone in urine) 140
Ehrlich's Haematoxylin...... 140
Eosin...... 140
Ethanol, 70%...... 140
Formalin, Buffered...... 140
Formalin, Neutral...... 140
Formol Calcium 141
Formol Milk...... 141
Gelatin-Phenol...... 141
Hanks' Balanced Salt Solution...... 141
HEPES Buffer...... 141
Hydrochloric Acid...... 141
Lowy's Fixative...... 142
Methylene Blue...... 142
Phosphate Buffer, pH 7.4...... 142
Phosphate-Buffered Saline...... 142
Picric Acid...... 142
Polyethylene Glycol Palmitate...... 142
Saline, Physiological...... 142
Sodium Hydroxide...... 143
Sodium Nitrite...... 143
Tribromoethanol...... 143
TRIS-Buffered Saline, pH 8.0...... 143
Trypan Blue Solution...... 143
Trypsin 143
BIBLIOGRAPHY...... 144
INDEX ..... '...... 154 WHO/CDS/LEP/86.4 page 1
PREFACE
The manual on laboratory techniques for leprosy was conceived of several years ago when a workshop on the mouse footpad technique, organized by the Scientific Working Group on Chemotherapy of Leprosy (THELEP) of the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR) , revealed that the source materials on laboratory techniques in leprosy were scattered throughout several publications which were not easily accessible to laboratory workers, particularly in the countries where leprosy is endemic. Dr H. Sansarricq, then Chief of the Leprosy Unit in WHO, therefore took the initiative of promoting a comprehensive manual covering all the important laboratory techniques used in leprosy. Dr L. Levy, then Chairman of the THELEP Steering Committee, agreed to put together all the available material and edit a manual on the subject. The editorial task of Dr Levy was not easy in spite of excellent contributions received from several contributors. In this connexion our grateful thanks are due to Dr H. Bercovier, Dr T.M. Buchanan, Dr P. Draper, Dr G.A. Ellard, Dr J. Guld, Mrs C. Lowe, Dr R.J.W. Rees, Dr D.S. Ridley, Mr T.K. Sundaresan, Mr H.G. ten Dam and Dr D.B. Young for their contribu tions, and to Mrs F. Perry and Dr Ji Baohong for their editorial assistance. Particular mention should be made of the contribution of the late Dr Charles C. Shepard, whose monu mental work, particularly on the mouse footpad technique, revolutionized leprosy research and led to the technique becoming one of the most widely used research tools in leprosy.
The manual aims to be as comprehensive as possible. Even so, it is felt that the researcher should refer to the original references cited in the bibliography section of the manual if he or she wants to become an expert in any specific technique.
It is hoped that the manual will stimulate and promote leprosy research particularly in the leprosy-endemic, developing countries, and facilitate the transfer of technology, to the overall benefit of leprosy control. WHO/CDS/LEP/86.4 page 2
INTRODUCTION
Leprosy is a chronic infectious disease caused by Mycobacterium 1eprae, an acid fast bacterium. The disease is characterized by a long incubation period, usually measured in years. Although the disease was prevalent in Europe during the Middle Ages, today leprosy patients are found almost exclusively in developing countries. The number of leprosy patients in the world is estimated to exceed 10 million (216). Even in those areas in which the disease is now endemic, the prevalence is low, rarely exceeding 50 patients per 1000 population.
Although leprosy may affect many organs in the body, those chiefly affected are peripheral nerves and skin. Involvement of nerves results frequently in pain, and in loss of sensation, primarily of the extremities. This renders the patient vulnerable to repeated small injury, with consequent deformity and disability.
In fact, the response to infection with M. 1eprae exhibits a broad range, from the individual harbouring sub-clinical (unapparent) infection with M. 1eprae to the patient with advanced disease. In addition, the manifestations have been said to describe a continuous spectrum, from tuberculoid leprosy (few bacilli; brisk cellular immune response) at one pole to lepromatous leprosy (many bacilli; little cell-mediated immune responsiveness) at the other. Untreated patients with lepromatous and near-lepromatous leprosy frequently exhibit involvement of the nasal mucous membrane, and may excrete huge numbers of M. 1eprae in nasal secretions and sputum.
The number of M. 1eprae harboured by the patient may be estimated from the "bacter iological index" (BI), which is calculated from the numbers of organisms seen in smears of skin lesions that have been stained by means of the acid-fast stain. Measurement of the BI is useful in classifying the patient's disease, in terms of its position in the spec trum from the tuberculoid to the lepromatous poles, and is a very important consideration in the management of the patient's chemotherapy. Patients with polar lepromatous leprosy may harbour truly enormous populations of M. 1eprae; this has given rise to the concept of "multibacillary" leprosy. Similarly, patients with tuberculoid leprosy harbour small bacterial populations, giving rise to the concept of "paucibacillary" leprosy.
An estimate of the proportion of the patient's organisms that are living is provided by the "morphological index" (MI) or "solid ratio". Viable M. 1eprae appear both brightly and solidly stained, when stained by means of a carefully standardized acid-fast stain, and observed under optimal microscopic conditions, whereas pale or non-uniformly staining organisms are assumed to be dead - i.e., they are unable to multiply in the mouse footpad. This measurement, although difficult to perform with accuracy and repro ducibility, has given important information with respect to the rate with which lepromatous and near-lepromatous patients respond to chemotherapy. It is of considerable interest that the majority of the lepromatous patient's M. 1eprae do not appear to be viable, even before the first dose of an effective drug has been administered.
In addition to the important differences of the size of the bacterial populations harboured by typical patients with lepromatous and tuberculoid leprosy, there are impor tant differences of immunological responsivity. Thus, patients with lepromatous leprosy typically do not respond to a skin-test antigen, "lepromin", prepared from heat-killed M. 1eprae. Moreover, early in the course of the disease, the lepromatous patient may fail to react to a considerable variety of antigens, both mycobacterial and non-mycobac terial, employed as skin-test antigens or in lyrnphocyte transformation. After some period of effective chemotherapy, when the size of the patient's bacterial population has been greatly reduced, he appears to have recovered the ability to respond to virtually all of these antigens, with the exception of antigens derived from M. 1eprae. This persisting immune deficit has not yet been explained. WHO/CDS/LEP/86.4 p~e3 .
Leprosy occurs in an unknown proportion of individuals infected by M. Leprae, which is transmitted from infectious patient to uninfected contact. Transmission is thought to be accomplished via airborne droplet nuclei (152, 164), the patient discharging viable organisms in his respiratory secretions, and the contact inhaling viable organisms into his respiratory tract. In addition, transmission of M. 1eprae by inoculation appears certainly to have occurred (116, 145), and the possibility of transmission by arthropod vectors (130, 131) has not been excluded. Although an organism very similar to M. 1eprae has been isolated from a number of feral armadillos captured in the south central region of the United States, to which these animals are native, no evidence has been obtained to suggest that these infected armadillos represent a source of human infec tion. On the other hand, the weight of evidence suggests that the only important source of human infection is the infectious leprosy patient.
Leprosy patients have been treated for many years with dapsone (4,4'-diaminodiphenyl sulfone) as monotherapy. This treatment has been difficult to apply, as it requires good patient compliance for many years, and relapse with dapsone resistance has become alarm ingly frequent, even among cooperative patients. Nevertheless, substantial reductions of the numbers of leprosy patients have resulted in a few areas,from careful application of dapsone monotherapy, coupled with intensive case-finding activities. It is hoped that currently recommended treatment for a defined period with combinations of several anti microbial drugs, at least some of them administered under supervision, will prove more successful in the control of leprosy.
Although M. 1eprae, the first human bacterial pathogen to be identified, was recog nized by Hansen in 1873 (72), it has not yet been cultivated in artificial media. How ever, the organism produces a limited infection of immunologically normal rodents, and more extensive involvement of the nine-banded armadillo (Dasypus novemcinctus) , and of certain immune-deficient rodents. Because the organism cannot be cultivated, much of our understanding of the biology of M. 1eprae, and our knowledge of the activity of anti microbial drugs on the organism have come to be based on a unique set of labcratory tech niques. The development of these techniques, most of them within the last 20 years, has transformed leprosy research from an empirical to an experimental science.
Before 1960, the research worker in leprosy had very few laboratory techniques avail able to him. By means of the BI, he could estimate the concentration of organisms in the skin, and, with the assistance of the lepromin reaction, he could learn something of the immune status of the patient. In 1960, Charles C. Shepard of the Centers for Disease Control, Atlanta, Georgia, USA, published his first papers on the mouse footpad technique for cultivation of M. 1eprae in vivo (171, 172); this technique has proved to be a major contribution to leprosy research. The technique has made possible the screening of drugs and assessment of their efficacy in chemotherapy in a quantitative and specific manner, has provided means of detecting drug-resistant strains of M. 1eprae, and also has been exploited for immunological studies. In chronological order, the next important contributions to leprosy research were the development of the thymectomized-irradiated mouse (by R.J.W. Rees, National Institutefut Medical Research, London, UK), improvement of the means of classifying leprosy patients (by Ridley and Jopling), and the demonstration of unlimited multiplication of M. 1eprae in a proportion of nine-banded armadillos (by Kirchheimer and Storrs). More recent important contributions to the armamentarium of the leprosy researcher have been Draper's procedure for purification of armadillo-derived M. 1eprae, and demonstration of the great susceptibility to M. 1eprae infection of the nude mouse (by Colston and by Ito). More recently, there has been a spate of new tech niques for immunological study and molecular biology, based on the availability of large numbers of M. 1eprae from the armadillo through IMMLEP (IMMLEP is the Scientific Working Group on Immunology of Leprosy set up under the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases), and on the tremendous advances in these two disciplines that have occurred during the last few years. Thus, at the present time, leprosy research appears to be entering a period of extraordinarily rapid advancement. Leprosy remains uncontrolled in most areas of endemic disease, and it appears unlikely that the disease can be controlled until we have gained much more knowledge of many aspects of the disease. New techniques - laboratory, clinical, and epidemiological - are WHO/CDS/LEP/86.4 page 4 required, in order to make possible the acquisition of additional information, and devel opment of the new tools - vaccine, skin-test antigens, serological tests, chemotherapeutic regimens - needed to control leprosy.
Because these techniques are unique to leprosy, and of so recent development, they have not yet been collected in a single volume. This volume seeks to fill this gap. It is designed primarily for the researcher working in leprosy-endemic countries. Recogniz ing that those at whom this volume is aimed require more than simply a collection of recipes, an attempt has been made to provide a background of theory, and to describe applications of many of the techniques included. This book should also be of interest to the clinician, as he should have a thorough understanding of what the laboratory can offer in the interpretation of the disease process and cure.
This volume begins, in Chapter 1, with considerations of safety in dealing with M. leprae, proceeds in Chapter 2 with the use and care of the microscope, and continues in Chapters 3 and 4 with the procedures related to the smear and the biopsy. Chapter 5 deals with histopathological examination and classification, and Chapters 6, 7 and 8 consider techniques requiring equipment and expertise ordinarily available only in research laboratories. Chapter 9 deals briefly with the monitoring of patients for compliance with treatment. In several appendices, there appear descriptions of production of M. leprae in the armadillo, purification of M. leprae, preparation of lepromin and preparation of reagents. Finally, the volume includes a large bibliography, which should be useful to the researcher working in leprosy-endemic countries, who may have found it difficult to keep abreast of the more recent literature.
For some of the techniques presented in this manual, the presentation cannot provide sufficient information to enable the reader immediately to perform the technique with expertise. To become expert, the interested reader should review the literature cited, and should be alert to opportunities to work for a brief period of time in a laboratory experienced in the technique, or to attend a workshop on the technique; such workshops have, in the past, been sponsored by WHO. For information on new techniques and research tools of value to research in leprosy, the reader is encouraged to approach the Chief Medical Officer of the Leprosy Unit, Division of Communicable Diseases, World Health Organization, and the Secretaries of the Steering Committees of the Immunology (IMMLEP) and Chemotherapy (THELEP) of Leprosy Scientific Working Groups of the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases, World Health Organiza tion, 1211 Geneva 27, Switzerland. WHO/CDS/LEP/86.4 page 5
CHAPTER 1 - SAFETY WITH HYCOBACTERIUM LEPRAE
Only a few of those exposed to leprosy and infected with H. leprae develop overt disease (216). However, the factors that determine who of those infected will subse quently develop leprosy are unknown. The one certain means by which leprosy may be pre vented is by prevention of infection with H. leprae. This principle should be borne in mind, both when dealing with infectious patients, and when handling materials - clinical or experimental - containing viable H. leprae. Therefore, it is important that leprosy workers at every level and in every setting take precautions against infection.
In brief, protection of the medical or laboratory worker against infection by H. leprae appears important, and can be readily provided. That the clinic or laboratory is situated in a leprosy-endemic area is no reason for not taking safety precautions. It is clear that the worker himself bears considerable responsibility for his own safety, which is as much a matter of practice as of equipment. Therefore, at the same time that a new worker is trained to perform the work of the clinic or laboratory, he must be imbued with good safety habits. Moreover, he must see that the safety precautions are applied meticulously by everyone.
In this first chapter, which deals primarily with the methods by which one may work safely with H. leprae, whether in a clinical or a laboratory setting, the properties of H. leprae that are relevant to prevention of infection with the organism are reviewed; the subject of aerosols, probably the vehicle by which H. leprae is most often trans mitted, is considered; safety precautions to be taken in the clinic and in the laboratory are dealt with in detail; the problem of accidental inoculation is reviewed; and, finally, methods of cleaning and of sterilization, not unrelated to the matter of safety, are presented.
Properties of M. 1eprae
H. leprae is an acid-fast bacillus (AFB) that has not yet been cultivated in cell free culture media, and attempts to cultivate the organism in cell culture have been largely unsuccessful (62, 168). When suspended in saline, the organism survives for several weeks at 00 , 20 0 , or 3loC (176, 190), and in nasal secretions exposed to drying for several days (35, 151). H. leprae survives slow freezing and storage in liquid nitrogen for an indefinite period of time (23). Survival in this context means that, although some fraction of the organisms may have been killed, the proportion of viable organisms remains large enough to infect normal mice (i.e., approximately 1:1000; as is stated on p. 48, although one viable organism is theoretically sufficient to initiate infection, probably four or five viable organisms are actually required, because as much as 80% of the inoculum may be lost from the site of inoculation within one hour following inoculation. If the standard inoculum contains 5000 H. leprae, then a proportion of viable organisms smaller than 1:1000 may not provide the four or five viable organisms required for infection).
H. leprae is killed by autoclaving for 20 minutes at 121°C, by heating for 30 minutes at 750C (55), by pasteurization (99), and by 2.5 Mrad (2.5 x 104Gy) gamma irradiation (from a 60Co source) (40). Killing of H. leprae, in the sense in which the word is employed here, means that the proportion of viable H. leprae has been greatly reduced, and inocula of autoclaved or irradiated organisms as large as 10 7 (10 million) will not give rise to multiplication in immunosuppressed rodents (40, 55 see p. 49 - 57).
Resistance of H. leprae to ultraviolet irradiation, solvents and antiseptic solu tions has not been studied systematically. Wang and Wang reported (206) that H. leprae lost infectivity for mice after exposure of a suspension of the organisms to ultraviolet light (wave-length not stated) for 30 or 60 minutes.Similarly, suspensions of H. leprae WHO/CDS/LEP/86.4 page 6
lost infectivity after exposure to summer sunlight for 2 - 4 hours at an ambient temperature of 32°C.
Aerosols
Aerosols may be important in the spread of H. 1eprae (35, 152), as they are for the spread of many infectious agents, especially H. tuberculosis, that are transmitted via the respiratory route. Therefore, a consideration of aerosols is important in planning measures to be taken to prevent spread of H. 1eprae. Both infectious leprosy patients and many laboratory procedures produce aerosols.
A study of laboratory-associated infections caused by a variety of pathogens revealed that only 20% of the infections had resulted from recognized accidents (144); many of these accidents had resulted in the production of aerosols. For many of the remaining 80% of laboratory infections, the only available information was that the victim had worked directly with the infectious agent, or in a room or building in which the agent was handled. Exposure to infectious aerosols was believed to be the source of infection in a considerable proportion of the cases. That the prevalence of tuberculosis among labora tory workers varied from two to 28-fold that in the general population provides support for this hypothesis (144).
Two distinct types of aerosol are recognized (164). One is composed of particles greater than 5 ~m in diameter; these particles settle slowly, contaminating laboratory surfaces and the skin of laboratory workers. When inhaled, these particles are usually captured in the upper respiratory passages, where they do no harm, at least in the case of tuberculosis. The other type of aerosol consists of particles smaller than 5 ~m in diameter; these dry almost instantaneously, producing tiny, solid "droplet nuclei", which may remain suspended almost indefinitely, and which are readily dispersed by air currents. Upon inhalation, some of the droplet nuclei, which may contain viable microorganisms, reach the pulmonary alveoli, the site from which they may initiate infection.
Aerosols may be generated by a large number of laboratory procedures. Laboratory procedures known to generate aerosols composed predominantly of particles larger than 5 ~m in diameter include opening containers, use of pipettes without obvious spill, use of test-tube mixers, and centrifugation without breakage. Laboratory procedures known to produce aerosols composed predominantly of smaller particles include careful pouring, . sonication, heat-fixing slides, use of automatic pipettors, pipette-mixing, high speed blenders, breakage, centrifuge accidents, pipette spills, and flaming wire loops.
Safety Precautions
PRECAUTIONS IN THE CLINIC
The doctrine of low contagiosity of leprosy has so permeated medical thinking and practice that, in many treatment centres, no infectious precautions whatever are taken. Not only is leprosy believed to represent but a small threat of contagion to leprosy workers, especially with regard to leprosy workers stemming from the same community as the patients, but it is also said that, because there are so many sources of infection in the leprosy-endemic community in addition to the patients being attended, it makes little sense to seek protection from one source while one is at the same time continuously exposed to other sources, from which the worker cannot be protected. Finally, to counter the stigma of leprosy, it has been the practice in many communities to deal with leprosy patients as if leprosy were not an infectious disease.
In fact, in a study of subclinical infection by H. 1eprae, employing the FLA-ABS test (see pp. 63 - 66 ), significant antibody titres suggesting subclinical infection were demonstrated in 42% of staff members at a hospital for smear-positive leprosy patients. The risk of subclinical infection appeared to be highest among those staff members with at least 15 years' seniority (89). WHO/CDS/LEP/86.4 page 7 .
The leprosy patient is infectious whenever and for as long as he broadcasts viable M. 1eprae into his environment. It appears certain that untreated and relapsed leproma tous patients serve as sources of contamination of the environment with viable M. 1eprae. Such patients frequently manifest ulcerated lesions of the nasal mucosa (6), from which organisms are shed into the nasal secretions. Organisms aspirated with the nasal secre tions into the lower respiratory passages during sleep are coughed up in the sputum, a source of contamination certainly recognized in tuberculosis. Patients probably broadcast M. 1eprae into the environment also when sneezing and talking (164).
It was formerly thought that patients with lepromatous leprosy were infectious as long as one could detect AFB in smears and biopsy specimens of skin lesions. In fact, there is epidemiological evidence that treated lepromatous patients are non-infectious (222, 223). Also, the weight of laboratory evidence now suggests that the great majority of the organisms seen in smears and sections of biopsy specimens and in smears of "nose-blows" and sediments from nasal washings of treated patients are dead (81, 185, 195). With the onset of effective treatment, the proportion of viable M. 1eprae in skin lesions and nasal secretions decreases with greater or lesser rapidity, depending upon the specific drugs employed in treatment (19, 20, 114, 115, 153, 185, 187-189). For example, the proportion of viable M. 1eprae had become too small to infect normal mice after an average of 100 days of dapsone monotherapy (114), and viable organisms disappeared at a much more rapid rate during treatment with rifampicin (19, 115, 153, 188, 189). In addition, after beginning effective treatment, there is a rapid decrease of the absolute number of organisms in nasal secretions (81, 185, 195). Thus, not only are viable organisms killed as the result of therapy, but also the total number of M. 1eprae shed into the environment from the nasal mucosa decreases, probably the result of healing of the mucosal lesions.
To this point, the discussion has been limited to patients who are infectious because they broadcast viable M. 1eprae from their nasal secretions into the environment. Not yet considered are the possibilities of infection by direct inoculation, by &ccidental puncture during a surgical procedure, or by the bite of an infected arthropod. However small the importance of these routes of infection may be, they would appear even less important when the source of the organisms contaminating the scalpel blade or arthropod mouth parts is a treated patient.
In short, the infectious patient is essentially the patient with untreated or relap sing lepromatous leprosy, who transmits viable M. 1eprae through the medium of bacilli laden nasal secretions. If infection by M. 1eprae is to be prevented, it will be pre vented by protecting the uninfected worker from contact with the droplet nuclei derived from the patient's nasal secretions and sputum.
With respect to the preventive measures to be taken by those attending infectious leprosy patients, one may be guided by the precautions prescribed for personnel attending patients with untreated pulmonary tuberculosis (134). These precautions fall into two broad categories: (1) preventing microbial contamination of the air; and (2) elimina ting contamination of the air.
Contamination of the air around the patient is most effectively prevented by an early start of therapy, and by showing the patient how to control his sputum. Effective chemo therapy, which quickly renders leprosy patients non-infectious (19, 81, 114, 115, 153, 185, 188, 189, 222, 223), is to be started as soon as possible after the diagnosis is made. In addition, the concentration of M. 1eprae-containing droplet nuclei in the air around the patient decreases rapidly with increasing distance from the patient (164).
Indoors, eliminating contamination of the air is best accomplished by ventilation (134); out-of-doors, no special efforts need be made because of the high dilution.
LABORATORY PRECAUTIONS
The same arguments advanced to justify the failure to employ preventive measures against infection of medical attendants are employed to excuse the failure to take even WHO/CDS/LEP/86.4 page 8 the most elementary of precautions against infection of laboratory workers. In some laboratories, smears are fixed, biopsy specimens homogenized, and suspensions of M. 1eprae inoculated into mice on the open laboratory bench, with no attempt made to contain the aerosols that may be produced in the course of these procedures. Just as an aerosol containing viable M. 1eprae may be produced when the infectious patient talks, coughs or sneezes, so may heat-fixing a slide, flaming a loop, or clearing the needle lumen of air bubbles (144), if any of these objects has been in contact with a suspension of the organisms. The laboratory worker is also exposed to other hazards. Pipetting a bacterial suspension by mouth carries the risks of contaminating the oral mucous membrane and of ingesting the organisms (144). And the risk of accidental inoculation by means of a contaminated hypodermic needle is probably greater for the laboratory worker than for the medical attendant. Bearing in mind the principle that leprosy is best prevented by preventing infection by M. 1eprae, the adoption of precautions against infection appears mandatory for laboratory workers. Thus, the arguments in favour of protecting personnel who attend infectious patients with leprosy from infection with M. 1eprae are also valid for laboratory personnel, who deal with biological specimens that contain viable M. 1eprae.
The same precautions against infection prescribed for the clinical situation - preven ting and eliminating contamination of the air - are relevant to laboratory safety. Pre venting contamination of the air of the laboratory should be much more easily accomplished than preventing air-contamination of the spaces in which health workers attend patients.
Generation of aerosols may be limited by a number of simple precautions: draining rather than blowing out pipettes; mixing by mixer rather than by pipette; protecting working surfaces by impervious coverings; withdrawing a needle from a diaphragm "vaccine bottle", and clearing the needle of air bubbles by wrapping the top of the bottle and the needle in an alcohol-soaked pledget; using sealed centrifuge buckets; and working in a microbiological safety cabinet.
In addition to measures designed to prevent, contain and eliminate aerosols, there are general measures, appropriate to laboratories dealing with any bacterial pathogen, that should be adopted (218):
(1) mouth pipetting is prohibited. Instead, a suitable bulb, such as a PropipetteR (Fig. 1), or other remote pipette filler (e.g., Pipette-AidR), should be used; (2) eating, drinking, smoking and storing food in the laboratory is not permitted; (3) personnel must wash their hands thoroughly after handling infectious materials. and animals, and upon leaving the laboratory. The laboratory should be equipp~d with a wash-basin - preferably foot- or elbow-operated; (4) suitable protective laboratory clothing must be worn in the laboratory, and must not be worn outside the laboratory. The purpose of protective clothing is to prevent contamination of the clothing which is worn outside the laboratory. Therefore, one might simply take off street clothing and don, in its place, clothing which is worn only in the laboratory. Or one might elect to wear over street clothes a gown that closes in back. A front-buttoning laboratory coat worn over street clothes is certainly not suitable. The protective clothing should be laundered at least weekly; (5) use of hypodermic needles should be restricted to injecting animals and aspira ting sterile fluids from diaphragm vaccine bottles; (6) procedures with high potential for creating aerosols must be conducted inside a Class 1 microbiological safety cabinet, exhausted to the outside. The design of a suitable cabinet that can be constructed locally is shown in Fig. 2; (7) centrifugation of materials containing viable M. 1eprae should be performed in safety cups, which are opened only in the safety cabinet; (8) all contaminated wastes are decontaminated before disposal. This requires thefr temporary storage in a leak-proof container, and subsequent autoclaving or Figure 1. Safety pipette-filler, to be used instead of pipetting by mouth.
ACTUAL SIZE SIMPLE TO OPERATE WITH ONE HAND
--- .!!Iil ~ Using thumb and fore- - . finger, press on valve"A" and squeeze bulb with other fingers to produce a vacuum for aspiration. Releasevalve"A" leaving bulb compressed.
Insert pipette into liquid. z Press on valve "S". Suc tion draWl; liquid to de sired level.
:5 Press on valve "E" to expel liquid.
10 deliver the last drop, -g ~ maintain pressure on ()Q 0 valve "E", cover "E" inle! (1) ...... with middle finger, and CO) "'t::l squeeze the small bulb. Cf.l ...... t'" t"l "'d <, ex: (j\
",.. WHO/CDS/LEP/86.4 page la
Figure 2. Plan for a simple microbiological safety cabinet. WHO/CDS/LEP/86..4 page 11
incineration before opening the container; (9) work surfaces are decontaminated at least once daily, and after every spill of material containing viable H. 1eprae. Bench tops should be fabricated of, or covered with, material that is impervious to water and resistant to acids, alkalis, organic solvents and moderate heat. Decontamination is readily accom plished with a 1:5 dilution of household bleach; (10) access to the laboratory should be restricted as much as possible to the labora tory staff. Visitors should be provided with protective clothing; (11) gloves, surgical or disposable, may be used when handling materials containing H. 1eprae. However, their use must not be permitted to result in inadvertent contamination of the surroundings. Great care must be taken so that objects outside the safety cabinet - door-knobs, drawer-pulls, pipette cans, light switches - are not contaminated by unintentional touching with contaminated gloves during or after the procedure. Before taking them off, reusable gloves are rinsed in an antiseptic solution, washed with soap under running tap water and dried with a towel, followed by an application of talc. Damaged or disposable gloves should be washed in antiseptic solution followed by soap and water before being discarded; (12) ultraviolet irradiation, employing light at a wave-length of about 254 nm, is of some value in decontaminating smooth surfaces, such as the interior of the microbiological safety cabinet (164). However, because the effectiveness of irradiation decreases in proportion to the square of the distance from the light source, ultraviolet irradiation will be of little use in decontaminating entire rooms. Moreover, the minimal effective duration of irradiation is measured in hours. Thus, ultraviolet irradiation is effective for decontaminating small, closed spaces such as that within the safety cabinet; the ultraviolet source is turned on when the day's work is complete, and left on for at least two hours and even over-night. Because light at this wave-length can be injurious to the eye, the ultraviolet source should be turned off when the cabinet is in use; (13) arthropod and wild rodent infestation should be controlled.
Accidental Inoculation
Every effort must be made to prevent accidental inoculation of laboratory personnel with viable H. 1eprae. The use of hypodermic needles in the laboratory should be strictly limited. Certainly, a needle-and-syringe should not be employed in place of a pipette. Because accidental inoculation is particularly likely to occur during inocula tion of animals, this procedure should be carried out only after careful planning and much practice of the technique. Inexperience and fear of mice are factors that may contribute to accidental inoculation.
When accidental inoculation has occurred, usually of the finger or hand, the entry wound should immediately be thoroughly cleansed and treated like any other puncture wound. The accident should be recorded, so that, if the organisms with which the worker was inoculated were at the same time inoculated into mice, the results of mouse footpad inoculation can be used, at least in retrospect, to guide treatment. If accidental inocu lation occurred in the course of some activity other than mouse footpad inoculation, it would be prudent to inoculate mice with the same suspension (see pp. 63 - 66).
It appears unlikely indeed that the worker should have been inoculated with more H. 1eprae than were the mice, simply because the volume of bacterial suspension acciden tally inoculated into the worker should be smaller than that deliberately inoculated into the mouse. If, therefore, it is later noted that the organisms did not multiply in mice, then the likelihood that the worker had been infected with H. 1eprae could be assumed to be much reduced. If, on the other hand, the organisms multiplied in mice, then their susceptibility to drugs could be measured. In instances of accidental inoculation, the possibility of appropriate chemoprophylactic treatment might be considered. WHO/CDS/LEP/86.4 page 12
Cleaning and Sterilization
In the leprosy research laboratory, as in any microbiology laboratory, there is a constant need to clean equipment and working surfaces, and to sterilize materials and equipment prior to use, so as to decontaminate materials and equipment that have become contaminated by contact with H. 1eprae, an important aspect of laboratory safety, and to prevent contamination with unwanted microorganisms.
CLEANING
Cleaning - the removal of dirt - is a prerequisite to sterilization. Dirt both pro tects microorganisms from contact with lethal agents, and reacts with these agents so as to neutralize their activity. Moreover, removal of dirt also removes many of the organ isms associated with the dirt. Cleaning - the removal of chemical contamination - is also important in the microbiology laboratory.
Cleaning Glassware
New glassware is often slightly alkaline, and should be soaked in a 2% solution of HCl and rinsed in tap water and then in demineralized or distilled water and finally dried before use. Dirty glassware should first be rinsed in tap water, then soaked and scrubbed in a detergent solution, and finally rinsed in tap water and then in demineralized or distilled water before drying. Glassware that has been in contact with protein-containing fluids should not be allowed to dry before it can be placed in the detergent solution. Glassware that has been in contact with water-immiscible organic solvents may require rinsing with alcohol before washing. Finally, glassware that has been in contact with pathogenic microorganisms - e.g., H. 1eprae - should be decontaminated by autoclaving (v.i.) before any cleaning is attempted. Unfortunately, exposure to heat, as during autoclaving, may make cleaning more difficult; a practical solution to this problem is immediately to place the contaminated glassware - e.g., pipettes, tubes, small flasks _ directly into an antiseptic solution - e.g., 1:5 household bleach, and subsequently to place the container of antiseptic solution, together with other articles to be decontamin ated, into the autoclave. Cages that have housed mice infected with H. 1eprae are decontaminated and then cleaned in the same way.
New microscope slides should be cleaned before use by washing in tap water, rinsing several times in distilled water, and storing them in 1:1 (v/v) acetone and alcohol. Alternatively, the slides are washed in soap and water, scrubbed with gauze squares, rinsed in tap water and dried with a clean towel. In some laboratories, it is the prac tice to clean and re-use slides, after the AFB have been counted. However, the slides represent irreplaceable records, and storing them will permit comparison with subsequent specimens. Moreover, the cost of repeating an experiment is usually very much greater than that of the purchase of the slides themselves. Therefore, at least for important experiments, the used slides should be filed. In fact, some useful work has been carried out on stored slides (97, 107, 111, 191). If, nevertheless, slides must be reused, the used slides must first be cleaned, preferably by boiling in dilute nitric acid, according to the following procedure:
(1) oil present is first wiped off with a dry cloth; the slides are then soaked in 5% nitric acid for 24 hours; (2) the slides are next boiled in Decon 90R, 2%, (3) washed in tap water, (4) boiled again in 5% nitric acid, (5) washed again in tap water, and (6) rinsed several times in distilled water; finally, (7) the slides are stored in 1:1 (v/v) acetone and alcohol. WHOjCDSjLEPj86.4 page 13
Cleaning Working Surfaces
Because of the association between microbial contamination and dirt, particular atten tion should be paid to cleaning laboratory bench-tops and floors. Walls are subject to much less contamination, and may be cleaned as demanded by aesthetic rather than other criteria. As noted in the introduction to this section, physical removal of dirt is accompanied by removal of the associated microorganisms. Therefore, laboratory bench-tops and floors are best cleaned by scrubbing with a detergent solution, to which an antiseptic - e.g., 1:5 household bleach - may be added. Working surfaces and floors should be cleaned at least once daily, and after every spill of contaminated material.
STERILIZATION
Sterilization is the destruction of all living organisms in, or their removal from, materials. The usual methods employed for sterilization of laboratory materials involve heat, which may be dry or moist. Dry heat, provided by hot air, sterilizes by oxidation, whereas moist heat sterilizes by coagulation of protein. Sterilization by moist heat is more efficient, partly because wet materials conduct heat better than do dry.
Autoclaving
Autoclaving is the process of sterilizing by steam under pressure, which permits the attainment of temperatures higher than 100oC. An autoclave is essentially a vessel con structed to withstand a pressure of 2.05 x 105 Pascals (2.0221 atmospheres or 2.089 kgjcm2); at this pressure, the boiling point of water is l2loC, a temperature at which all microorganisms are killed within 20 minutes. Autoclaves are of two general types - those equipped with a reducing valve, for attachment to a central source of high pressure steam, and those equipped with self-contained steam-generating units, energized by electricity or cooking gas.
A wide range of materials and equipment may be sterilized by autoclaving, including solutions and articles of paper, cloth, glass, metal, and certain kinds of plastic. A second advantage of the autoclave is its capacity; all but the smallest will admit mouse cages, for example. Another advantage of many autoclaves is that they are capable of drying by a vacuum process; articles sterilized by autoclaving become wet in the process, and after sterilizing, it is desirable to dry such materials as cloth and bedding for cages.
Generally, exposure to l2loC for 15 - 20 minutes is sufficient for sterilization. However, one must pay particular attention to the autoclaving process. It is not suffi cient simply to place the materials to be sterilized into the autoclave, close the door, and admit steam to achieve the desired pressure and temperature for the time indicated. Likewise, the use of a sterilization indicator, such as Time-TapeR, on the outside of the container does not guarantee that the conditions necessary for sterilization have been met. Steam may have difficulty penetrating thick or securely closed wrappings, and may be blocked from entry by air trapped in the material - especially bedding - being sterilized. It is important to:
(1) use wrappings and closures that are readily penetrated by steam; (2) evacuate the autoclave before introducing steam; (3) place sterilization indicators within the materials being autoclaved. Certain indicators, such as Temp-TubesR, may be immersed in water to be autoclaved, and filter-paper strips impregnated with spores of Bacillus sp and sealed in plastic film (Spore-StripsR) may be used to monitor sterilization by any method.
Hot-Air Sterilization
Hot-air sterilization (1800C for 3 hours) is less damaging than autoclaving. It is suitable for sterilizing dry articles, and the equipment is less expensive and simpler to WHO/CDS/LEP/86.4 page 14 operate than is the autoclave. On the other hand, hot-air sterilization is more time consuming, and is less certain than autoclaving to kill spores and certain viruses. Moreover, hot-air sterilization may not be used for solutions and media, which will simply boil away.
Ethylene Oxide Sterilization
It is sometimes necessary to sterilize materials - e.g., certain plastics, drugs and chemicals in crystal or powder form - that do not tolerate the moisture or heat of autoclaving. For articles of small size, exposure to ethylene oxide may be the ideal method of sterilization. Several small pieces of apparatus are commercially available for sterilization with ethylene oxide. An advantage of this method is that ethylene oxide will penetrate paper and thin polyethylene films, so that articles may be wrapped before sterilization, and afterwards be maintained sterile indefinitely. A disadvantage of this method is the time required - several hours, as a minimum, and sometimes overnight, depen ding on the nature of the wrapping. It is as important to monitor sterilization with ethylene oxide as it is with steam.
Irradiation
Sterilization by exposure to gamma rays (usually from a 60Co source) is widely used industrially. Most disposable sterile syringes, needles, surgical gloves, and surgical dressings being marketed currently have been sterilized in this manner. A great advantage of this method is its minimal effect on the articles being sterilized. The great disad vantage, of course, is that irradiation facilities are not available everywhere. However, because 60Co is long-lived, and commonly used as a source for therapeutic irradiation, the necessary radiation facilities may be more generally available locally, in industry and in hospitals, than is commonly supposed. The researcher should inquire in his own area. The dose of irradiation required for sterilization is 2.5 Mrad.
Other Methods
Immersion in water boiling at 1000C (at sea level) for 20 minutes will kill virtually all microorganisms, including almost all spores and viruses; however, this method is only suitable for sterilizing those pieces of equipment that will not be damaged by the proce dure. "Cold sterilization" may be accomplished by immersion in certain antiseptic solu tions, such as formalin or glutaraldehyde (CidexR); this method is particularly suitable for certain optical and surgical equipment (e.g., cystoscopes), but has very limited usefulness in the microbiology laboratory. Finally, solutions that are damaged by heat may be sterilized by filtration through sterile membrane filters with a pore-size of 0.22 ~m. WHO/CDS/LEP/86.4 page 15
CHAPTER 2 - MICROSCOPY
The Microscope
One of the most commonly used pieces of laboratory apparatus, the microscope is sometimes not understood by its users. An ill-adjusted, badly-illuminated microscope can give completely misleading information. For this reason, a discussion of microscopy must begin with a description of the microscope (Fig. 3).
The modern microscope is "compound" - that is, it consists of a system of lenses so arranged as to project an enlarged image onto the retina. Light, preferably from a built in substage illuminator, passes, in order, through diaphragm, substage condenser - itself a system of lenses, the object to be examined, objective lens, and ocular lens.
All systems of lenses possess some defects. The lenses have spherical surfaces, and spherical surfaces cannot form a perfect image. Certain defects -"aberrations" - are inherent. Spherical aberration results from the fact that the outer portion of the spher ical surface has more power than the central portion. As a consequence, when the central portion of the microscope field appears to be in focus, the outer portion will not. Chromatic aberration results because the spherical lens behaves as a prism, refracting light of shorter wave-length (blue) more than that of longer wave-length (red). As a consequence of chromatic aberration, the image is focussed at a different level for light of each colour. By judiciously combining lenses of different shapes, sizes and types of glass, manufacturers have generally been able to counteract defects of one surface by introducing equal but opposite defects on other surfaces. This, in all likelihood, explains why, in general, the more nearly perfect the lens system, the more expensive it is. Objective lens systems characterized as "p1ano" have been maximal1y corrected for spherical aberration; those characterized as "apochromatic" have been maxim.s.l1y corrected for chromatic aberration. An apochromatic objective should be used with "compensating" ocu1ars, and an achromatic substage condenser with "swing-out" lens and numerical aperture (NA) matched to that of the objective.
For the purpose of observing M. leprae, the optimal lens system consists of 12.5 x wide-field compensating ocu1ars, apochromatic 100 x objective with NA approximately 1.30, and an achromatic condenser also with NA 1.30. During use, immersion oil is placed both between the objective and the upper surface of the slide, and between the undersurface of the slide and the swing-out lens of the condenser~ This system permits the maximal poss ible magnification and resolution when all other conditions are optimal.
Illumination
In order to obtain maximal optical resolution, Kohler illumination must be employed. By this method of illumination, the diameter of the light beam is so regulated that, when focussing on the object, the image of the field or lamp diaphragm coincides with the field viewed by the objective. Koh1er illumination is accomplished by first raising the conden ser until the swing-out lens has contacted the oil-drop on the underside of the slide. Then, one focuses on the object, employing the 100 x oil-immersion objective with oil both above and below the slide, The field diaphragm is then closed, and the condenser is raised or lowered until the image of the field diaphragm surrounding the object is sharp. This image is then centred by means of the two centring screws on the condenser, and the field diaphragm is opened until the beam of light just fills the field.
Contrast is controlled by the condenser diaphragm. This diaphragm is adjusted while observing the back of the objective lens with an ocular removed. The condenser diaphragm is closed so that no more than two-thirds of the objective lens is illuminated. Bright ness is controlled by adjusting lamp voltage, and not by means of either field or conden ser diaphragm. Figure 3. Research microscope. 'g ~ (IQ 0 (1) ...... e') t-'t; Labelled are the following parts: "'en<, f;; "d 1) Eyepiece, slipped into upper end of tube. <, The engraved value, e. g., 12.5X, indicates the magnification a-00 of the eyepiece; multiplied by the magnification of the ~ objective, this gives the total magnification of the microscope.
2) Tube (an inclined tube for monocular observation also available) The tube is interchangeable. When the screw collar is loosened and the tube pressed against a spring in it, it can be lifted out.
3) Nosepiece for qulck exchange of objectives.
4) 'Objective designation Plan 40/0.65 160/0.17 means: objective magnification 40 x, numerical aperture 0.65
computed for mechanical tube length of 160 mm and 0.17 mm cover-glass thickness.
5) Specimen stage, here a square mechanical stage
6) Iris lever (condenser or aperture diaphragm).
7) Condenser (correct position is always just below the upper stop).
8) Lever for swinging out the condenser front lens, for illumination of larger object fields.
9) Knobs for centering the condenser (for Kohler illumination).
10) SWing-out holder for filters of 32 mm diameter.
11) SWing-out holder for centerable auxiliary lens.
12) Fine adjustment knob. On STANDARD microscopes this allows unlimited rotation; on other microscopes its rotation is limited by two stops (index beside the knobt Coarse adjustment knob.
13) Diaphragm insert with field iris diaphragm.
14) Base with built-in low-voltage illuminator (6v, 15w, 2.5 a). On the underside, tapped holes for screwing down in cabinet as well as two holes for mounting the connecting brackets of separate illuminators. WHO/CDS/LEP/86.4 page 17
Modern microscope-objectives are "parfoca1" - that is, having focussed on the object through one objective, one can substitute another objective without the need to repeat the focussing procedure. Nevertheless, a small adjustment of focus is usually necessary when substituting, for example, the 100 x for the 10 x objective, and it is usually also neces sary to adjust the centring.
Micrometry
In order to calculate the number of M. leprae present in a measured drop placed on the slide, when only a portion of the resulting "spot" or "circle" is to be examined, it is necessary first to measure the diameters of the area on a slide and of the microscope field.
DIAMETER OF AREA ON SLIDE
On modern microscopes, the movable stage is equipped with two scales graduated in millimetres, one along one side of the stage, and the other at right angles to the first. Both scales are usually equipped with a vernier (Fig. 4), which permits measuring with accuracy to 0.1 scale division (0.1 mm). While making this measurement, the observer must be careful to take into account the diameter of the microscope field. Thus, if he locates one end of the diameter at the extreme right of the microscope field, he should then traverse the diameter until its opposite end comes to rest at the same point - at the extreme right of the microscope field.
Figure 4. Schematic drawing of a vernier scale.
;ointer
1 ~ ~ t ~ ~ I 8 ~ I I I I I I T 101 lb2 103 104 105 lb6 107 I ) I i 'i WHO86633 I : ~- 4 division ..I Coincidence
For example, in measuring the diameter of a circle on a counting slide of the type employed by Shepard (see p. 63), one places the upper (as seen through the microscope) inner margin of the ceramic circle just at the upper margin of the microscope field, and notes the position of the movable stage as shown on the scale at the side of· the stage (see Fig. Sa). One then moves the stage until the lower inner margin of the ceramic circle comes to rest just at the upper margin of the microscope field, and again notes the position of the movable stage (Fig. Sb). The diameter of the circle is simply the differ ence between the two measurements. WHO/CDS/LEP/86.4 page 18
These measurements of the position of the movable stage are made more accurate by use of the vernier with which the graduated scales of the stage are equipped. A length of the vernier equivalent to nine divisions of the graduated scale is divided into ten equal parts. When the pointer of the vernier rests exactly opposite a division of the scale,
Figure 5. Measurement of the diameter of a circle on a counting slide with one of the micrometers 'on the edge (usually one side, and at the back) of the stage.
Panel a: The starting point is found by placing the edge of the microscope field so that it exactly overlies the inner edge of the black ceramic material, and the measurement, in this example, of 20.4 mm is recorded. Panel b: Having traversed the length of the diameter of the circle, one stops when the same edge of the microscope field overlies the inner, opposite edge of the circle, and the measurement of 31.8 mm is recorded. The diameter of the circle is the difference between the two measurements - 11.4 mm.
0 10 20 30 ~O 50 60 70 80 0 10 20 30 ~O 50 60 70 BO 1,,,1 ",I, . 1. ! , .1 .1 't".I,,,.I, ! . "I., .'I.. , ,1. ,1 ..• 1. ,,1 .. 1 ,I."." I" .,!""Ip"I! "".I 1".",.1.. "r hl'i Wo 5 10 lIDQ]
a b
the measurement is easy. However, when the pointer rests between two divisions, one may estimate "by eye" the fraction of the distance between the two divisions indicated by the pointer. But this fraction is shown with much greater accuracy by that division of the vernier which rests exactly opposite any division of the scale. In the example in Fig ure Sa, the pointer of the vernier rests between 20 mm and 21 mm on the scale; only the fourth division on the vernier coincides with a division on the scale. This indicates that the pointer of the vernier rests at 20.4 mm, which is the desired measurement.
DIAMETER OF MICROSCOPE FIELD
For many purposes, including that of counting M. leprae by Shepard's method, it is important to know the diameter of the area "seen" by the microscope when any system of ocular and objective lenses is used. The area will certainly differ from system to sys tem; the smaller the degree of magnification, the larger will be the area. The area may also differ from one lens to another, even when the lenses have the same nominal magnifi cation. WHOjCDSjLEPj86.4 page 19
The measurement is made with the assistance of a "slide micrometer", a slide on which has been etched a graduated scale (Fig. 6a). Typically, the scale (Fig. 6b) is 2 mm in length, and has been divided so that the distance between two marks is 0.01 or 0.02 mm. To carry out the measurement, the slide is positioned on the movable stage, so that one mark rests exactly at the margin of the microscope field. One then counts the number of divisions between this point and that opposite it in the microscope field, at which the scale disappears from view because it is not within the field. It will usually be necessary to estimate the distance between the last mark before the scale disappears, and the point on the scale at which it actually disappears; this can usually be done to the nearest 0.002 mm, when employing magnification of 1250 x (12.5 x oculars, 100 x objective). In the example shown in Figure 6c, the diameter is slightly larger than 14 parts (0.14 mm), and is estimated to be 0.144 mm. The orientation of the scale in the microscope field is important, if one is to measure the diameter and not a shorter chord. One can ensure measurement of the diameter by varying the placement of the slide until the measurement becomes maximal. With some microscopes, the magnification varies with the interpupillary distance, which differs among observers.
Care of the Microscope
The microscope must be kept clean. Difficulties in obtaining a sharp image of the field diaphragm may frequently be traced to a dirty lens. After use, the objective lens and the swing-out lens of the condenser must be wiped free of oil. This is best accom plished, without danger of scratching the lenses, by lens paper. The top surface of the ocular lens is exposed to dust, dander and mascara; examination through a hand magnifying lens will reveal its state. A few well-directed, light brushes with a camel's-hair brush may be sufficient to clean it, but greasy particles may require careful use of lens paper, which has been moistened with xylol when necessary.
The microscope must be kept free of dust. This is best accomplished, when the micro scope is not in use, by keeping the tubes of the microscope closed with oculars or dust plugs, and by placing a hood over the microscope.
In tropical climates, lenses are sometimes ruined by growth of fungus within the lens. In some laboratories, this is prevented by placing the entire microscope in a large desiccator; in others, the microscope is stored in a small cupboard that is continuously heated by means of a 40 W incandescent bulb. WHO/CDS/LEP/86.4 page 20
Figure 6. a. Stage micrometer, approximately one-half actual size. b. Stage micrometer scale, enlarged approximately 35 times. c. Stage micrometer scale, as seen through the microscope in the course of measuring the diameter of the microscope field. To show the scale more clearly, the micrometer has been moved slightly, so that it is not in its final position. To make the measurement, the micrometer should be moved so that the line representing 0.00 (that nearest the top of the figure) comes to lie exactly under the upper (in this example) edge of the microscope field. Once this has been accomplished the field diameter will be seen to measure 0.144 mm in length.
"~1~2~J~~S"~7M~1~'.lUt31A151~1.71Jl~~_ 0,1 mm b. a. e O,Olmm I ililiiliiiillliilllillllllllllllhl~lIi!illliliillllllil!lIlllliililliilllllllllllllilllllIIl1illll~lIIll1l1ifllll!IIIIIii:!lllliIlIIIIlIlI1l91I1!Mml~lIIilllljll!!11l1l11l1l11ll1[llIIjllll! '------I
----0.05
0.10 WHO/CDS/LEP/86.4 page 21
CHAPTER 3 - THE SMEAR
Laboratory investigation of the patient with leprosy, whether for diagnosis, classi fication, or monitoring response to treatment, depends primarily on the examination of smears and tissue specimens. The subject of tissue specimens will be dealt with in Chap ter 4. Smears are prepared of material scraped from the edges of a small slit incised into a skin lesion and, sometimes, into normal-appearing skin. Smears are also made of nasal secretions, obtained by swab, by washing the nose and collecting the sediment by centrifugation, or by having the patient blow his nose. These smears are subsequently fixed, stained and examined under the microscope. Detection of AFB and assessment of their number and morphological appearance requires a carefully standardized acid-fast stain and skilful use of an adequate microscope.
Smears of scrapings from skin lesions stained for AFB are obtained for the purpose of measuring the BI, a method for estimating the total number of M. 1eprae, both viable and dead, in the patient's lesions, and the MI, also termed the solid ratio, a method for estimating the proportion of viable organisms in the patient's bacterial population (see pp. 25 - 29).
Involvement of the nasal mucous membranes of patients with lepromatous leprosy has been recognized since the writings of Danielsen, Boeck and Hansen (33, 72). After the advent of modern chemotherapy, rapid clearing of nasal lesions was observed to follow institution of therapy. Subsequently, Shepard demonstrated the presence of viable M. 1eprae in the sediments of nasal washings from untreated patients, and the progres sive disappearance of organisms from the sediments in response to effective treatment (185, 195). More recently, Rees and his co-workers have demonstrated that organisms were present in the nasal secretions obtained as "nose blows", and that M. 1eprae disappeared from the nose blows in the course of therapy (81).
In this chapter, the techniques of smears of skin scrapings and "nose blows" are described, preparation of the smear for examination is considered at length, and the techniques for measurement of the BI and MI are presented.
Sites for Smear
MULTIBACILLARY LEPROSY
In previously untreated patients with multibacillary leprosy (BI ~ 2 at any site), coalescing papules, plaques or areas of heavily infiltrated skin are usually present. These are typically situated over the face and extensor surfaces of the trunk and limbs; in general, the most suitable sites for smears are to be found in the lumbar and lower thoracic areas of the back, the middle and upper parts of the arms, and over the extensor surfaces of the thighs. Because infiltration of the ear lobes is very common, these are among the most suitable sites for smear. Thus, smears are generally taken from six sites: both ear lobes, and four active skin sites on extensor surfaces, such as the arm, back (the lumbar region is usually the most suitable) and thigh.
For previously treated lepromatous patients who appear to have responded to therapy, smears should be made from sites previously found to be positive, lesions, both ear lobes and representative areas of skin. In dapsone-resistant patients, relapse lesions may be scattered and in unusual sites; it is essential to choose lesions that are clinically active.
The claim has been made (91, 92, 163) that, in long-treated lepromatous patients, the skin sites at which AFB are most frequently detected by smear are the dorsa of the fingers, and that smears from these sites give the earliest indication of impending relapse. WHO/CDS/LEP/86.4 page 22
PAUCIBACILLARY LEPROSY
Many patients with paucibacillary leprosy (BI ~ 1 at all sites) may have only one or two skin lesions, with the result that the choice of smear sites is severely limited. In general, clinically more active lesions are preferred to less active lesions. Skin smears should be taken from a minimum of three sites, including one ear lobe and two representa tive active skin lesions. When the chosen lesion is annular, the smear should always be taken from the active raised rim, near the outer edge. Similarly, for a macule or a plaque, it is also preferable to take the smear from just inside the edge of the lesion. Should there be only a single skin lesion, two smears may be taken from its active edge at sites diametrically opposite one another.
Smear Techniques
SMEARS OF SKIN SCRAPINGS
Skin smears should always be taken in a good light, so that the lesions can be easily seen, and their colour and activity evaluated. It is essential for both the patient and the operator to be comfortable. This can easily be achieved by both sitting on identical stools.
Equipment required includes a spirit lamp and spirit, sterile cotton-wool swabs and sterile gauze squares in metal containers, a pair of surgical rubber gloves for the opera tor, sterile scalpels, glass microscope slides, slide box, and a diamond pencil (if the slides are frosted at one end, a graphite pencil is sufficient). The shape of the scalpel blade depends on the operator's preference; the blade must be sharp, and disposable surgical blades are preferred. Sterile scalpels can be easily and safely transported, if each one is autoclaved in a test-tube with the point of the blade resting on a pad of cotton-wool, and the end of the handle projecting through the middle of a cotton-wool plug at the mouth of the tube.
The skin area chosen for the smear is cleaned by rubbing briefly but vigorously with a small cotton-wool swab dipped in spirit, and allowed to dry. Next, the skin is pinched up into a fold between the index finger and thumb (of the left hand of a right-handed operator). To stop or minimize bleeding, enough pressure should be applied so that the skin-fold blanches after a few seconds; if it fails to do so, excess blood may be massaged away by rubbing a few times with a sterile gauze square in one direction along the crest of the fold. Then a cut about 5 mm long, and deep enough (about 2 mm) to penetrate into the infiltrated layer of the dermis, is made with the sterile scalpel. If blood or tissue fluid exudes, it should be wiped off with a dry cotton-wool swab. Next, the blade of the scalpel is turned transversely to the line of the cut. Using the blunt edge of the blade, the side of the cut is scraped two or three times, sufficiently firmly to obtain a little tissue pulp from below the epidermis. This material is then transferred on the tip of the scalpel blade to a glass microscope slide, where it is spread in a circular motion by the flat of the blade to produce a uniform and moderately thick smear over an area 5 to 7 mm in diameter. Two to four smears from the same patient may be placed on a single slide; the slides must be carefully labelled. The cut may be either compressed with a small dry piece of cotton-wool until oozing stops, or dressed with a small sterile dressing.
Between each smear from the same patient, the scalpel blade is wiped on a cotton-wool swab or gauze square, and re-sterilized by flaming in the spirit lamp. If the same scal pel must be used for more than one patient, it is essential for the blade to be carefully wiped with spirit and flamed before proceeding to the next patient.
Good quality smears can usually be prepared from the lesions of new tuberculoid patients. When lesions are in reaction, however, the dermis may be both desquamating and very friable; the rather copious, thin tissue fluid must be wiped off before the side of the cut is scraped. Treated lesions are likely to be flat, with the skin thin and atro phic, so that scraping the side of the cut yields very little tissue to smear on the microscope slide. WHO/CDS/LEP/86.4 page 23
SMEARS OF NASAL SECRETIONS ("NOSE BLOWS")
Smears of nasal secretions are best prepared from early morning nose blows. The patient blows his nose thoroughly into a clean, dry, small sheet of plastic film. Either the smear may be made beside the patient, or the plastic may be folded, labelled and sent to the laboratory in a sterile container.
To make the smear, an aliquot of the nasal discharge is transferred to a labelled glass microscope slide, and spread as evenly as possible. If the discharge consists of both watery and opaque material, the latter should be selected. A platinum loop is not rigid enough to spread thick nasal discharge, especially if crusts are present. It is better to use a small cotton-wool swab, slightly moistened in normal saline and held by f,orceps.
TRANSPORT OF SMEARS
If a smear of skin-scrapings or of nose blows is obtained in the field, and must be transported to a laboratory, it should first be fixed (v.i.). Smears fixed in the field and clearly labelled are placed carefully in slide boxes for transport to the laboratory. Slide boxes are available in a range of sizes, and some are quite suitable for mailing.
Preparation of the Smear for Examination
Because of the importance of the smear to leprosy control and research activities, the fixation and acid-fast staining of smears must be carried out with utmost care, to ensure reliable and standard interpretation of the specimens.
FIXATION OF SMEARS
Fixation both kills the H. leprae present in the smear and preserves the structure of the organisms and cells, serving to protect the smear from damage during handling. Smears are most commonly fixed by heating. However, heat-fixation is difficult to con trol, and excessive heating, as may occur if the smear is held over the flame of a bunsen burner, can affect adversely the acid-fastness of H. leprae. Smears of skin scrapings and nasal secretions should first be allowed to dry, preferably under cover of a Petri dish, immediately after which they should be fixed before further handling.
In the field, fixation is most readily accomplished by placing the smear in an atmo sphere of formalin fumes. The smears are placed for at least 15 minutes in a Coplin jar (Fig. 7) containing enough formalin (37-40% formaldehyde) to cover the bottom of the container. If the smear is obtained near the laboratory, fixation by formalin fumes and heat is preferred. An acceptable standard method for fixation in formalin fumes is the following. Air-dried smears are:
(1) exposed to formaldehyde fumes for three minutes; (2) heated (on the lid of a boiling water bath or on an electric hot-plate set at 600C) for two minutes; (3) covered with gelatin-phenol (added with a pipette); the slide is then drained momentarily; (4) heated for two minutes; (5) exposed again to formaldehyde fumes for three minutes; and, finally, (6) heated for two minutes. WHO/CDS/LEP/86.4 page 24
Figure 7. Coplin jar, for staining and other procedures on microscope slides.
ACID-FAST STAINING
Because the staining quality of H. 1eprae is compromised by uncontrolled heating, staining with an acid-fast stain is best accomplished by staining at room temperature, rather than by the traditional Ziehl-Neelsen technique. The slides (not more than four at a time) are flooded with freshly-filtered carbolfuchsin and allowed to stand for 20 min utes. After the first 10 minutes, fresh stain is added to replace that lost by evapor ation. The slides are then washed gently in tap water, placing them in a beaker with the smear away from the stream of water. Then, the slides are allowed to stand wet until destaining, which is done, one slide at a time, by gently streaming 1% HCl in 70% ethanol over the slide until the destaining fluid is colourless. As soon as destaining is com pleted, the slides are washed in tap water and allowed to stand wet until counterstaining, which is done with methylene blue for one minute. (Preparation of stains is described in Appendix 4.)
PERIODATE OXIDATION
It has been claimed that exposure of H. 1eprae to periodic acid greatly increases the number of organisms that can be stained by the acid-fast stain. This property has not been described as one unique to H. 1eprae, but has also been reported to be characteris tic of H. tuberculosis (135, 136) and the cultivable "NQ bacillus" (73). However, other studies suggested that stainability with the acid-fast stain only after periodate oxidation may be a property of dead or senescent H. 1eprae, somewhat like pyridine extractability (v.i.). The numbers of AFB counted in bacterial suspensions prepared from multibacillary patients who had received effective treatment were some 30% greater than those counted in duplicate preparations not subjected to periodate oxidation. On the other hand, although prior periodate oxidation resulted in brighter and more uniform staining of the AFB, the numbers of H. leprae counted in oxidized preparations made from the footpads of mice during logarithmic multiplication were not different from those in preparations not subjected to oxidation (107).
Periodate oxidation is carried out by immersing formalin-fume-fixed smears of H. 1eprae-containing suspensions in a freshly prepared 10% (w/v) aqueous solution of periodic acid for 4 - 24 hours at room temperature, rinsing with water, air-drying and staining by the standard acid-fast stain. WHO/CDS/LEP/86.4 page 25
The Bacteriological Index
The BI represents an estimate of the size, in relative terms, of the patient's bac terial population. The BI is high in untreated patients with lepromatous and borderline lepromatous leprosy, and low in patients with tuberculoid leprosy. During effective treatment of multibacillary leprosy, the BI decreases, at a rate of about 90% (1 BI unit) per year for lepromatous patients (159, 185), and perhaps more rapidly for borderline lepromatous patients (159).
The BI is measured by estimating the concentration of AFB in a number of microscopic fields (magnification approximately 1000 x, employing oil immersion) of smears taken from four to six representative sites on the body. Reliable quantitative estimates of the concentration of AFB in smears are best obtained by employing Ridley's logarithmic scale (160). The concentration of AFB in a smear is scored after examining 25-100 representa tive fields by a number ranging from 0 to 6, each number representing, on the average, 10 times as many organisms as the number smaller by 1, as shown in Table 1. When the BI is 1+ or 0, at least 100 fields must be examined. The BI for the patient is the average of the BIs at the individual sites.
To carry out the measurement, a drop of immersion oil is placed on the smear, and it is examined first under low power. Erythrocytes should be few.
Table 1. Ridley's logarithmic scale for scoring the BIl
Average number of AFB BI
>lOOO/field 6+ 100-1000/field 5+ 10-100/field 4+ l-lO/field 3+ 1-10/10 fields 2+ 1-10/100 fields 1+ 0/100 fields 0+
If possible, areas with well scattered macrophages are selected for examination under immersion (100 x objective). About three to five macrophages per oil-immersion field is ideal. From new lepromatous patients, very good smears can be prepared, but after sane years of treatment it may become very difficult to obtain a good spread of cells in the smear.
The Morphological Index and the Solid Ratio
A number of leprosy workers, beginning with Hansen himself (72), had called attention to the variable morphology of M. 1eprae, even in untreated patients. And after effective chemotherapy became available, Muir (129), Lowe (117) and Davey (34) called attention to the morphological changes of M. 1eprae that accompanied effective treatment of patients with lepromatous leprosy. Rees and his co-workers (154, 155) inferred, from electron-microscopic observations of M. 1eprae and other organisms, that morphologically imperfect M. 1eprae were, in fact, dead. Subsequently, Waters and Rees (211) proposed the quantitative assessment of bacterial morphology as a technique for
1 Taken from reference no. 160. WHO/CDS/LEP/86.4 page 26 measuring the response of lepromatous patients to treatment with an antimicrobial drug (Fig. 8).
Figure 8. Changes of the mean BI and MI as a function of the duration of dapsone mono therapy and patients' classification. The BI for the patients with LL (mean of 32 patients) may be seen to be higher initially, and to fall more slowly than that for patients with BL leprosy (mean of 7 patients). For both groups of patients, the MI falls much more rapidly than does the BI.2
60 ,. -' '0 , l.L
-='.J , .. , ~.O ., \ o , ~ ~O , , :< \ ... .---.. BACTtR.IAL .. , \ rx ocx > \ , ... -' o" :2 , , a: lO ~ o , \ ... ..J \ \ z e l ).0 ~ :> \ \ ';. ...0: \ \ l V ~ 20 .. ' \ ...... \ , ., I- \ \ ..Z 2.' u \ ' 0- _ -0 UNlrOR"''-'t sT ....INeD SAC'L!-I a: '~ ... , "- .. 10 \ " a..- "-,,- 2.0 -- ...... "-~ ====.t: ==:::: =:------
• , 12 IS 10 PIRIO() Of TRI,A.T'IEHT ("ONTHS)
Shepard and McRae (121, 191) subsequently demonstrated correspondence between the solid ratio and the proportion of organisms in a suspension of H. 1eprae that was cap able of multiplying in the mouse footpad. Their demonstration rested on two lines of evidence. First, as shown in Figure 9, the generation time of H. 1eprae in the mouse footpad was reasonably constant for inocula with widely varying solid ratios, when calcu lation of the generation time was based on the number of solid organisms rather than on the total number of organisms inoculated into each footpad. Second, as may be seen in Table 2, examination of the infectivity in the mouse footpad of serially-diluted inocula (all prepared from human skin biopsy specimens with small proportions of solidly-stained organisms) of H. 1eprae revealed that the minimal number of organisms required to initi ate multiplication was between 50 and 500, in terms of the total of solid plus non-solid organisms, but as small as 3 to 40, in terms of solidly-stained organisms.
2 Taken from reference no. 211. WHO/CDS/LEP/86.4 page 27
Figure 9. Gs the doubling time, in mice, of H. 1eprae isolated from a number of speci mens, calculated from the number of solidly stained AFB in each inoculum) assuming that multiplication proceeded at a constant rate from inoculation to harvest.
o BIOP', • Mouo. PouoQe · u I I , ... . ._ 0 • -· ,... .-
o 10 la 40 S/(N.SI (~l
Although the terms "morphological index" and "solid ratio" have much the same mean ing, they are by no means identical, as is evident from a comparison of the data in Table 2 with those in Figure 8. Thus, the MI of 32 previously untreated patients in Malaysia ranged from 8 to 88%, with a mean of 54% whereas the solid ratios of the four patients whose biopsy specimens were studied in Atlanta were 3 to 4 solids per 50 organ isms examined. Other experience has suggested that the MI of H. 1eprae recovered from skin-biopsy specimens of patients with previously untreated lepromatous leprosy varies widely from laboratory to laboratory, and may be greater than 50%, whereas the solid ratio of H. 1eprae recovered from similar specimens varies over a much narrower range, and values greater than 10% for such specimens are rare.
The solid ratio, as defined by Shepard and McRae (121, 191), is measured under optimal conditions - formalin-fume fixation; carefully standardized, room-temperature staining procedure; 1250 x magnification, employing compensated 12.5 x oculars and a 100 x apochromatic objective; immersion oil both above and below the slide; K6hler illumination; and a rigid definition of a solidly-stained organism. The MI, on the other hand, is generally measured under somewhat less favourable conditions, which may well vary from laboratory to laboratory. Organisms that appear solid under less-than-optimal conditions may be recognized to be non-solid when examined under more favourable conditions. As a result, the MI may overestimate the proportion of viable H. 1eprae. Indeed, the solid ratio, which should be reproducible from one laboratory to another, appears to correspond much more closely to the proportion of viable organisms, as measured by inoculation into mice of serially-diluted suspensions of H. 1eprae (214).
Shortcomings of these morphological measurements are illustrated by the results of clinical trials, in which morphological measurements were employed to determine the effi cacy of the chemotherapy. A series of trials of chemotherapy among patients with leproma tous leprosy at the Sungei Buloh Leprosarium, reported between 1962 and 1967, demonstrated the regular decrease of the MI during effective chemotherapy. However, the rate of decrease of the MI did not discriminate clearly between more and less effective chemother apeutic regimens. Thus, by this criterion, dapsone, 50 mg twice weekly, appeared as
3 Taken from reference no. 191. WHO/CDS/LEP/86.4 page 28
Table 2. Titration of infectivity of H. 1eprae in four inocula4
Inoculum Solid No. AFB No. solids No. AFB ratio inoculated inoculated harvested5 (x 10 5)
1 3/50 5000 300 12.4 500 30 18.9 50 3 6
2 4/50 5000 400 4.70 500 40 11.9 50 4
3 4/50 5000 400 10.8 500 40 7.97 50 4
4 3/50 5000 300 5.52 500 30 8.10 50 3 1/87
effective (142) as the drug administered in a daily 100 mg dose (207). And clofazimine, 100 mg twice weekly, appeared as active (208) as clofazimine 300 mg daily (143). On the other hand, Shepard and his co-workers (188) showed that, whereas the solid ratio changed at the same rate during treatment with rifampicin 600 mg daily or dapsone 50 mg daily, killing of H. 1eprae as shown by mouse foot-pad inoculation was much more rapid during treatment with rifampicin than during treatment with dapsone.
Thus, the MI and the solid ratio are useful to demonstrate the response of the indi vidual patient with untreated or relapsed lepromatous leprosy to effective chemotherapy. But as criteria of efficacy of a chemotherapeutic regimen for use in clinical trials, these measurements lack sufficient sensitivity. The reasons are the following. First, the morphological changes may lag significantly behind bacterial killing (188). Second, it is not feasible to examine many more than 100 individual organisms in the course of the measurement. Because the average untreated patient with lepromatous leprosy presents with a solid ratio no greater than 10 per 100 AFB, the solid ratio will fall to 0 per 100 AFB after no more than 90% of the viable organisms have been killed, at a time when the patient's organisms are still capable of infecting mice. Finally, assessment of bacterial morphology is standard only under the optimal conditions described for measurement of the solid ratio - conditions that cannot easily be met by many laboratories, because of the high cost of the equipment and the high level of technician-training required.
4 Adapted from reference no. 121. 5 Harvests were performed 203 - 327 days after inoculation. 6 No multiplication noted. 7 Multiplication noted in only one of eight mice harvested. WHO/CDS/LEP/86.4 page 29
MEASURING THE SOLID RATIO
The smear has been fixed by formalin-fumes, and stained by the standard procedure described. The microscope lenses are clean, and consist of 12.5 x compensated oculars and a 100 x apochromatic objective or its equivalent. The slide is placed on the stage, drops of immersion oil are placed both on the slide and on the uppermost, swing-out lens of the sub-stage condenser, and K6hler illumination is achieved.
When these conditions have been met, examination of the smear is begun. Only organ isms, the entire outline of which can be seen, are considered. Organisms that are touch ing or superimposed, one on another, are ignored. Solid bacilli must not only be uni formly stained; they must also be intensely stained, and their length at least four times their width. Uniformly stained organisms that are pale are considered to be non-solid. In examining a smear in which many non-solid organisms are encountered, the examiner must take care to score as the first solid organism only one that is uniformly and intensely stained; otherwise, he may tend to score as solid those organisms that are uniformly stained but pale. Finally, one may occasionally encounter an organism that is not uni formly stained, but is nevertheless intensely stained, and would be considered solid if the organism were uniformly stained with .the intensity of the least intensely-stained portion; such an organism should be scored as a solid. The examination is continued until at least 50, and preferably 100, organisms have been scored, a careful count being made of the solids and non-solids in the course of the examination. The solid ratio is the ratio of solids to the total of organisms examined.
The measurement of the solid ratio is not a rough estimate. Measurement of the solid ratio under these standard conditions is so demanding a procedure that few laboratories will wish to undertake it. On the other hand, attempting the measurement under less stringent conditions, as is the practice in many laboratories, can only lead to confusion, and should be discouraged.
It must be recognized that measurement of both the BI and the MI depend upon the examination of tiny fractions of the patient's bacterial population. Thus, because of large sampling error, one may expect considerable variation from measurement to measurement. Moreover, a BI of ° does not mean that the patient no longer harbours M. leprae, nor does a solid ratio of ° indicate that he no longer harbours viable organisms. WHO/CDS/LEP/86.4 page 30
CHAPTER 4 - THE BIOPSY
Biopsy is one of the most important techniques employed in leprosy work. Histopatho logical examination of the tissue is of crucial importance to differential diagnosis and classification. And the results of animal inoculation are of great value in the detection of drug resistance, and in monitoring the patient's response to antimicrobial chemother apy. Tissues, most often those of a skin lesion, but sometimes of peripheral nerve or another tissue, are biopsied. The biopsy specimens are fixed and further processed for histopathological examination, or the fresh tissue is employed as a source of H. leprae for animal inoculation. This chapter considers biopsy sites and techniques. Preparation of the biopsy specimen for histopathological examination, the histopathological examina tion itself, with emphasis on the Ridley-Jopling classification, and the measurement of the MI in tissue sections are considered in Chapter 5. Finally, employment of the biopsy specimen as a source of H. leprae for inoculation of animals is considered in Chapter 6.
Sites for Biopsy
The preferred sites for skin biopsy are those already described for smear (see pp. 21 _ 22). Those sites with a high BI are preferred. In addition, the individual patient's wishes should be taken into account; many patients prefer the lumbar region, and very few wish a biopsy from the face.
When the chosen lesion is annular, the biopsy specimen should always be taken from the active raised rim, near the outer edge. Similarly, for a macule or a plaque, it is also preferable to take the biopsy specimen from just inside the edge of the lesion. The lesion should be large enough to permit at least one follow-up biopsy.
Biopsy Techniques
SKIN BIOPSY
Two aseptic techniques are used for performing skin biopsies.
Incision Method
The skin of the biopsy site is carefully swabbed, first with an antiseptic, and then with surgical spirit, and allowed to dry. Next, 3 to 4 ml of local anaesthetic, either 1% lignocaine (lidocaine), or 2% procaine with 0.001% adrenaline, are infiltrated intra dermally in the chosen area. The site is then covered with a sterile surgical towel with a hole in the centre, and two to three minutes are allowed to elapse for the anaesthetic to be fully effective.
Using a sharp sterile scalpel, and with the skin slightly stretched between the index finger and thumb of the left hand (of a right-handed operator), two semilunar incisions are made to mark out an elliptical piece of skin. The longitudinal axis should be paral lel to the direction of tension in the skin. The size of the specimen depends upon whether the biopsy specimen is required for a single purpose (for which 4 x 12 mm will suffice), or for both inoculation of mice and histopathological examination (for which the specimen should measure 6 x 15 mm). In the latter circumstance, it is important to divide the tissue in situ, in which case a full-skin-thickness transverse incision is next made across the middle of the biopsy specimen, down to the subcutaneous fat. Then the semi lunar incisions are deepened down to the subcutaneous fat, care being taken to keep the scalpel blade at right angles to the skin surface. The elliptical specimen (or each of the two pieces separately) is lifted gently with sterile forceps, and the attaching sub cutaneous fat is cut off with the scalpel. The wound is sutured with two or three skin stitches, cleaned with the antiseptic solution, and dressed with a sterile gauze dress- ing. WHO/CDS/LEP/86.4 page 31
Punch Method
A 5 mm punch is used for a single biopsy, and a 6 mm punch when it is necessary to divide the tissue. The specimen should range in weight from 50 - 100 mg. After cleansing, anaesthetizing and covering the site, a sterile silk suture is inserted through the superficial layer of the corium in the centre of the anaesthetized area. The free ends of the suture are threaded through the centre of the punch, which is then firmly placed in the centre of the biopsy site, encircling the stitch.
Using a screwing motion, and keeping the instrument at right angles to the skin surface, the punch is forced down to the level of the subcutaneous fat. The punch is withdrawn, the biopsy specimen lifted by gently pulling on the stitch, and the attaching subcutaneous fat cut off with a scalpel or sharp pair of scissors. When two pieces of tissue are required, it is advisable to make first a superficial incision with the larger punch, which is then withdrawn. Then a single cut is made with a sharp scalpel across a diameter of the specimen, down to the subcutaneous fat. The punch is reapplied, and the superficial circular incision is extended down to the subcutaneous fat. The piece of tissue to which the thread is attached is removed first, and the second half is lifted gently with sterile forceps to allow the attaching subcutaneous fat to be cut off. A single skin suture is usually sufficient for a 5 mm biopsy, but two may be required when the larger punch is used.
NERVE BIOPSY
Of the named peripheral nerves commonly affected in leprosy, the radial cutaneous nerve at the wrist is most suitable for biopsy. It is one of the sites at which nerves frequently sustain damage, and it lies superficially, making the procedure technically straightforward. At this level, the nerve is composed of 10 or more bundles; the removal of one or two does not produce appreciable loss of function. Moreover, the nerve contains only sensory and autonomic fibres. Although biopsy might have an adverse effect on a named peripheral nerve already damaged by leprosy, experience has shown that the procedure is safe and unlikely to impair sensory recovery. However, nerve biopsy is to be performed only by an experienced surgeon, and with full justification.
Procedure
The skin is cleaned and draped with a sterile towel. The nerve is then located by palpation and infiltrated with local anaesthetic (preferably 2% lignocaine with added hyaluronidase) over and around a 1-2 cm segment, taking care to avoid the adjacent vein. About five minutes are allowed for the anaesthetic to penetrate the nerve.
Again the nerve is located, and a 1-2 cm incision is made directly over it, avoiding the nearby vein. The incision is deepened just into subcutaneous fat, and the nerve is located and separated from adjacent structures by very gentle blunt dissection. When the nerve has been isolated, a "V"-shaped piece of plastic or wax (sterilized by immersion in 70% alcohol) is inserted under the nerve (Fig. 10). A longitudinal incision is made through the nerve down to the wax, so that not more than 30% of nerve is separated (Fig. 11). The cut segment is then excised, using very fine-tipped forceps to steady the cut end of the nerve if necessary (Fig. 12). If the specimen has been obtained for histo pathological examination, the damaged nerve ends are trimmed off, the wound closed and the biopsy specimen is straightened on thin cardboard and fixed. WHO/CDS/LEP/86.4 page 32
Figure 10. Isolated exposed nerve, with wedge of dental wax slipped under it.
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Figure 11. A longitudinal incision has been made in the nerve. WHO/CDS/LEP/86.4 page 33
Figure 12. Separating the nerve biopsy-specimen.
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DARTOS MUSCLE BIOPSY
It has been stated (212) that a few viable M. 1eprae may be demonstrated in the dartos muscle, and that one may demonstrate viable organisms in this tissue by mouse inoculation, when it is no longer possible to demonstrate the organisms in skin biopsy specimens. Thus, biopsy of the dartos muscle may be useful. However, when there are psychological or cultural obstacles to this procedure, these should be considered.
This procedure may be undertaken in the same way as skin biopsy, with scalpel and stitches. Only small pieces of tissue are usually required, and these can be obtained conveniently by a biopsy punch 3 to 4 mm in diameter.
Procedure
The skin is cleaned and infiltrated with local anaesthetic. A fold of anaesthetized skin is taken, and, with gentle pressure and rotation, the punch is pushed through the fold of skin and into a soft pad (a piece of wax sterilized by immersion in 70% alcohol is suitable). The biopsy specimen will consist of a cylinder of tissue with epidermis at both ends. There is seldom much bleeding. Pressure is maintained at the biopsy site for a minute or two, after which a light dressing is applied. WHOjCDSjLEPj86.4 page 34
Preparation of the Specimen for Examination
STORAGE AND SHIPMENT OF SPECIMENS
It is sometimes necessary to obtain specimens for study at a site at some distance from the patient, whether a few kilometres by road or thousands of kilometres by air. Because the specimens most commonly obtained will be biopsy specimens for histopathological examination or animal inoculation, the procedures recommended for storage, preparation for shipping, and shipping will be confined to these specimens.
Biopsy Specimens for Animal Inoculation
In order to preserve the viability of the M. leprae contained in biopsy specimens taken for animal inoculation, and to prevent growth of contaminants, the fresh tissue specimen must be maintained at 0 - 40 c until its arrival in the laboratory. Therefore, obtaining the biopsy specimen must be related to airline flights in such a way that no more than seven, and preferably no more than five days elapse between performance of the biopsy and animal inoculation. Within this period of five to seven days, 48 hours should be allowed for processing the specimen and animal inoculation, once the specimen has arrived at the reference laboratory. The specimen should be shipped within 24 hours, if at all possible. When several suitable international flights are available, the biopsy specimen should be shipped on a flight arriving at its destination early in the week and never, when avoidable, on flights arriving at their destination near the end of the week or on the weekend.
Immediately following exc~s~on of the whole or the part of the biopsy allocated for animal inoculation, it must be placed without any preservatives in a suitable sterile container which can be sealed against leakage. Many such suitable containers are now available, either of glass or plastic, with screw-caps or snap-on, "leak-proof" lids. Plastic containers are preferred, as they are smaller and lighter than those made of glass, and are unbreakable. If the timing of the biopsy immediately precedes dispatch by air, the container should be placed directly into a suitable thermos flask with wet ice; the flask should be capable of preserving wet ice for at least 30 hours. If there is to be a delay, the container should be placed in a refrigerator at 0 - 4oC. To meet international air transport regulations, these fresh tissue consignments must be marked as "Diagnostic Specimens". Before shipment, customs regulations and quarantine requirements should be ascertained from airport and other authorities both locally and at the destination. To facilitate handling of the specimens at the destination, it is essential that the receiving laboratory be informed by cable of both the flight number and air-way bill number.
Biopsy Specimens for Histopathological Examination
Biopsy specimens to be processed for histopathological examination must be fixed prior to shipment, and must not be permitted to dry out during shipment. If there is only one specimen to be sent, it may be left in its bottle, with the identifying name or number on the outside of the bottle, along with the name and address of the sender. If there are several biopsy specimens, each specimen should be wrapped separately with its identifying name or number, and the specimens should be sent together in one bottle. For this pur pose, pieces of gauze 5 cm square and pieces of stiff paper 1 x 2 cm are cut. The identi fying name or number of the biopsy specimen is written on a piece of the paper with a soft lead pencil and placed together with the corresponding specimen on a square of gauze. Ink or a ball-point pen should not be used for writing on the paper, as these will wash out in the alcohol. The corners of the gauze are brought together and tied with with cotton to make a loose bundle, which is immediately placed in 70% alcohol in a suitable bottle with a plastic screw top or rubber stopper (never a metal cap). Fingers cut from a rubber glove should not be used as containers for specimens as these frequently leak. Sufficient alcohol is added to keep the gauze-wrapped tissues moist. The bottle top is firmly closed, and, if necessary, fixed with adhesive tape. The bottle is wrapped in corrugated WHO/CDS/LEP/86.4 page 35 cardboard to protect against damage, and the package completed according to postal regula tions. The parcel should be marked "Pathological specimens. No value", for customs purposes, and mailed to the reference laboratory by air mail.
If one cannot ship the biopsy specimen in a bottle, one may use a plastic bag that fits into an ordinary-sized envelope. The fixed tissue specimen is placed in a small plastic bag, together with a pledget of cotton-wool moistened with fixative and a label written in pencil for identification. The bag is then sealed with heat and placed in the envelope for mailing. Heat-sealing may be accomplished by means of an ordinary soldering iron or heated knife-blade. WHOjCDSjLEPj86.4 page 36
CHAPTER 5 - HISTOPATHOLOGICAL EXAMINATION AND CLASSIFICATION8
Fixation, Processing, Sectioning and Staining Tissue Specimens
FIXATION
The most satisfactory fixative for good preservation of cytological detail, and for producing sections that are easy to cut, is a modification of Lowy's fixative (118). The specimen should be placed in a small amount of the fixative (3 to 5 ml for a small piece of skin) for 2 hours. More prolonged fixation harms the tissue, and also makes sections more difficult to cut. At the end of this period, the specimen should be transferred without washing to 70% ethyl alcohol. It can then be left indefinitely for transport to a laboratory and processing.
If this fixative is not available, 10% buffered formalin may be used. Phosphate buffer (pH 7.0) should be used according to the directions. Marble chips may be used as a substitute for the buffer if necessary, but they are not so good. The specimen should be left in the formalin for transport to the laboratory.
PROCESSING
The examination of tissues with the microscope requires a slice (section) of tissue thin enough to transmit light. Usually, the tissue must undergo preparatory treatment, involving impregnation of the specimen with an embedding medium, to provide support and a consistency suitable for cutting sections. This preparatory treatment is known as process ing. The fixed tissue specimen is processed by exposing it to the solutions for the per iods of time indicated in Table 3.
Table 3. Processing schedules for fixed tissue specimens9
Duration of exposure Solvent Manual Automatic processing processing
70% alcohol 1 hr + 1 hr 95% alcohol 2 hr 2 hr Absolute alcohol 110 2 hr 2 hr Absolute alcohol 11 3.5 hr 3 hr Cedar wood oil I overnight 1 hr Cedar wood oil 11 2 hr Cedar wood oil III 2 hr Cedar wood oil IV 3 hr Xylol 20 min 30 min Paraffin I 3 hr 3.5 hr Paraffin 11 overnight 10 hr +
8 Except for the section on measuring the solid ratio in sections, this chapter has been contributed by D.S. Ridley. 9 Adapted from reference no. 162. 10 The Roman numeral indicates exposure to a fresh batch of the same solvent. WHO/CDS/LEP/86.4 page 37
SECTIONING
For cutting sections from skin biopsy specimens, a rotary or Cambridge rocker micro tome is preferable to a sledge. With a rotary microtome, a small knife of the Heiffor pattern is used rather than the more usual wedge knife -i.e., of the type normally supplied with the rocker. The weight of the rotary microtome makes it easier to cut serial sections in ribbons, although they are not necessarily of superior quality. On the other hand, sections that do not come off in ribbons are probably irregular in thickness. Sections are cut from dermis to epidermis with a thickness of 4-5 ~m for tissue stain ing, and of 6 ~m for staining the AFB.
STAINING
Haematoxylin and Eosin
The following method gives optimal nuclear detail, especially with the fixative recommended:
(1) the paraffin is removed by immersion for 2 minutes in each of two changes of xylol; then the xylol is removed, and the specimen "brought to water" by immer sing the section for 1 minute in each of two changes of absolute ethanol, after which the section is immersed for 2 minutes in 90% ethanol followed by 2 minutes in 70% ethanol; (2) the residual mercury pigment, resulting from the mercuric chloride, is removed by immersion for 3-5 minutes in a solution of 0.5% .iodine in 70% ethanol, after which the residual iodine colour is bleached by immersing the section in an aqueous solution of 2.5% sodium thiosulfate (Na2H2S03) for 30 seconds to 2 min utes. Finally, the section is washed in running tap water for 5 minutes; (3) the section is stained in celestine blue for 3 minutes; (4) the section is rinsed in tap water, and then distilled water; (5) the section is stained in Ehrlich's haematoxylin for 20 minutes; (6) the section is once again rinsed in tap water; (7) the section is decolourized (nuclei are "differentiated" from cytoplasm) by immersing the section for a few seconds in 1% HCl in 70% ethanol. Because acid changes the colour of the haematoxylin from blue to red, the section should be "blued" (i.e., the blue colour should be regained) by washing in running tap water for at least 5 minutes; (8) the section is next counterstained with eosin; (9) the section is rinsed in tap water; (10) finally, the section is dehydrated by blotting on bibulous paper and immersion in absolute ethanol, and the opacity of the section is "cleared" by immersing it in xylol. Then, the excess xylol is wiped off, and a coverslip is mounted with a suitable mounting medium.
Acid-Fast Stain
The paraffin is removed by placing the slides in a Coplin jar containing peanut oil and xylol (1:2) for 12 minutes, after which they are transferred to a second Coplin jar containing peanut oil and xylol in the same proportions for another period of 12 minutes. Then, the slides are blotted with bibulous paper, placed in carbolfuchsin (also in a Coplin jar) for 30 minutes at room temperature, and washed in tap water five or six times. Decolourization is carried out by immersing the slides in two changes of 1% HCl in 70% ethanol for one minute (by a stop-watch) each change. The slides are finally counterstained by immersion in methylene blue for one minute.
Histopathological Examination
The histopathological examination of tissue specimens obtained by biopsy represents an important laboratory procedure for research purposes as well as for diagnosis. Careful WHO/CDS/LEP/86.4 page 38 classification of the patient's disease by a standard method is essential for much clini cal research, and histopathological evaluation of the degree of "activity" of a lesion may be very helpful in trials of therapy.
PATHOGENESIS OF THE SKIN LESION
Host Cells of M. leprae
M. leprae is an intracellular parasite. Under unfavourable conditions, it may for a time find a retreat in nerve, for which it appears to have an unexplained affinity, and in which it is not readily detected by immunological mechanisms. Muscle may sometimes serve the same purpose. But once the organism establishes itself, it multiplies to a point at which it is detected, and the body then responds by sending in numbers of motile phagocytic macrophages, the normal function of which is destruction or removal of invading organisms or foreign particles. Unfortunately, unless immunity is high, M. leprae is neither destroyed nor removed. After ingesting the organisms, the macrophages become immobilized and, at the same time, they allow the organisms to multiply freely. They are the ideal hosts to the organisms they should have destroyed, and are the most important constituent of every lepromatous lesion.
When immunity is high, as in tuberculoid leprosy, the situation is different. Young macrophages (mononuclear cells, histiocytes), which are derived from the monocytes of the blood, are able rapidly to destroy or remove M. leprae, and then transform into non motile epithelioid cells and perhaps giant cells. Unfortunately, unless the infection is quickly eradicated, there is usually a heavy over-reaction, and the mass of epithelioid cells becomes the main source of damage. The function of these cells is not clear, but they are the counterpart of the macrophages of the lepromatous lesion. Epithelioid cells and macrophages in lesions are always seen in tightly knit clumps, which are collectively called the granuloma. l l
"Activity" and "Regression"
Macrophages. In a macrophage granuloma, the more the multiplication of organisms, the greater is the call for an influx of healthy young macrophages. These cells are not yet well differentiated; they have only a small amount of rather solid cytoplasm, and they may even show some resemblance to a small epithelioid cell. This is not surprising; at this stage, they have still the capacity of differentiation in either direction - to epithelioid or lepra cell. Those macrophages that have not yet ingested bacilli may multiply to produce a further increase of young cells, which replace those that succumb to their heavy bacterial load. The combination of influx and multiplication leading to a progressive expansion of the lesion continues for as long as the organisms continue to multiply. This phenomenon is conveniently referred to as cellular activity.12
11 "Granuloma", which means a tumour-like granule, can be legitimately applied to an accumulation of macrophages or any of the cells derived from them. These are the only inflammatory cells which produce compact congregations of this sort, although their appearance of permanence is somewhat illusory. Some authors restrict the term to epithelioid cell granuloma; in leprosy, this is inconvenient; and even in an epithelioid granuloma, there will be a proportion of young macrophages undergoing evolution. 12 For leprosy, the concept of "cellular activity" in the granuloma is preferred to that of cell-turnover, the subject of much recent study. Cell-turnover implies a state of equilibrium between influx and multiplication on the one side, and emigration and death on the other. But the most active lesions are often not in equilibrium, cellular increase far outstripping decay. Cell-turnover is usually related to life-span. The life-span of the granuloma cells, in order from shortest to longest, is: giant cell, epithelioid cell, macrophage in an active lesion, macrophage in an inactive lesion. Thus, the rate of cell turnover is greater in tuberculoid than in lepromatous granulomas; but the rate of influx and multiplication combined (cellular activity) may well be as high in some lepromatous as in tuberculoid lesions, and possibly higher. WHO/CDS/LEP/86.4 page 39
When multiplication of bacilli slows down, so does the influx of cells. At the same time, with the death and degeneration of many organisms, the fat content of the phagolyso somes in which they have earlier multiplied gradually increases. It is likely that most of the lipid is of bacterial origin, although some may be derived from the macrophage itself. The cytoplasm gradually acquires a soapy appearance, and as the fat content con tinues to increase, small vesicles develop and coalesce to produce more definite vacuoles. Sometimes, one may also see giant vacuoles, about 100 ~m in diameter, formed perhaps by the fusion of several macrophages, but this does not always happen. The larger the vacu ole, the higher the proportion of fat and the lower the proportion of organisms in its contents. Later, there may be a fresh influx of macrophages to remove the fatty content of dead vacuolated cells. The new cells are round, self-contained (unlike those in the granuloma), and probably motile. A lepromatous granuloma in which the majority of macro phages show marked fatty change is said to be in regression.
Epithelioid cells. These have a shorter life-span than macrophages. They do not undergo degenerative changes, but simply die and disappear. The character of a tubercu loid granuloma, therefore, is the same, whether or not the infection is active.
Evolution of the Lesion
In "early" lesions - i.e., in the stage before the infection is fully established, the granuloma is most likely to make its first appearance near the epidermis or one of the deep nerve bundles. But when the lesion becomes fully established, the granuloma is most prominent around the subpapillary blood vessels, situated in the upper dermis below the subepidermal zone.
A granuloma can increase in size, until it occupies almost the whole of the dermis at one site. This comes about in one of two ways.
(1) Infiltration. Young macrophages can infiltrate between the collagen bundles, producing spurs around the main granuloma mass. It is also possible that the organisms may sometimes colonize fibroblasts. If the infection is very active, small satellite foci of granuloma develop at the tips of some of the spurs. This may happen with either macrophage or epithelioid cell granulomas, and provides a method of determining whether the infection is active, even when there are no organisms. After treatment, the spurs resolve and disappear sooner than do the satellite foci. The granuloma mass is then left with a smooth edge, and, if lepromatous, will soon show the signs of regression already described. Infiltrative spread of this sort is more prominent in the superficial part of the dermis than in the deep part. It is likely to be confused with a flare-up in the dermis that often occurs in reactions. (2) Expansion. A granuloma may also increase by expansion from the interior of the mass, the smooth-edged periphery being pushed outwards so that it compresses the dermis. This is what happens in histoid lesions. Expansion appears to be associated with cell-division of young macrophages, rather than with influx of new cells from the circulation, and is probably more pronounced in the deep dermis. But in most very active lesions, there is probably a mixture of both expansile and infiltrative spread.
Cellular Infiltrate
The other inflammatory cells that may be encountered in leprosy lesions are lympho cytes, which cooperate with macrophages in the immune response, plasma cells, which are producers of antibody, neutrophilic polymorphonuclear leucocytes, which are seen usually in erythema nodosum leprosum (ENL) , and occasionally eosinophils in small numbers or mast WHOjCDS/LEPj86.4 page 40 cells, the significance of which in leprosy is uncertain. These inflammatory cells differ from those of the mononuclear cell series, in that they are not host cells to H. leprae. This is an important distinction. Of these other cells, only polymorphs have the capa city to ingest organisms; they do not often do so, or, if they do, they do not linger at the site of the lesion. The inflammatory cells, therefore, are less precise markers of the site of H. leprae, either of its presence or its destruction, than is the granuloma. Nevertheless, these cells are essential components of the inflammatory reaction, making their appearance on the scene of the lesion before the granuloma, and persisting for a time after its final resolution. The most important among them is the lyrnphocyte.
Nerve Involvement
Sequestration of H. leprae in a nerve bundle in the skin is frequently, if not always, the first step in the inception of a skin lesion, and the lesion that develops in the nerve is a microcosm of that which later develops elsewhere in the dermis. The habi tat of the organism is the Schwann cell, or occasionally the axon which is ensheathed by it. In the presence of a foreign particle, the Schwann cell assumes a phagocytic func tion, and it is thought that, in an infection, it may eventually evolve as either a macro phage or an epithelioid cell. However, when a granuloma eventually develops in a nerve in response to H. leprae, it is not clear to what extent the component cells are derived from Schwann cells, or from mononuclear cells that have entered the nerve via the blood stream. If organisms reach the periphery of the nerve bundle, invasion of the endoneurium will follow. This also appears to be an immunologically protected site, and the whole endoneurial zone may become occupied by granuloma cells, with or without organisms. Breaching of the perineurium occurs as a result of colonization of perineurial cells by H. leprae, followed by infiltration of lyrnphocytes and plasma cells through the peri neurial layers, and followed in turn by the invasion of granuloma cells. In the course of these events, the perineurium becomes laminated, and presents an onion-skin appearance. This is particularly noticeable in some lepromatous infections, in which the cellular component may retreat or disintegrate, leaving spaces between the layers.
EXAMINATION AND INTERPRETATION OF A SKIN SECTION
The first point to note about a section is its thickness. If a section is double its proper thickness because of faulty technique, it will contain twice as many cells - e.g., lyrnphocytes - as well as twice twice as many organisms as it should. Interpretation is difficult unless sections are of standard and even thickness.
The examination of a section must always cover the whole of its area, and should conform to a routine. This avoids omissions. Points for observation are the presence of a granuloma, its character, the' cellular components of the infiltrate, the position of granuloma and infiltrate in relation to skin structures and blood vessels, the degree of pathological involvement of nerves, hair-follicles, sweat-glands, epidermis and subepi dermal zone, arrector pili muscle, the presence of oedema, the state of the collagen and elastic tissue of the dermis, and the number of organisms present in the various situ ations. Of course, one should not neglect any evidence that the disease may not be lep rosy.
Granuloma and infiltrate. The characters of the various types of granuloma and cellular infiltrate have already been dealt with. It must be emphasized that the nuclei of the various sorts of granuloma-cells are all essentially similar, and that the distinctive points of the various granuloma-cell-types lie in their cytoplasm. By contrast, the cells of the infiltrate each have a characteristic nucleus. It is pointless to attempt to identify the precise form or stage of evolution of each cell. What matters is the overall picture, the direction in which the granuloma appears to be evolving, and the cell-types of which the infiltrate is composed and their relative numbers.
Nerve bundles. In haematoxylin-eosin stained tissue, nerve bundles in longitudinal section in the dermis appear as wavy cords surrounded by a straight sheath of perineurium. The oval nuclei oriented in the direction of the bundle are those of Schwann cells. The WHO/CDS/LEP/86.4 page 41 first pathological response to leprosy is an even proliferation of Schwann cells, in which orientation of the nuclei is maintained. This probably represents regenerative activity, which in leprosy is most likely to occur proximal to the lesion. Near the lesion, the nuclei become disoriented, so as to produce disorganization of nerve structure. Granuloma production may follow, or the perineurial changes already described may be more promi nent.
In the nerve bundles of the skin, it is not possible to distinguish endoneurium from perineurium, and there is no epineurium. Therefore, the nerve sheath is referred to simply as perineurium.
Nerves occupied by granuloma can often still be identified by their perineurium, but in tuberculoid leprosy they may be destroyed beyond recognition. Unfortunately, the absence of nerves from a section of average size does not necessarily signify destruction, as nerves are not present in every section.
Hair-follicles and sweat-glands. Loss of hair and impairment of sweating are fea tures of tuberculoid leprosy better assessed clinically than histopathologically. Cellu lar infiltration or partial destruction of hair-follicles or sweat-glands should be noted whenever they are detected, but because there may not be a hair-follicle in every section, and sweat-glands may be destroyed beyond recognition, histopathological evaluation is not always conclusive. Inflammation of hair-follicles may be caused by a fungal infection rather than by leprosy, but, in such a case, neutrophils are usually present.
Epidermis and subepidermal zone. Erosion of the basal and squamous layers of the epidermis to varying depths by epithelioid cell granuloma is one of the most important aspects of tuberculoid leprosy. Usually, this is seen in a segment of epidermis corres ponding to two or three papillae; less often, the erosion is shallow but more extensive.
Less significant but worthy of note is a wedge of infiltration by lymphocytes, which is sometimes seen in early or indeterminate lesions. Diffuse cellular infiltration of the epidermis is not a typical feature of leprosy.
Thinning and atrophy of the epidermis with loss of rete pegs is a feature of many large lepromatous lesions with expansile granulomas, and is presumably a pressure effect. The subepidermal zone is similarly compressed. If the basal layer is also destroyed, the end-result is the same as erosion. Thickening (acanthosis or hyperplasia) of the epider mis is not a feature of leprosy. The epidermis will not be eroded without infiltration of the subepidermal zone, but there is often loss of clear zone without epidermal involve ment.
Blood vessels. Localization of granuloma around the blood vessels of the upper dermis is the hallmark of an established infection, which is usually already disseminated or becoming so. Once a granuloma has become extensive, its situation in the dermis ceases to be of much significance. Localization of cellular infiltrate, as opposed to granuloma, around the blood vessels of the upper dermis, as opposed to other skin structures, is more likely to indicate some form of dermatitis other than leprosy. Vasculitis in leprosy is nearly always associated with ENL. The affected vessels may be of any size from small artery to capillary. The inflammation may be acute, subacute or chronic.
Oedema. Oedema is present in a leprosy lesion in the acute stage of all reactions, unless the patient is on immunosuppressive drugs. It is also common in mild degree in the lesions of untreated patients in the BT - BB portion of the spectrum, probably indicating either an incipient reaction, or a mild immunological imbalance, with downgrading of the patient's immune status.
Oedema is indicated by dilatation of the lymphatics just below the subepidermal zone and perhaps the mid-zone of the dermis, and also by a watery appearance of the collagen around the granuloma. In addition, the granuloma cells may be separated in a way that makes them difficult to recognize. All of this results from an increase of extracellular WHO/CDS/LEP/86.4 page 42 fluid. A similar appearance may be produced by formalin fixation, which causes shrinkage of collagen, pulling the collagen away from the granuloma, and pulling the granuloma cells apart. This is the main disadvantage of formalin fixation.
Intracellular oedema may also be seen in severe reactions. It appears as vacuoles in foreign-body giant cells when there are no organisms present. The appearance is indis tinguishable from that of fat vacuoles, and its main importance is that a vacuolated giant cell from a reacting lesion has not necessarily been a host cell to H. leprae. But intracellular oedema can be excluded if extracellular oedema is not profuse.
Connective tissue. The collagen and elastic tissue of the dermis are disturbed in a mild way by the process of infiltrative spread already described. But widespread or severe connective tissue involvement is indicative of a reaction. A mild or incipient reaction, sometimes clinically inapparent, may produce dermal changes that mimic those caused by infiltrative spread; but the two can be readily distinguished, in that the changes resulting from infiltration are localized around the periphery of the granuloma, whereas the damage caused by reactions is widespread throughout the dermis, and unrelated to the granuloma.
In mild reactions, there is proliferation of fibroblast nuclei between the collagen bundles. In severe reactions, the proliferating cells become more primitive, with large pale nuclei; and there may be marked oedema, which diffuses between and into the collagen bundles. Although the staining reactions of the collagen are not altered, the injury to the collagen must often be serious, to judge from the extent of the fibrosis that follows. However, it is not clear whether the collagen damage has been caused mainly by separation of fibrils by the oedema, or more directly, by an immunological insult. Associated with the "dermal reaction", there is always some swelling and disruption of elastic fibres, which is quite prominent and readily apparent in good haematoxylin-eosin stained sec tions.
Fibrosis and scarring of the dermis may be severe as a sequel to a serious reaction, particularly one with severe dermal involvement. Such scarring sweeps across the dermis like a keloid, but nevertheless the normal pattern of the dermis is eventually reconsti tuted without permanent damage. A less common cause of fairly widespread but less severe fibrosis is local medication. More circumscribed fibrosis may result from a previous biopsy at the same site. Resolution and healing of a granuloma is a silent process which does not leave apparent fibrosis. But heavy proliferation of fibrocytes within a portion of the granuloma is a common finding of uncertain significance.
Organisms. Examination of a section for the diagnosis or assessment of leprosy is never complete without a search of at least one section for organisms. Their situation and morphology must be noted, and their number estimated.
Activity of the Process
Leprosy, like any other chronic infection, may be active, quiescent or in regression, according to whether the causative organism is viable and multiplying, or its numbers are stable or decreasing. Active leprosy is characterized clinically by erythema and perhaps oedema of the lesions, as well as by spread of the lesions.
Similarly, there are histopathological characteristics associated with active dis ease: a preponderance of young and relatively healthy macrophages with few vacuoles (cellular activity); and an increase of the granuloma, either by infiltration or expan sion. These processes have already been described. In lepromatous lesions, these two histopathological features always go together. In general, they correspond also to bac terial activity, although not exactly. In relapse, for example, it is possible to have a high level of histopathological activity in response to a lower level of bacterial activ ity, or vice-versa. The activity of an infection is best judged from the state of the organisms. Histopathological activity is certainly worth noting, but its main importance is that failure to recognize it will lead to errors of interpretation. Furthermore, it is WHO/CDS/LEP/86.4 page 43 histopathological rather than bacteriological activity which is the source of clinical evidence of advance or resolution of the disease.
DIAGNOSIS
Early Leprosy
Leprosy lesions containing readily detectable AFB do not present a problem. However, early skin lesions and lesions of tuberculoid leprosy, in which it is not always possible to detect AFB, may present problems of differential diagnosis. The more sections that are examined, the greater the likelihood of finding AFB or histopathological evidence of leprosy.
Early lesions, in which a granuloma has not developed, must be distinguished from other forms of dermatitis. The presence of AFB in one of several characteristic sites - a nerve bundle, the subepidermal zone, or an arrector pili muscle - is diagnostic of lep rosy. Bacilli are most readily detected in nerves cut longitudinally, because they usu ally lie parallel to the axons. In the sub~idermal zone, they usually lie parallel to the epidermis, just below the basal layer. Later in the evolution of the lesions, AFB may be found in capillary endothelium, and still later in perivascular macrophages in the upper dermis.
In the absence of AFB, the histopathological picture may suggest the diagnosis of leprosy with a degree of probability;
(1) an epithelioid cell granuloma in one of the sites of predilection of H. leprae is almost as reliable a diagnostic criterion as the presence of bacilli. On the other hand, a macrophage granuloma without AFB is not diagnostic of leprosy; (2) a cuff of lymphocytes around a normal-appearing nerve, or disorientation of Schwann cell nuclei with disorganization of nerve structure or infiltration of perineurium, is good evidence of leprosy. Proliferation of Schwann cells with preservation of nuclear orientation represents a non-specific change; (3) cellular infiltration without granuloma is not diagnostic, but suggests the possibility of leprosy if it involves the sweat ducts or arrector pili muscle; (4) a patchy infiltrate that involves primarily the epidermis or neurovascular bundles is a little suggestive. However, involvement of these sites is of no significance if the main infiltrate is perivascular; (5) normal skin or a mild non-specific infiltrate of lymphocytes may be the only finding in an early lesion, and does not exclude leprosy. However, in lesions of more than one year's duration this is not common and is against leprosy. Neutrophils and much oedema are not found in leprosy except during reactions.
Tuberculoid Leprosy
The lesions of tuberculoid leprosy must be distinguished from other pathological processes that produce tuberculoid granulomas. Because tuberculoid granuloma may be caused by many diseases other than leprosy, one should have good evidence for the diag nosis. The general principles are the same as for early lesions. The finding of AFB or of definite nerve involvement is diagnostic. Also helpful is the finding of massive swelling of a nerve, or an area of necrosis in its centre. The presence of normal nerve bundles within a granuloma almost certainly excludes the diagnosis of leprosy unless the nerve is surrounded by a cuff of lymphocytes. However, normal or nearly normal nerves lying outside a granuloma or infiltrate have no diagnostic significance.
A tuberculoid granuloma that has eroded deeply into the epidermis is typical of TT leprosy, especially if the granuloma is associated with nerve involvement or if nerves are absent. The diagnosis is unlikely to be leprosy if an epithelioid or giant-cell granuloma WHO/CDS/LEP/86.4 page 44 is accompanied by pseudoepitheliomatous hyperplasia of the epidermis, caseation except in the centre of the nerve, massive necrosis, or numerous plaslna cells.
THE RIDLEY - JOPLING CLASSIFICATION
Classification employs clinical, bacteriological and histopathological criteria to discern the immune response of the patient. The histopathological appearance of the lesion is at present the best index of a patient's immune status, at least in established disease. Clinical findings are more conveniently employed than are the histopathological; on the other hand, histopathological findings change much more rapidly than do the clini cal in response to a change of the immune status. This is particularly true in the event of "down-grading" and "reversal" reactions (see pp. 46 - 47).
To classify the individual lesion or patient, it is essential to employ the criteria shown in Table 4. In considering the histopathology, it is important to note the number of bacilli in the lesions, and to know if the patient has been treated.
The histological classification of active lesions is as follows:
TT (Full Tuberculoid)
The histopathological picture is that of an epithelioid cell granuloma distinguished by one or more of the following characteristics:
(1) numerous lyrnphocytes around the periphery of the granuloma; (2) deep, extensive erosion of the epidermis by granuloma; (3) central caseation in a nerve bundle, or massive swelling of the nerve caused by granuloma; (4) many large Langhans-type giant cells.
BT (Borderline Tuberculoid)
This form of leprosy, which is seen much more frequently than either TT or BB, is characterized by an epithelioid granuloma accompanied by giant cells or a moderate number of lyrnphocytes; obliteration of the subepidermal clear zone is sometimes present. Giant cells are small or not of the Langhans type. Nerves are typically only moderately swollen, with slight lamination of the perineurium having resulted from infiltration of lyrnphocytes.
BB (Mid-Borderline)
This relatively uncommon form is characterized histopathologically by an epithelioid granuloma without giant cells or aggregates of lyrnphocytes. Oedema is common. The sub epidermal zone is clear. Nerves are not greatly swollen, and may appear quite normal, although lamination of the perineurium may be noted.
BL (Borderline-Lepromatous)
There is a macrophage granuloma with, in addition, one of the following features:
(1) numerous lyrnphocytes packed densely over the whole of at least one segment of the granuloma, or around a nerve bundle; or (2) a solitary clump of epithelioid cells among the macrophages, with or without lyrnphocytes.
The macrophages have fairly solid cytoplasm with little foam and no vacuoles. Nerves commonly show an onion-skin perineurium, with some cellular infiltrate that may mask the perineurium and penetrate the nerve. WHO/CDS/LEP/86.4 page 45
Table 4. Guide to the histology and performance of the classification groups13
TT BT BB BL LLs LLp
+ Epithelioid cells .. ++ ++ ++ -/- Non-vacuolated giant cells ++/- +/- Histiocytes/foamy macrophages ++ ++ ++ Small vesicles ++/- ++/- ++/- + Vacuolated giant cells ++/- -/- Giant vacuoles +/- ++/- + + + + + + Lymphocytes . +-/+ +-/- ++/+ +/- -/- Dermal nerve, maximum diameter (pm) 1000 400 250 200 200 80 + + Onion-skin perineurium -/- +/- ++/- ++/- + Clear subepidermal zone -/- ++/- ++ ++ ++ ++ + Erosion of epidermis ++/- -/- Acid-fast bacilli in granuloma (BI) 0/1 0/2\ 3/4\ 4/5\ 5/6\ 5\/6\ + Acid-fast bacilli in nose ++ ++ Lepromin (Mitsuda) reaction 3+ 2/1+ Lymphocyte transformation test (% transformation) 15 6.0 2.8 0.9 0.6 0.4 Leucocyte migration index 0.76 0.83 0.88 0.92 0.92 0.95
Histological index, fall ~n 6 months (%) 100 78 23 14 5.5 + + Immunological stability ++ + ++ + Reversal and downgrading reactions + ++ + + Erythema nodosum leprosum .. . ++ ++
LL (Full Lepromatous)
This form is characterized by macrophage granuloma with no epithelioid cells and few lymphocytes. Foamy change is present, and small vacuoles may be found. Nerves may show an onion-skin perineurium, without cellular infiltration. Two sub-types may be distin guished.
LLs (sub-polar lepromatous). The macrophages show mild foamy change but without much swelling of the cytoplasm, so that the nuclei are fairly close together. There are a number of diffusely scattered lymphocytes, and usually plasma cells. Nerves may be swol len, and often show an empty laminated perineurium, without lymphocytes.
LLp (polar lepromatous). The macrophages have a rather more bulky, foamy cytoplasm, with a few more small vacuoles, so that the nuclei are further apart. Lymphocytes are scanty, although plasma cells may be found. The distinction between LLp and LLs cannot be
13 Adapted from reference no. 149. WHO/CDS/LEP/86.4 page 46 made without taking account of the state of activity or regression of the lesion (v.i.).
Indeterminate (I)
The lesions of indeterminate leprosy fit into none of these categories. The diag nosis is not in doubt, but histopathological classification is impossible because there is no granuloma. There may, however, be features suggestive of tuberculoid or lepromatous leprosy. 14
Classification of Regressing Lesions
In untreated patients the lesions gradually become less active; with treatment, the changes of regression are more marked and affect classification.
TT, BT. The epithelioid cells lose their differentiating features and gradually disappear, leaving a lymphocytic infiltrate which is unclassifiable.
BB. Treated patients usually "upgrade" to BT or TT before the lesions resolve, so that it is uncommon to see regressing BB.
BL. There is increasing foamy change. Vacuoles are sometimes, but not always, large, and the macrophages may be multinucleate.
RELAPSE
Relapse occurs first at one site and then at another, so that lesions may vary histo pathologically, in terms of activity and appearance of the AFB. Relapse is characterized by the presence of brightly stained AFB, and the presence of young, non-vacuolated macro phages. Histoid lesions are extremely active and more often seen in relapse than in untreated patients. The young macrophages are so uniform in appearance that they may resemble tumour cells.
REACTIONS
ENL
ENL, which occurs almost exclusively in patients with LL leprosy, consists histopath ologically of acute inflammatory reactions in a lepromatous granuloma. The lesions are usually small and subcutaneous, but may involve predominantly the dermis, and may ulcer ate. Necrotizing vasculitis may also be seen in severe lesions. Acutely, neutrophils predominate, and later, there is an increase of the number of lymphocytes, which may make the distinction from BL difficult.
Lepra (Borderline, Reversal or Upgrading) Reactions
These reactions, which have been characterized as delayed hypersensitivity reactions, occur mainly in BT - BL leprosy, and in a few patients with LLs leprosy, typically during treatment. Early histopathological findings are mild oedema around the granuloma and in the superficial dermis, and diffuse proliferation of fibroblasts in the dermis. In strong
14 Other descriptions of the histopathological characteristics of I leprosy may be found in the literature. For example, Khanolkar (94) states that "histologically the biopsy specimen shows foci of chronic inflammatory cellular exudate, mainly arranged around the finest nerve-fibres in plexuses of the dermis. These fibres are richly disposed around the pilosebaceous apparatus and the nerve-bundles which travel down along the same blood and lymph-vessels towards the subcutis. The cellular exudate is mainly concentrated as strands or groups of cells around these structures. The exudate consists of small round cells (lymphocytes), histocytes (macrophages), plasma cells, and, very occasionally, eosinophilic, basophilic, and neutrophilic polymorphonuclear leucocytes." Editor WHO/CDS/LEP/86.4 page 47 reactions, these features become more marked, and there is occasionally necrosis and ulceration. Later, one sees foreign body giant cells, which may contain vacuoles. The number of lymphocytes is variable, and eosinophils or foci of neutrophils may be present. Except in the late stage, it is not possible to determine from a biopsy specimen whether the reaction is associated with upgrading or downgrading of the patient's immune response.
Measurement of the Solid Ratio in Tissue Sections
The solid ratio has been measured in acid-fast-stained tissue-sections (105, 106, 110). The procedure employed is that described in Chapter 3 for measurement of the solid ratio in smears. However, the measurement is even more difficult in sections than it is in smears, and should not be attempted unless there is no alternative - i.e., the only available material consists of sections. WHO/CDS/LEP/86.4 page 48
CHAPTER 6 - YORK YITII EXPERIMENTAL ANIMALS
In the absence of a method for cultivation of 11. 1eprae in vitro, its "cultivation" in the footpad of the mouse must remain a laboratory technique of paramount importance to research in leprosy. As will be seen, multiplication of 11. 1eprae in the mouse footpad is the primary criterion of viability of the organism; when organisms have multiplied in mice, the inoculum must have included viable 11. 1eprae. When, however, organisms have failed to give rise to multiplication, one may not conclude that there were no viable 11. 1eprae in the specimen.
11. 1eprae multiply to a limited degree in immunologically intact rodents of several species, and to a greater degree in immunologically disabled rodents and in the armadillo. The experimental species to be inoculated depends upon the purpose of the work. Consid ered in this chapter are the species of experimental animals studied, the characteristics of bacterial multiplication in several species, both intact and immune-deficient, hus bandry of experimental mice, the techniques for cultivation of 11. 1eprae in the mouse footpad, the techniques needed to produce immune-deficient mice, some important applica tions of the inoculation of experimental animals with 11. 1eprae, some important calcula tions and basic statistical techniques, and, finally, the preparation of drug-containing mouse diets.
Immune-Competent Rodents
In work based on the demonstration by Fenner (52) of multiplication in the hind footpads of mice of 11. marinum and 11. u1cerans, for which the optimal temperature for growth is lower than 37oC, Shepard demonstrated (171, 172) that 11. 1eprae multiply to a limited extent in the hind footpads of immunologically intact mice. Following inocula tion into a hind footpad of fewer than 10 5 (100 000) 11. 1eprae, obtained from harvested mouse foot-pad tissues, or from skin biopsy specimens or sediments of nasal washings of patients with untreated lepromatous leprosy, there was very little change during the first several months. Histopathologically, granulomatous lesions consisting of large round cells, some containing AFB, appeared and gradually increased in size during the next several months, accompanied by an increase of the number of organisms. Grossly, a lesion was almost never evident. Inoculation of 10 5 or more 11. 1eprae was followed by the early appearance of granu10mas without evidence of bacterial multiplication. This most significant finding was subsequently confirmed, and has since been exploited by many workers.
GROWTH OF 11. LEPRAE IN THE IMMUNE-COMPETENT RODENT
Employing the methods described (see pp. 121 - 127) for inoculating, harvesting, and enumerating AFB, 11. 1eprae may be demonstrated to multiply in footpads of immuno1ogi ca11y intact mice with the following characteristics:
(1) the minimal infecting dose of 11. 1eprae is of the order of five viable organ isms (104, 191, 214). Because the inoculum is distributed to tissues, both contiguous and distant, not ordinarily encompassed in the harvest, a fraction of the inoculum is effectively lost (109), suggesting that the minimal infecting dose may be as small as one or two viable 11. 1eprae; (2) multiplication proceeds in a characteristic manner. One may readily identify 1ag, logarithmic and stationary phases of multiplication (171, 172) (Fig. 13); WHO/CDS/LEP/86.4 page 49
Figure 13. Growth curve of H. 1eprae in the hind footpad of the immunologically normal BALB/c mouse. Harvests were made from pools of 4 (.), 6 ( .. ) or 8 (.) footpads .15
•
6.0
LOG NUMBER AFB/FOOTPAD
5.0
4.0
o 100 200
DAYS AFTER INOCULATION (3) the doubling time16 of H. leprae measured during logarithmic multiplication is 11 to 13 days (104); (4) the stationary phase or "plateau" represents, in fact, the effect of the immune response of the mouse. Evidence for this is failure of multiplication when three immunologically intact mice are inoculated with 10 5 or more organisms (171, 172); the resistance of H. leprae-infected mice to a second challenge with H. leprae (93, 102, 184); the activated appearance of the macrophages in which the H. leprae reside, beginning from the time at which multiplication of the organisms is maximal (50); and the higher ceiling to multiplication in immunosuppressed rodents (22, 26, 53 - 57, 96, 98, 150).
15 Adapted from reference no. 100. 16 The "doubling time" or "generation time" (the average time required for each two-fold increase of the number of organisms),
G = number of days between inoculation and harvest number of doublings between inoculation and harvest
The number of doublings is the logarithm to the base 2 of the fold-increase of the number of organisms. Thus, if 5 000 H. leprae have been inoculated, and 100 days later, 10 6 AFB are harvested, then the number of organisms has increased 200-fold (106/5 000) in the course of 100 days. log 200 Log2 200 10 2.301/0.301 7.644;
G = 100/7.644 = 13.08 days per doubling.
The doubling time may be measured directly from the straight line representing the phase of logarithmic multiplication - i.e., from the straight line, calculated by the method of least squares, that best fits the experimentally observed values. Alterna tively, the doubing time may be calculated from the average distance between the growth curves resulting in mice inoculated with serial dilutions of H. leprae, as was done in reference no. 104. WHO/CDS/LEP/86.4 page 50
The maximum of bacterial multiplication varies among strains of mice, and of M. leprae, and is approximately 10 6 (1 million) to 10 6. 3 (2 million) per footpad in immuno10gical1y intact CFW, CBA, and BALB/c mice and those of several other inbred strains, and fails to reach this level in the mice of yet other strains (183). A later, secondary growth phase of M. leprae has been described in intact CFW mice (191).
Although multiplication of M. leprae in the footpads and ears of rodents other than the mouse has not been so extensively nor so carefully studied as that in the footpads of mice, there appear to be no important differences of bacterial multiplication among rodent species. Thus, the same "ceiling" to multiplication of M. leprae is encountered in the immunologically intact rat (57, 77), hamster (Mesocricetus auratus) (7, 172, 210), gerbil (Meriones unguiculatus) (172), and mystromys (Mystromys mystromys) (7) as in intact mice. This is so despite, for example, the much larger mass of the footpad of the rat than of the mouse.
Immune-Deficient Rodents
Inoculation, either systemically or locally, of immunosuppressed or natively immune deficient rodents with M. leprae results in a greater maximum of multiplication, multi plication after inoculation of a larger number of M. leprae, dissemination of the infec tion, prolonged survival of the organisms, and grossly evident lesions in a variable proportion of the ani~als studied. Most of the published work has involved the adult thymectomized, lethally irradiated, and bone-marrow reconstituted (T900R) mouse originally employed by Rees (150). Binford studied, as an alternative, an adult-thymectomized mouse that was subsequently irradiated with one femur shielded (B), so that bone-marrow reconstitution was not required. Fieldsteel worked with mice thymectomized three or four days after birth and not additionally immunosuppressed (53). Finally, Gaugas described work with an adult-thymectomized mouse subsequently treated with antilymphocyte globulin (63). All of these animals permitted enhanced multiplication of M. leprae, but none of them appeared to offer an advantage over the T900R mouse.
Fieldsteel also worked extensively with the neonatally thymectomized rat (54 - 57). This animal survives well under conventional animal-house conditions, and permits multi plication of M. leprae from a larger inoculum, and to a higher maximum.
Because it has appeared that the degree of immunosuppression required to permit unlimited multiplication of M. leprae does not permit the rodents to survive long enough for experimental work with M. leprae, investigators have turned to the specific pathogen-free (SPF) or germ-free congenitally athymic, "nude" mouse (22, 26, 96, 9B). Greater enhancement of the multiplication of M. leprae has been reported in these ani mals than in T900R mice or neonatally thymectomized rats. Another interesting immunologi cally deficient rodent is the congenitally athymic rat, with which work with M. leprae has only recently begun (36).
GROWTH OF M. LEPRAE IN IMMUNE-DEFICIENT RODENTS
The "ceiling" to multiplication of M. leprae in immunologically intact mice and rats has already been described. Much of the evidence that this ceiling is the result of a cell-mediated immune response is based on work in rodents depleted of thymus-directed lymphocytes ("T-lymphocytes"), in which M. leprae multiply to a much higher limit, and in which bacterial multiplication is sometimes accompanied by gross evidence of disease.
Thymectomized-Irradiated Mice
By far the best understood of the immune-deficient rodents is the CBA mouse that has been subjected to adult thymectomy followed by whole-body irradiation. Virtually all of the work with this experimental animal has been carried out at the National Institute for Medical Research (NIMR) in London. During the first years of work with this animal, the radiation was administered in a single dose of 900 rad (9 Gy); because this dose is WHOjCDSjLEPj86.4 page 51 sufficient to destroy the functioning bone marrow, it was necessary to transfuse the mice with syngeneic bone marrow immediately after irradiation. In those years, these mice, termed T900R mice, survived well in clean but otherwise conventional animal-house condi tions. In more recent years, T900R mice have not always survived well at the NIMR. To improve survival, the dose of radiation is sometimes delivered in five doses of 200 rad (2 Gy) each over a period of eight weeks. This fractionated dose of radiation is less destructive to the bone marrow, and "T200X5R" mice do not require bone-marrow transfusion. They are not as immune-deficient as T900R mice, a fact that probably accounts for their better survival. 17
A typical growth curve of H. leprae in T900R mice is shown in Figure 14. In addi tion to the higher ceiling to multiplication in these mice, in comparison to that in immunologically intact mice, multiplication of H. leprae occurs when the inoculum con tains as many as 10 6 organisms. H. leprae do not multiply in intact mice when the inoculum contains 10 6 organisms, presumably because the quantity of antigen represented by the large inoculum is sufficient to trigger the cell-mediated immune response, that
Figure 14. Growth curve of H. leprae in the ears and hind footpads of normal and T900R mice inoculated with 10 4 or 10 6 AFB. Each point represents the mean number of organisms in both ears and both hind footpads. 0------0: total of solid and non-solid AFB; x------x: solid AFB.18
9
6
Cl 9 ""'7 ell I Z :::> o u ~ j 61':...r-----II----!.='-'-'=-='--'~--_I_-----':>.....- .J \j « co ,," I ell-c " 11..5. / " o / " u -c / " /
. . 2 4 6 8 10 12 14 16 TIME (MONTHS)
17 Alternatively, these workers might have attempted to reduce bacterial contamination of their mouse colony by several weeks' administration of combinations of non-absorbable antibiotics (27). Editor
18 Taken from reference no. 157. WHOjCDSjLEPj86.4 page 52 normally limits multiplication of M. leprae in immunologically intact mice - i.e., the large inoculum immunizes immune-competent mice. Finally, it was reported that swelling of the infected foot occurred in about 10% of the T900R mice. T200X5R mice are less immune deficient than T900R mice, as demonstrated by the facts that the ceiling to multiplication of M. leprae is somewhat lower in T200X5R mice, and that multiplication does not regu larly occur when these animals are inoculated with more than 10 5 organisms.
Nude Mice
Much less work has been done with the congentia11y athymic, "nude" mouse infected with M. leprae (22, 26, 96, 98). Experimentation with M. leprae infection of this very interesting experimental animal was initiated only within the last decade. These animals are difficult to breed and to rear, making work with them more difficult than with thymectomized-irradiated mice.
Congenital absence of the thymus gland, which is for some reason accompanied by hair lessness, appears to have arisen as a mutation, and is transmitted as an autosomal reces sive trait. These animals are much more fragile than are thymectomized-irradiated mice, a fact consistent with a total lack of mature T-lymphocytes. They are extremely susceptible to many bacterial and viral infections that are non-pathogenic for immuno1ogically intact mice, and, consequently, do not survive when housed under conventional animal-house condi tions.
As shown in Figure 15, such mice will permit multiplication of M. leprae from inocula ~ 10 6, and permit multiplication to a ceiling of 10 10 (ten thousand million) or more organisms per footpad. Grossly evident lesions are a regular feature of M. leprae infected nude mice.
Figure 15. Growth of M. leprae in the left hind footpads of nude mice after inoculation with 10 6. 9 (.),104. 7 (0) or 10 2. 7 (0.) M. leprae. 19
10
q o -< := 8 8.... ee It
• 3
j I I I I I I o 100 200 300 400 SOO 600 700 DAYS AFTER INOCULATION
19 Taken from reference no. 98. WHO/CDS/LEP/86.4 page 53
Neonatally Thymectomized Rat
Studies of M. leprae infection of the neonatally thymectomized rat began from the presumption that neonatally thymectomized animals would be more immune-deficient than would adult-thymectomized, whole-body-irradiated animals (54 - 57). The rat was selected because its newborn offspring are much larger than those of the mouse, thus rendering thymectomy much easier. These animals survive well when reared under clean but conventional conditions.
In fact, the neonatally thymectomized rat appears to be very much like the T900R mouse in its response to infection with M. leprae. As shown in Figure 16, the maximum of bacterial multiplication is between 10 8 (100 million) and 10 9 (1 thousand million) organisms per footpad, and multiplication often occurs when 10 6-107 M. leprae are inocu lated. Gross evidence of disease has never been reported in the M. leprae-infected, neonatally thymectomized rat. A problem peculiar to these animals is important animal-to animal variation of the degree of immune-deficiency, the reason for which is not under stood (58).
Figure 16. Maximal growth of M. leprae in the hind footpads of BALB/c mice inoculated with viable organisms only (open bars), neonatally thymectomized rats inoculated with viable organisms only (striped bars), neonatally thymectomized rats inoculated with viable M. leprae plus 10 7 dead organisms (stippled bars), and neonatally thymectomized rats inoculated with 10 7 dead M. leprae only. The number above each bar represents the time between inoculation and harvest. 20
109
'" '">- >- « « 0 ....0 ... w . s ~ 0>= 108-- w .... 0 "'ww .... >« '"«>- a:u 0 «-0 M :r Z ... lD- '" ~ ~ 10'-- "'00« a:"- w .... lDO '">- ~O « ;:) ...... :" Zffi l 06- - e,
1 S. 10 S. 10° S. 10-1 10' dead AFB NUMBER OF AFB INOCULATED
Congenitally Athymic Rat
Of great interest is the congenitally athymic rat (36). As in the nude mouse, the absence of a functioning thymus gland appears to have resulted from a mutation; the trait
20 Taken from reference no. 55. WHO/CDS/LEP/86.4 page 54 is inherited as an autosomal recessive. This animal appears to be at least as immune deficient as the neonatally thymectomized rat, but much les3 so than the nude mouse. It survives reasonably well in clean, conventional conditions, especially if the cages are covered with filter-caps. Studies of its susceptibility to infection with H. leprae are still in progress. Although comparison with the neonatally thymectomized rat is not yet completed, there appears to be much less animal-to-animal variation of susceptibility than has been found among neonatally thymectomized rats.
Production of Thymectomized-Irradiated Mice21
Although these immune-deficient, T-Iymphocyte-depleted mice are more susceptible to infection by H. leprae, thus extending the use of the mouse as an experimental host for the study of leprosy, they are also more susceptible to infections in general. Therefore, the successful production of these mice is dependent upon breeding, husbandry and care of the animals at all stages, under the cleanest conditions possible. As many as possible of the lIideal ll conditions referred to in the sections on breeding and husbandry (see pp. 61 - 62) should be adopted; in particular, breeding stocks of SPF mice should be employed. All the mice set aside for immunosuppression and subsequent infection with H. leprae should be housed separately, in order to reduce to a minimum the risk of cross-infection.
T900R MICE
In mice, nearly maximal depletion of T-Iymphocytes is obtained by thymectomy followed by total-body irradiation administered in a single lethal dose of 900 rad. Thus, this treatment is ideal for the purpose of immunosuppression. However, this dose of irradia tion destroys completely the functioning bone-marrow cells, necessitating a life-saving transfusion of syngeneic bone-marrow cells immediately following irradiation. To ensure the survival of these bone-marrow cells, inbred strains of mice must be used, and trans fusion with syngeneic cells must be carried out immediately following irradiation. Because this lethal dose of irradiation also damages the intestinal mucosa, a prophylactic course of oxytetracycline should be administered in the drinking water, beginning immed iately following irradiation, for three weeks. The drinking water containing oxytetracyc line (125 mg oxytetracycline + 6 g sucrose per I sterile water) is freshly prepared daily.
Inbred mice are thymectomized at four to eight weeks of age. Two weeks later, they are irradiated with 900 rad, and a syngeneic bone-marrow cell transfusion is administered. The three-week course of oxytetracycline is then administered. Thus, T900R mice are ready for H. leprae inoculation five weeks after thymectomy.
If survival of the T900R mice is poor in spite of all reasonable precautions against intercurrent infections, one should resort to a sub-lethal course of total-body irradia tion.
T200X5R MICE
Thymectomized mice that have been subjected to whole-body irradiation of five doses of 200 rad each, referred to as T200X5R mice, are less immune-deficient than are T900R mice, a fact that presumably accounts for their better survival. These mice are more susceptible to H. leprae than are normal animals, however, and may be used when the T900R method cannot be successfully established because of an unacceptably high mortality. Because the dose of irradiation used is sub-lethal, no bone-marrow cell replacement is required; therefore, inbred strains of mice are not required. Similarly, the sub-lethal dose of irradiation is less damaging to the intestinal mucosa, and a course of oxytetra cycline is not required.
21 Contributed by C. Lowe and R.J.W. Rees. WHO/CDS/LEP/86.4 page 55
The mice are thymectomized at four to eight weeks of age. Two weeks later, irradi ation is begun; a series of five, 200-rad doses is administered at two-week intervals. Thus, T200X5R mice are ready for H. leprae inoculation two weeks after last dose of irradiation (i.e., 12 weeks after thymectomy).
PROCEDURES
Mice Four-to-eight week-old female CBA or BALB/c mice are employed for the T900R method. Mice of the same strain or those of any out-bred strain susceptible to H. leprae are employed for the T200X5R method. Female mice are preferred because they fight less than do male mice.
Anaesthesia Thymectomy requires a general anaesthetic. Although ether or pentobarbital (NembutalR) can be used, tribromoethanol (AvertinR) is preferred because of its wide range of safety. The anaesthetic is administered by intraperitoneal injection in a dosage of 0.01 ml/g mouse body weight + 0.01 ml. Deep anaesthesia is obtained within five minutes.
Thymectomy
The simplest and safest technique for removing the thymus from young mice is by aspiration, and the same technique is used for mice to be irradiated by either the T900R or the T200X5R method. It is essential to undertake adult thymectomy within the age range four to eight weeks, as in older mice the thymus is too firmly adherent for removal by aspiration. Materials needed are:
Waxed cork board 10 x 15 cm Ethanol, 70% v/v in distilled water Scissors, small Forceps, small Half-curved triangular suture needle, size 19 2 black braided sutures (size 3/0) Pasteur pipette with cut end of 2 mm internal bore Vacuum pump
Thymectomy is carried out with sterile surgical instruments and the cork operating board having been swabbed with ethanol; the same instruments may be used for all of the mice to be thymectomized. The mouse must be deeply anaesthetized before commencing thy mectomy. The rate of success depends on the speed of operation, and on preventing col lapse of the lungs; when the technique has been mastered, a surgical mortality as low as 2% can be achieved. The fully anaesthetized mouse is laid on its back on the cork board and harnessed in place by a simple arrangement of pinned elastic bands, as shown in Figure 17. The head of mouse is placed across a 0.5 cm-thick wedge of cork mounted on the board in order to extend the neck. The fur is swabbed with ethanol from chin to upper thorax. Then, the skin is cut with scissors in the mid-line from below the chin to just below the top of the sternum, after which the trachea is exposed by blunt dissection with forceps. Then a 2 mm mid-line incision is made through the top of the sternum to expose the upper end of the thymus gland. The thymus has two lobes; the right lobe, being slightly above the left, is visible after splitting the upper end of the sternum. Therefore, suction should be applied first to this lobe, by means of a Pasteur pipette connected with rubber tubing to a vacuum pump; this may result in the removal of this lobe alone or both lobes together. WHO/CDS/LEP/86.4 page 56
It is important to determine the outcome, as it is essential to achieve a complete thymec tomy. This can be accomplished by employing a Pasteur pipe~te in which a small, bulbous expansion has been blown to catch the thymus. If only one lobe has been removed, suction should be re-applied to the left side below the sternum, to ensure the removal of the left lobe. If a vacuum pump or water aspirator is unavailable, the thymus can be removed by mouth suction, using the same type of pipette with a cotton-wool plug in the distal end. Immediately following thymectomy, the edges of the surgical wound are pinched together with the finger and thumb to prevent collapse of the lung, and the incision is closed by three or four continuous silk sutures. There is no need to remove the sutures; the mouse will do it.
Irradiation
This requires a therapeutic X-ray generator or a gamma-ray source, either cobalt (60 Co) or caesium (137Cs), of the type employed in radiotherapy. The times of exposure required for doses of 200 or 900 rad total-body irradiation will depend on the available source and apparatus.
Figure 17. Harness for thymectomy, with the anaesthetized mouse in position. WHO/CDS/LEP/86.4 page 57
Bone-Marrow Replacement
The materials needed are: Syngeneic 6 - 8 week-old donor mice Scissors, small Forceps, small Trypan blue 0.16% (w/v) in Hanks' balanced salt solution (HBSS) lml syringes and needles Pasteur pipettes Blood-cell counting chamber (Neubauer)
Bone-marrow replacement is undertaken immediately following 900R irradiation. The bone-marrow cells are obtained from the femurs and tibias of syngeneic donor mice. Mice are sacrificed, these bones are removed, both ends are cut off, and the bone-marrow is blown out with HBSS, using a syringe and needle. The marrow cells are dispersed with a Pasteur pipette, and counted after adding an equal volume of trypan blue solution. Only the unstained cells are counted, as these represent the viable cells; the plasma membrane of viable cells does not ~ermit the entry of trypan blue. Each irradiated recipient mouse is injected with 1-2 x 10 viable marrow cells into a tail vein. In general, the viable bone-marrow cells obtained from one donor mouse are sufficient for three recipients.
Other Experimental Animals
ARMADILLO
Inoculation with H. 1eprae of the nine-banded armadillo (Dasypus novemcinctus Linn.) , initially selected because of the low core temperature (30 - 36 0C) characteris tic of this animal species, has resulted in generalized, progressive disease in a large fraction of the animals (95), and subsequent studies (2) have demonstrated the susceptibility to H. 1eprae infection of some armadillo species, and the apparent insusceptibility of others.
Armadillos cannot be bred in captivity, but must be obtained from the wild in the Western Hemisphere, to which they are native. Before inocttlation with H. 1eprae, they must be first quarantined for three to four months in a holding area, in which they are isolated from contact with wild armadillos, rodents and arthropods. During this time, they must be examined by serological and histopathological methods to exclude as far as possible the presence of mycobacterial infections. Not all nine-banded armadillos are susceptible to H. 1eprae; however, as many as 60% develop progressive and heavy systemic infection 12 - 24 months after intravenous inoculation of 108 H. 1eprae.
OTHER SPECIES
Infection with H. 1eprae of a few other animal species is of interest. Successful experimental disease (as opposed to infection without disease) of the European hedgehog (Erinaceus europaeus) has been reported (2). A few studies of non-human primates have been described. Gunders, working in Liberia, reported (70) the first successful experi mental infection of a chimpanzee, and Waters and his co-workers (209), described suc cessful experimental infection of a gibbon (Hi1obatus 1ar) in Malaysia. Finally, suc cessful inoculation of the mangabey monkey (Cerocebus sp) has recently been reported (124, 205).
Animal Husbandry and Breeding
Simply inoculating viable H. 1eprae into footpads of mice is not sufficient to ensure multiplication of the organisms. Among other requirements, it is necessary that the mice be "clean", uniform, and of a suitable strain. Mice infected with any of a number of pathogenic organisms may resist multiplication of H. 1eprae, as a consequence of an increase of non-specific immunity. On the other hand, the mice may be in such poor WHO/CDS/LEP/86.4 page 58 health that they do not survive long enough to permit multiplication of the organisms. Moreover, variation from mouse to mouse must be minimal. FInally, all other things being equal, strains of mice vary in their ability to sustain multiplication of M. leprae.
Thus, a continuing supply of uniform mice of good quality is essential to the success of a laboratory dedicated to the mouse foot-pad technique. The means of ensuring such a supply are considered under two major headings - husbandry and breeding.
HUSBANDRY OF IMMUNE-COMPETENT MICE
The husbandry of immune-competent laboratory mice includes the elements of housing, nutrition and care.
Housing
The mice must be housed in clean, vermin-proof quarters, which should be air conditioned, if possible. The practice, common in the tropics, of housing caged laboratory animals entirely in the open, except for a roof, exposes the animals to arthropods and wild rodents, with resultant contamination by pathogenic bacteria, viruses, and parasites. To exclude vermin to the maximal degree possible, the animal quarters must be fully enclosed. In order to expedite cleaning, the mouse cages should be placed above the floor, on fixed shelves or movable racks; the interior of the building in which the animals are housed should be finished; and there should be an abundant supply of clean, running water.
Ideally, the animal quarters should be maintained at a temperature of 20 - 250C. Not only do M. leprae multiply better in mice housed at this temperature, but both the health and fertility of the animals are improved. Also, humidity should be maintained constant at 80%
The breeding colony and the mice infected with M. leprae should be housed in sepa rate rooms.
Mice inoculated with M. leprae may be caged, separated by sex, in groups of five to ten" -dependi.ng upon the size of the cage; a commonly used cage suitable for housing five mice is 15 cm wide, 15 cm high, and 30 cm long (6 x 6 x 12 inches). Avoidance of crowding aids breeding, and minimizes the needed frequency of cleaning. Cages of seamless con struction, ideally fabricated from stainless steel or heat-resistant plastic (polycarbo nate) , are easily cleaned. However, cages fabricated locally from aluminium or galvanized metal are certainly suitable. Cage lids may similarly be locally fabricated from stain less or galvanized wire mesh. Shelves and racks may also be locally fabricated - from galvanized metal or laminate-on-wood (FormicaR) - to permit easy cleaning.
A final consideration is the requirement for clean bedding and water. It makes little sense to provide, at great expense, animal quarters from which vermin are excluded, and at the same time to use vermin-infested bedding. Any number of locally available materials may be used as bedding - wood shavings, sawdust, ground corn-cobs, chopped straw or hay, shredded paper or rice husks, for example. Ideally, the bedding should be auto claved shortly before use, especially if it is stored in bulk for any length of time prior to use. A layer of bedding 2-3 cm in depth should be employed. The water should be free of Salmonella sp. and other enteric organisms; if a source of clean water cannot be depended upon, chlorination [1-3 mg per 1 or parts per million (ppm) as C12] or auto claving should be considered. Addition of HCl to a final concentration of 0.003 - 0.006 N helps to minimize bacterial contamination of the water. Water is usually administered in a sterilizable bottle of 500 ml capacity, equipped with a one-hole rubber stopper contain ing a metal (stainless steel) sipper tube.
Nutrition
Mice require a diet composed of 20% protein, 10% fat, various minerals, vitamins and unsaturated fats. A nutritious diet is shown in Table 5. Locally available components WHO/CDS/LEP/86.4 page 59 should be substituted where possible. A laboratory mouse requires, on average, 3 - 5 g diet daily.
Table 5. Nutritious diets for the laboratory mouse22
Composition (g per kg diet) A. Ingredients Diet 1 Diet 2 Diet 3 Diet 4
Ground milling wheat 515.00 329.50 560.00 602.00 Nonfat skim milk, edible 200.00 120.00 200.00 200.00 50% dehulled soybean meal 112.50 67.50 112.50 25.00 Corn oil, edible grade 102.50 34.50 57.50 102.50 Dried brewer's yeast 40.00 24.00 40.00 40.00 Sodium chloride 13.75 8.75 13.75 13.75 Dicalcium phosphate 10.00 10.00 10.00 10.00 Ferric citrate 1. 25 0.75 1. 25 1. 25 Vitamin premix (calculated from C,below) 5.00 5.00 5.00 5.00 Wheat germ meal, edible 400.00
B. Components Diet 1 Diet 2 Diet 3 Diet 4
Protein 21.2 24.9 21.3 17.9 Fat 10.9 8.2 7.1 11.2 Fibre 1.7 2.1 1.1 1.1 Ash 4.7 4.7 4.8 4.3 Ca 1.4 0.9 1.0 0.9 P 0.9 1.1 0.9 0.8
Recommended daily intake C. Vitamins
per kg feed per 25-g mouse
A 242 - 5500 IU 1.1 - 2.0 IU E (a-tocopherol) 9.9 - 27 mg 1.0 JLg B12 3.9 - 5.5 mg Riboflavin 2.4-11mg Niacin 26.4 - 143 mg Pantothenic acid 9.9 - 55 mg Choline 495 - 1452 mg Folic acid 560 - 2750 mg Menadione (vitamin K activity) Pyridoxine (B6) 0.99 - 5.5 mg Biotin 19.8 - 165 mg 0.7 ID D 143 - 5060 ID Thiamine 2.2 - 9.9 mg
22 Adapted from reference no. 79. WHO/CDS/LEP/86.4 page 60
The diet can be provided in the form of pellets or mash. Administration of pellets is more convenient; these are placed in a depression in tha cage lid, or in a feeder suspended inside the cage. A mash diet possesses an obvious advantage, if a drug is to be incorporated. However, the mash must be placed in a container inside the cage, and is therefore more easily fouled and wasted by the mice. Because some components - especially vitamins - are unstable, the diet should be used within 30 days of manufacture, unless it can be stored in the cold. During storage, the diet must be protected from vermin. Finally, the diet must be free of contamination by Salmonella sp., common contaminants of foodstuffs, especially in the tropics.
Animal Care
Not only must the mice be free of contamination by pathogenic bacteria, viruses, and parasites when the mouse colony is first established, but the colony must also be main tained free of contamination. The most effective measures in maintaining a colony of disease-free mice are a high standard of cleanliness and the observance of a few simple precautions.
Cleaning cages - as a m1n1mum, replacing the soiled bedding - must be carried out once or twice weekly, depending upon the number of mice per cage. At least as frequently, water bottles must be exchanged. In addition, the cages should be inspected daily, to ensure adequate supplies of food and water. Periodically, the cages should be washed and, if possible, autoclaved. As a minimum, this must be done before a cage is used to house a new group of mice.
In addition to cleanliness of cages, food and water supply, cleanliness of the sur roundings must also be maintained. Mouse colonies produce a considerable quantity of litter, both that generated by the mice, and that created in caring for them - e.g., during cage-cleaning, and as a result of transferring feed from storage container to cage. The litter must not be permitted to accumulate, but should be removed periodically by sweeping and washing the shelves and floor.
Animal handlers should be provided with facilities for handwashing and several changes of clothing to be worn only in the animal quarters, and they should be required to use them. The clothing should be donned upon first entering the animal quarters in the morning, taken off upon leaving at the end of the work-day, and laundered frequently.
As a precaution against introducing a new pathogenic organism, it is essential that animals from another source not be introduced into the animal quarters.
Finally, cages must be clearly and unambiguously labelled. If the same cage is employed continuously, the label should be fixed to the cage by an adhesive tape that is resistant to wetting. If, on the other hand, cages are frequently exchanged, the label should take the form of a stiff card, and the cages must be fitted with card-holders that permit ready insertion and removal of the card.
BREEDING
Mice suitable for inoculation with M. leprae are widely available from commercial breeders, but it is assumed that, for reasons of economy, most laboratories will wish to breed their own. In a quantitative sense, mice breed readily. The duration of gestation is approximately 21 days, and the female is fertile immediately after parturition. The average productivity during the nine months or so of a female's active breeding life is of the order of six litters of six mice each. Thus, one can readily estimate one's require ments, calculate the number of breeding pairs or "families" needed to produce these, and add a proportion to be used to replace the breeders. The most common practice is that of "harem-mating"; one male is placed together with two or three females. WHO/CDS/LEP/86.4 page 61
If one wishes to breed mice of good quality, however, the matter is somewhat more complicated than is suggested by these quantitative considerations. As stated in the introductory paragraph, mice suitable for inoculation of H. leprae must be clean - that is, free of certain infections, uniform, and of a strain that sustains the multiplication of the organisms. In order to be certain of a supply of clean mice, one must begin with a clean breeding nucleus. This is to say, one must obtain the clean mice for a nucleus from an appropriate outside source.
A prime consideration in selecting the breeding system to be used is the uniformity of the mice. Ideally, mouse-to-mouse variation of multiplication of H. leprae should reflect variation among inocula, and not individual variation among mice. One method of obtaining uniform mice is inbreeding. This is accomplished by brother-sister or parent offspring matings. After 20 generations (about six years are required), the mice have become virtually homozygous -i.e., genetically identical.
A large number of inbred mouse strains have been developed, some of which have been tested for their ability to sustain multiplication of H. leprae. Certain strains were found more suitable than others. The two strains of inbred mice that have been most widely used are BALB/c and CBA. Several sources of clean BALB/c and CBA mice are avail- able to leprosy workers.
If one obtains a breeding nucleus of clean mice of one of these two strains, and can maintain the mice and their progeny free of certain pathogenic organisms, one is "in bus iness". However, it will be necessary to pay attention to one practical problem. Because the breeding nucleus will necessarily represent a very small fraction of the parent colony from which the nucleus was derived, random mutations may accumulate, and, in the course of many generations, cause the local strain to diverge genetically from its parent strain. One way to minimize this is to refrain from mixing, for purposes of breeding, progeny from the initially established families and the lines that result from these families. Rather, when it becomes necessary to replace breeding pairs, these should be replaced with pairs of littermates parented by the pairs being replaced. In this way, mutations will not be diffused throughout the colony, but will remain confined to single lines.
To prevent genetic drift away from the parent colony, it is also wise, from time to time, to obtain new breeding stock from the original source. These new mice should be used as a new breeding nucleus.
HUSBANDRY OF IMMUNE-DEFICIENT RODENTS
T900R and T200X5R Mice; Neonatally Thymectomized and Congenitally Athymic Rats
As has already been described, these animals are all immune-deficient to a degree; therefore, their successful husbandry imposes requirements in addition to those for hus bandry of good quality immune-competent mice. These additional requirements derive from the need to protect these animals from intercurrent -i.e., inadvertent - infection by organisms non-pathogenic for the normal rodent, but capable of producing fatal illness in the immune-deficient host. All of the requirements listed in the discussion of husbandry of the immune-competent mouse (see pp. 58 - 60) must be met.
Immune-deficient rodents must be derived from SPF if not from germ-free stock. Having started with sufficiently clean animals, one must maintain them in the same condi tion. It is particularly important that the immune-deficient rodent be protected from contact with all other rodents, including not only wild rodents, but also those brought in from another laboratory, and even those immune-competent mice raised in the same labora tory, unless one can be assured of their cleanliness.
Not only must the animals be protected from contact with other rodents, but also from infection by other agencies, such as infected animal handlers, air, food, water and bed ding. Animal handlers suffering from infections of the skin or respiratory tract should not enter the quarters in which immune-deficient rodents are housed. And the clothing, WHO/CDS/LEP/86.4 page 62
including foot-wear, worn while working with the immune-deficient animals must not be worn elsewhere; it is especially important not to wear the same outer clothing in which one has entered other animal quarters.
To prevent infection by air-borne organisms, immune-deficient rodents should be housed in rooms in which the air pressure is maintained at a higher level than that in the immediate surroundings. Air should be taken in by a system of ducts and blowers directly from the outside, preferably through a system of filters. Under no circumstances should ventilation be accomplished by an exhaust fan, which creates a vacuum in the room, allow ing air to flow in from adjacent rooms and corridors. Air should flow only in an outward direction from the room in which the immune-deficient animals are housed. Prevention of air-borne infection can be rendered more certain of accomplishment by housing the animals in cages fitted with filter-caps, or by placing the cages in laminar-flow racks, in which filtered air is directed from behind the cages outward.
The certain means of preventing food-borne infection is to sterilize the food. Sterilization may be accomplished by one of two means - irradiation and autoclaving. If the animal diet can be irradiated, this is preferred. If irradiation is not feasible, the diet should be sterilized by autoclaving. However, exposure of the diet to the heating involved in autoclaving is destructive to many of the vitamins, which must be replaced by addition either to the diet or the drinking water. Needless to say, sterilization must be carried out with the food sealed in plastic (not suitable for autoclaving) or tough paper or cloth bags, which will prevent subsequent entry of vermin during storage. More over, because storage at ambient temperature will also result in loss of vitamins, the time between manufacture of the diet and its use must be minimized. When the diet must be stored for longer than a few weeks, storage should be carried out at a temperature below lOoC.
Water should be sterilized by autoclaving. This may be most conveniently accom plished by autoclaving the water in the drinking-bottles. Adequate chlorination of the water may suffice as a substitute for autoclaving, and acidification of the sterile water (see p. 58) will help to limit contamination of the water during use, which is inevitable unless one is working under germ-free conditions. Finally, as an aid in maintaining immune-deficient animals, oxytetracycline may be administered in the drinking water in a concentration of 125 mg per 1. Moreover, treatment of the animals for periods of a few weeks with drinking water to which have been added mixtures of antibiotics that are not absorbed from the gastrointestinal tract has been proposed (27) as a means of decontam inating immune-deficient animals.
Bedding, sealed in vermin-proof paper or cloth bags, should also be autoclaved before use.
Among the four immune-deficient animals discussed in this section - T900R mice, T200X5R mice, neonatally thymectomized rats and nude rats, the first and the last are more immune-deficient than the remaining two, and, as a consequence, their husbandry is more demanding.
Nude Mice
Nude mice are more immune-deficient than any of the four animals just mentioned. As a consequence, they are even more suitable hosts of H. leprae, which appear to require for optimal growth a degree of immune-deficiency of the experimental host that is ordinar ily incompatible with life.
Nude mice ordinarily do not survive for more than a few months unless they are bred from very clean stock and housed under "barrier" (i.e., SPF or germ-free) conditions. Experiments involving H. leprae require much of the lifespan (approximately 2 years) of an immune-competent mouse, and nude mice survive to a normal lifespan only under the most rigorous conditions. Therefore, their husbandry is more difficult and more demanding than WHO/CDS/LEP/86.4 page 63 that of the other four immune-deficient animals, and requires considerable investment, in terms of equipment, man-hours, and operating costs.
Nude mice must be housed in laminar-flow racks or germ-free isolators, and work in germ-free isolators proceeds extraordinarily slowly. Everything that comes into contact with the nude mice must be sterile, including the inoculum (except, of course, for H. 1eprae). And "sterility" implies freedom not only from bacteria and fungi, but also from mouse viruses. Because inocula of H. 1eprae cannot be autoclaved or irradiated or sterilized by exposure to ethylene oxide or surface decontaminants such as peracetic acid, special measures are required (see pp. 66 - 67).
Good general references for work with germ-free and nude mice appear in the biblio graphy (61, 80, 133, 167).
Mouse Foot-Pad Techniques
The mouse foot-pad technique for "cultivation" of H. 1eprae has assumed a role of primary importance in leprosy research activities, since its description by Shepard in 1960 (171, 172). This is the only technique by which one can determine directly whether H. 1eprae are viable or dead. The technique has many applications, which are dealt with in later sections. The purpose of this section is to describe in detail two techniques those of Shepard and Rees. Both techniques are currently employed, each in a number of laboratories, with complete success, and there appears to be no scientific reason to promote the use of one over the other.
SHEPARD'S TECHNIQUE23
Recovery of H. 1eprae from Human Biopsy Specimens
Working under aseptic conditions in a microbiological safety cabinet, and employing sterile equipment and materials, the biopsy specimen is trimmed free of fat, weighed, placed in a tared Petri dish, and minced with sharp scissors after addition of 1 or 2 drops of HBSS, until lumps of tissue are no longer detected. The tissue mince is then transferred on the blades of the scissors to a cup of a Mickle tissue disintegrator; one layer (20 to 30) of 3 mm glass beads is placed in the Mickle cup before sterilization; pyrexR or KimaxR beads are preferred, as other beads may be too fragile. Two ml HBSS are added, and the cup is tightly stoppered and vibrated for 1 minute with an amplitude of 5 mm and a frequency of 50-60 cycles/sec. The volume of the resulting tissue-homogenate is measured in a pipette, and the homogenate is simultaneously transferred to a test-tube. HBSS and bovine serum albumin (BSA) are added to a final volume of 2.5 ml and a final albumin concentration of 0.1%. The suspension is then thoroughly mixed by means of a pipette and rubber bulb, to promote aggregation of tissue-fibres, and allowed to stand for exactly 2 minutes. The resulting supernate is removed carefully with a pipette, its volume is measured, and it is transferred to a test-tube. An aliquot of the supernate is taken for inoculation of bacteriological culture media (usually L6wenstein-Jensen medium and tryptose agar) and for preparation of smears for counting.
Preparation of Smears and Counting AFB
Microscope slides are available with three fused ceramic circles, each about 1 cm in diameter. The actual size of the circles must be measured on a few slides selected at random from each new lot; this can be conveniently done to an accuracy of 0.01 cm with the microscope, by use of the vernier on the mechanical stage (see pp. 17 - 18).
To prepare smears for counting AFB, a volume of 10 ~l of formol-milk is first applied to each of the three circles. Then, 10 ~l of the suspension to be counted are added to the circle, and the liquids are immediately mixed and spread over the entire area
23 Adapted from references no. 172, 192 and 219. WHO/CDS/LEP/86.4 page 64
of the circle with the tip of a platinum wire bent to an angle of 60 0 . One circle is completed before the aliquot of suspension is added to the next; the wire is flamed between circles. The 10-~1 volumes may be applied with disposable capillary micropi pettes, or with one of the several models of automatic micropipette that use disposable, autoclavable tips. These operations are carried out on a levelling table on which the slides remain until dry. The smears are then fixed by the method involving formaldehyde fumes and controlled heating, and stained by the acid-fast stain (see pp. 23 - 24).
The AFB are counted under optimal conditions, employing clean lenses, Kohler illumi nation, 100 x apochromatic oil-immersion objective and 10 - 12.5 x compensating oculars. AFB are counted in fields selected every 0.5 mm across the equator of all three circles, employing the scale on the mechanical stage; thus, about 20 fields per circle or a total of 60 fields per slide are examined. When 10 ~l of suspension have been spread over a circle of diameter D, and the suspension is viewed with a microscope of field-diameter d, the following formula applies, employing the same units for d as for D:
no. AFB no. AFB counted 1 ml x x
ml sample no. fields counted 10 ~l
no. AFB counted X 100 X (D/d)2.
no. fields counted
Inoculation into Mice
The suspension is diluted with HBSS to contain 5 x 10 3 (5000) AFB per 0.03 ml. Employing a l-ml syringe and sharp 27- or 30-gauge needle, the diluted suspension is then injected subcutan~ously into the mouse footpad, so that the tissues are filled. A meas ured volume is not injected. Rather, the initial volume of inoculum in the syringe is recorded; then, after each group of mice has been inoculated, the injected volume is measured by difference, and the average volume per footpad calculated. Ordinarily, this volume is close to 0.03 ml. Usually, 20 - 30 mice are inoculated, each in one hind foot pad.
Harvesting H. leprae from Mouse Footpads
Monthly, beginning usually 3 months after inoculation, one mouse is killed by cervi cal dislocation, and the inoculated foot is removed, fixed in 10% formalin, and decalci fied by a gentle procedure that permits the H. leprae to be stained by the acid-fast technique. After fixation in formalin for 3 days, the foot is placed in a flask contain ing a solution of 5% formic acid in 70% ethanol, and held at 370C for 7 days, with daily changes of the decalcification solution. For the last 3 days, the flask is shaken gently on a rotary shaker. The fixed decalcified foot is then processed for histopathological examination (see p. 36 - 38), and sections are stained for AFB.
When significant numbers of AFB (more than 30 per section) are seen in a monthly specimen, more mice (usually 4) are killed, and pinned conveniently on a dissecting board. The inoculated feet are then cleansed with soap and water with the aid of gauze squares, rinsed with sterile water, and dried with sterile gauze squares. The foot-pad tissues are then removed in three layers - skin and subcutaneous tissue, tendon, and muscle - with a sterile scalpel and haemostat. The tissues are minced and homogenized, as described for human skin-biopsy specimens (see pp. 63), smears are prepared from the resulting suspen sion of H. leprae, and the AFB are counted, as already described. WHO/CDS/LEP/86.4 page 65
REES' TECHNIQUE24
Recovery of M. leprae from Human Biopsy Specimens
The biopsy specimen, trimmed of fat, is minced with sharp scissors, placed in a glass tissue grinder of 15 ml capacity, together with 2 ml HBSS containing 0.1% BSA, and ground until clumps of tissue are no longer detected with the naked eye. New glass tissue grin ders release glass particles into the homogenate, so old grinders should be used; these should first be tested by grinding water or saline, and inspecting the liquid with the naked eye for glass particles.
Preparation of Smears and Counting AFB
Standard 76 x 25 mm microscope slides are used on which four circles, each 8 mm in diameter, have been placed by etching or sand-blasting, or the slides may be placed over a template of 8 mm-in-diameter circles drawn on paper. The slides must be chemically clean and free of fat.
The standard volume of bacillary suspension is spread over the 8 mm circle by means of a 2 mm platinum loop, made as for an inoculating loop, but bent to an angle of 45 0 , and the circle of the loop opened slightly so that the contents will be completely released on touching the slide. The average volume delivered by the loop is first calculated from the weight of 50 deliveries of 0.1% BSA in distilled water, assuming that 1 ml weighs 1 g.
The suspension is sampled by inserting and withdrawing the loop vertically through the surface, with the tube sloped so that the surface of the suspension and the loop are parallel. The sample is then transferred to the slide, by placing the loop horizontally at the centre of the circle, and spread so that it completely fills the circle. The loop is flamed after each sampling, and four smears (one slide) are made.
The smears are air-dried, heat-fixed for five minutes on a hot plate that has been adjusted to 600C with the aid of a thermocouple, exposed to formaldehyde fumes for five minutes and heat-fixed again for five minutes. The fixed smears are stained by exposure to steaming carbol fuchsin for two minutes, gently washed with tap water, and counter stained and decolourized simultaneously by exposure for one minute to a solution of 0.2% aqueous methylene blue in 4% sulfuric acid. The slides are again gently washed in tap water and air-dried.
Optimal conditions of microscopy are employed. The area of the microscope field is determined from the diameter, which is measured with a slide micrometer. The smears are mounted with immersion oil, and so placed on the microscope stage that the first field is on the maximal diameter of the smear. AFB are counted in this first field, and in a total of eight fields, 1 mm apart along the diameter of the smear, using the scale on the mech anical stage. The numbers of AFB counted in eight fields are totalled, and the totals of the four smears are averaged.
Having determined the volume of suspension delivered by the loop and the area of the microscope field, the conversion factor for the loop and microscope is calculated:
area of circle 1 ml 16 1 x x 8 x area of field volume delivered by loop v
24 Adapted from reference no. 219. WHO/CDS/LEP/86.4 page 66
where r = the radius of the microscope field in mm, and v = the volume delivered by the loop in ml. The concentration of organisms per ml of suspension is then:
Conversion Factor x average no. AFB/field x dilution of the suspension.
When the number of AFB is very small, one counts across the full diameter of each of the four smears, and calculates the average number per smear. In this case, the conver sion factor = 16 IT/8D x l/v, where D = the full diameter of the microscope field in mm.
Inoculation into Mice
The suspension of M. 1eprae is diluted with HBSS to a final concentration of 104 (10 000) AFB per 0.03 ml, and a volume of 0.03 ml, measured in a glass tuberculin syringe of 0.5 ml capacity, is inoculated into each hind footpad of 6-12 mice.
Harvesting M. 1eprae from Mouse Footpads
All procedures are carried out using sterile materials in a microbiological safety cabinet. The hind footpads are cleansed with ethanol, and the mouse is pinned out on its back with hind legs so held that the footpads are uppermost. Each footpad is harvested separately, using a fresh set of instruments. One drop of 0.1% BSA in water is placed in a Petri dish. The bulk of each footpad is cut off from heel to base of the toes with small, curved scissors and placed in the drop in the Petri dish. Then, with a sharp pointed scalpel blade, all of the remaining tissue of the footpad and that between the underlying bones is scraped off and added to the tissue in the Petri dish. With the same scissors, the pooled tissue is thoroughly minced, and, with a blade of the scissors, transferred to and spread over the piston of the tissue grinder, confining the tissue mince to the lower end of the piston. The grinder is assembled, and the tissue is thor oughly homogenized in an ice bath both before and after adding 2 ml 0.1% BSA in water. Smears of the resulting suspension of M. 1eprae are prepared, and the AFB counted, as already described.
PREPARATION OF INOCULA FOR DETECTION OF PERSISTING M. LEPRAE
An important application of immune-deficient rodents is their use in the attempt to detect persisting M. 1eprae (this application is discussed in detail on pp. 73 - 77). Briefly, at least 10 5 M. 1eprae, or as many as possible, are injected into each hind footpad of a group of immune-deficient rodents, taking advantage of the fact that these animals are not so readily immunized by large inocula. Organisms are harvested approxi mately one year later, by which time multiplication should be evident, even if the large number of organisms inoculated had included only a very few viable M. 1eprae.
Because the demonstration of persisting organisms is commonly attempted after some considerable duration (at least one year) of treatment, by which time the BI has decreased, and only small numbers of M. 1eprae can be recovered from the biopsy speci men, it may be difficult to prepare a concentrated inoculum. To maximize the number of organisms that can be inoculated, recovery of the organisms from the biopsy specimen must be carried out with minimal dilution. The following method has been proposed (201).
All procedures are carried out using sterile materials in a microbiological safety cabinet. First, the biopsy specimen is trimmed of fat and weighed, to determine the volume of suspending fluid (0.1% BSA in water) required, as shown in Table 6. The tissue is then thoroughly minced with small curved scissors and transferred in its entirety, by scraping up with the scissors, to the lower end of the piston of a tissue grinder that has been immersed in ice water. A grinder of 15 ml capacity is employed for specimens to be suspended in 2 ml. The minced tissues are very thoroughly homogenized, and smears are prepared as described for other biopsy specimens. Although the resulting homogenates are dense, they are adequate, without further dilution, for visualizing AFB in the stained smears, and for inoculation of footpads. The homogenates are inoculated into mice whether WHO/CDS/LEP/86.4 page 67
or not they are seen to contain AFB. The inoculum is 0.03 ml per footpad; groups of eight or more mice are inoculated in both hind footpads.
PREPARATION OF INOCULA FOR NUDE MICE
Nude mice must be inoculated with material that is sterile, except for its content of viable M. 1eprae. Inocula contaminated with murine viruses must be avoided, as viruses non-pathogenic for immunologically intact mice may be pathogenic for nude mice, and there exist no means by which such inocula may be decontaminated. Therefore, M. 1eprae har vested from intact mice should not be used to inoculate nude mice. Rather, the inoculum of M. 1eprae must be prepared from infected human or armadillo tissues, or from those of other nude mice. Even so, the suspension of M. 1eprae must first be checked for steril ity by culturing for bacteria and fungi, and decontaminated by digestion with NaOH if bacterial or fungal contamination is found.
Table 6. Dilution of biopsy specimens for inoculation of immune-deficient mice25
Weight of specimen Volume of suspending fluid (g) (ml)
< 0.04 0.5 0.05-0.07 0.8 0.08-0.10 1.0 0.11-0.15 1.5 0.16-0.20 2.0 0.21-0.25 2.5 0.26-0.30 3.0
Digestion with NaOH is carried out by adding NaOH to a final concentration of 0.25 N, and neutralizing the solution by the dropwise addition of O.5N HCl after incubation for one hour at room temperature. This treatment will also kill a proportion of the M. 1eprae.
Applications of Animal Inoculation
Inoculation of laboratory animals with M. 1eprae may be applied to both clinical and laboratory research. The more clinical applications to be discussed include (1) detection of viable M. 1eprae; (2) drug-susceptibility testing; and (3) detection of persisting M. 1eprae. The more laboratory-oriented applications are (1) screening of drugs for activity against M. 1eprae; and (2) the experimental chemotherapy of leprosy.
CLINICAL APPLICATIONS
Detection of Viable M. 1eprae
The application of Shepard's mouse footpad technique to measure the rate at which M. 1eprae are killed during effective dapsone treatment of lepromatous patients, repor ted in 1968 (185), provided for the first time a reasonably quantitative and sensitive means of evaluating the antimicrobial activity of individual drugs. In this application, mouse inoculation is employed in much the same way that sputum culture is employed in assessing the efficacy of an antituberculosis drug.
25 Adapted from reference no. 219. WHO/CDS/LEP/86.4 page 68
A skin lesion large enough to permit several biopsies is chosen. A biopsy is per formed before the start of treatment, the resulting 50 to 100 mg specimen providing 106 to 10 8 organisms if the patient is lepromatous and previously untreated. Employing one of the mouse-foot-pad techniques described (see pp. 63 - 66), H. 1eprae are recovered from the biopsy specimen, counted and diluted so as to provide an inoculum of 10 3. 7 to 104 AFB per footpad, and the hind footpads of 10 to 20 normal mice are inoculated.
According to Shepard's technique, one mouse is sacrificed every month, beginning about three months after inoculation, and the inoculated hind footpad is fixed, lightly decalcified, and processed for histopathological examination, after which paraffin sec tions are stained by an acid-fast stain. The presence in a section of a "significant lesion" - one filling at least one-fourth of a 540 x microscope field with brightly-stain ing AFB with or without a typical infiltrate - indicates the end of the "incubation per iod" (IP), the time elapsed between inoculation and appearance of an important number of AFB in a histopathological section, and the signal for performing a harvest.
Additional mice are killed, and H. 1eprae are harvested from the pooled tissues, usually of four inoculated footpads. The AFB are enumerated, and the "generation time" (G) is calculated as if multiplication of H. 1eprae had occurred at a constant rate from the day of inoculation until the day of harvest.
Additional biopsy specimens are obtained in the course of treatment, mice are inocu lated, and killing of H. 1eprae is indicated by progressive increases of the values for IP and G, as shown in Figure 18. This illustrates the application of the technique to a patient with previously untreated lepromatous leprosy who was treated with dapsone in a daily dose of 50 mg. An IP > 12 months or G ~ 100 days is evidence of failure of the H. 1eprae to multiply in the mice. In the uppermost panel of this figure, the number of AFB recovered from skin-biopsy specimens, obtained at intervals during treatment, may be seen to have decreased only slightly in the course of about one year of treatment. In the second panel, the solid ratio, very low to begin with, fluctuated between 0 and 5 solids per 100 AFB, before solid organisms finally disappeared about eight months after the beginning of treatment. Infectivity of this patient's H. 1eprae for the mouse decreased during the first several months of treatment, as shown in the lower panels. The result at 90 days is characteristic of the irregular results encountered as the infectivity of the organism for the mouse becomes marginal. H. 1eprae appear to have multiplied in the single mouse sacrificed for histopathological study 11 months after inoculation (there fore, IP 11 months), but not in the four mice sacrificed for harvest at that time, yielding G ~ 100 days.
Employing this technique, several clinical trials were carried out in San Francisco and in Cebu, the Philippines. These trials established that, on average, H. 1eprae recovered from patients lost their infectivity for mice after 100 days of treatment with dapsone, 50 to 100 mg daily (19, 114, 185, 187, 188); 150 days of treatment with clo fazimine, 100 to 200 mg daily or 100 mg three times weekly (20, 114); longer than 150 days with acedapsone (4,4'-diacetamidodipheny1su1fone, DADDS), 225 mg intramuscu1ar1y every 77 days (187), or c10fazimine, 600 mg every two weeks or 1 200 mg every four weeks (20); and within a few days of single 600 - 1 500 mg doses and daily 300 - 600 mg doses of rifampicin (19, 115, 153, 188, 189).
In addition to chemotherapeutic research in patients, inoculation of immuno10gically intact mice for the detection of viable H. 1eprae may be useful in other situations. Such a situation is presented by the "problem" patient, who appears with active lesions, having begun treatment elsewhere, and having taken the prescribed treatment only irregu larly. Is he responding normally, responding slowly, or relapsing? Inoculation of mice might provide the answer. Failure to detect viable H. 1eprae might suggest a normal response; slow response or relapse caused by failure to take sufficient quantities of an antimicrobial drug might be suggested by the finding of viable, drug-susceptible organ isms; and the demonstration of viable, drug-resistant H. 1eprae would be consistent with drug-resistant relapse. WHO/CDS/LEP/86.4 page 69 Figure 18. Results of treatment with dapsone of a patient with previously untreated lepromatous leprosy26
'0 .S 0 co u, g :f
~ 100 x X .L-J. X ,1-._-l----+.----'- X ..L---'-_-'- X .1...--'-_.1.---'------' - 100 200 300
TIME AFTER START OF DAPSONE (DAYS)
Drug-Susceptibility Testing
Measurement of the susceptibility to drugs of strains of H. leprae - i.e., the detection of drug-resistant H. leprae - recovered from patients is one of the most important applications of the mouse footpad-technique. In this application, drug-treated mice serve the same purpose as do drug-containing culture media in the laboratory study of patients with other infections. In order to carry out this measurement, organisms are inoculated in the footpads of mice, and several dosages of the drugs are administered to some of the mice, either incorporated into the diet, by gavage, or, less commonly, by injection, while some of the inoculated mice are not treated and serve as controls. Multiplication of H. leprae in both the control mice and those to which drug has been administered indicates that the organisms are resistant to the drug.
Procedure. An active-appearing lesion is biopsied, the biopsy specimen is minced and homogenized, H. leprae are counted, the bacterial suspension is diluted to provide the usual inoculum of 103. 7 - 104 organisms per footpad, and the hind footpads of immun~ logically intact mice are inoculated. The mice are divided among a number of groups, 10-20 per group; one group is held without treatment, and the mice of the remaining groups are administered drug in two or three dosages; for each drug, the smallest of these concen trations is the minimal effective dose (MED), the smallest concentration of the drug that
26 Adapted from reference no. 185. WHOjCDSjLEPj86.4 page 70 inhibits multiplication in mice of M. 1eprae from all previously untreated patients. The larger concentrations are usually 10- and lOO-fold multiples of the MED. The concen trations that have been employed in testing susceptibility of M. 1eprae to the most commonly employed drugs - dapsone, rifampicin, clofazimine and protionamide or ethionamide - are shown in Table 7.
Thus, to measure the susceptibility of strains of M. 1eprae to dapsone, a most important measurement now performed in many laboratories, organisms are inoculated in the footpads of mice fed drug-free diet and of mice fed diets containing dapsone in concentra tions of 0.0001, 0.001 and 0.01 g per 100 g diet. Strains isolated from previously untreated patients, at a time when dapsone resistance was rare and primary resistance had not yet been recognized, were uniformly unable to multiply in mice fed dapsone in a diet ary concentration of 0.0001 g per 100 g (186); this dosage was therefore taken as the MED. Dapsone-containing diets are fed from the day of inoculation. Harvests are made at intervals from control mice. When the organisms are noted to have multiplied in control
Table 7. Concentrations of drugs to be employed in testing of drug-susceptibility of M. 1eprae
Drug Concentrations (g per 100 diet)
Clofazimine 0.0001, 0.001, 0.01 Dapsone 0.0001, 0.001, 0.01 Ethionamide or Protionamide 0.01, 0.1 Rifampicin 0.003, 0.03
mice to the level of approximately 5 x 105, harvests are made from mice of all treated groups. Multiplication of M. 1eprae in both the control mice and those fed the dapsone containing diets indicates that the organisms are resistant to dapsone. M. 1eprae are said to be susceptible to a certain concentration of a drug if the organisms multiply in untreated control mice, but not in mice administered the drug in that concentration.
In Table 8 are shown the results of a study of four patients, in which sus ceptibility of the patients' M. 1eprae to dapsone was measured by Rees' technique (219; see also pp. 65 - 66), in which mouse footpads are harvested individually. The organisms of patients no. 1, 2 and 4 multiplied in mice administered 0.01 g% dapsone, as well as in the untreated control mice, and may be concluded to be fully resistant, because they multiplied in mice administered dapsone in the largest dosage. Only the M. 1eprae of patient no. 3 were fully susceptible - i.e., they were inhibited from multiplying even by the smallest dosage of dapsone - 0.0001 g%.
In Table 9 are presented the results of another study, in which susceptibility to dapsone of five patients' M. 1eprae was measured by Shepard's technique (186; see also pp. 63 - 64). In this study, the M. 1eprae of patients no. 1 and 5 were fully suscep tible to dapsone, as shown by their failure to multiply in mice administered the smallest dosage of dapsone. None of the patient strains of M. 1eprae was fully resistant; however, those of patients no. 2, 3 and 4 multiplied in mice administered the intermediate dosage of dapsone (0.001 g%). WHO/CDS/LEP/86.4 page 71
Table 8. Measurement of the susceptibility to dapsone of strains of M. leprae, in terms of the proportion of mice showing multiplication27
Number mice showing multiplication/number inoculated
Dapsone concentration (g per 100 g diet)
Patient no. o 0.0001 0.001 0.01
1 5/5 5/5 5/5 5/5 2 5/5 3/5 2/5 1/5 3 2/6 0/5 0/5 0/6 4 5/5 5/5 5/5 5/5
Table 9. Measurement of the susceptibility to dapsone of strains of M. leprae, in terms of the generation time, G28
G(days)
Dapsone concentration (g per 100 g diet)
Patient no. 0 0.0001 0.001 0.01
1 23.7 >100 >100 >100 2 14.9 33.6 32.1 >100 3 22.3 31. 2 52.3 >100 4 22.8 32.7 41.8 >100 5 25.4 >100 >100 >100
Testing M. leprae for susceptibility or resistance to drugs other than dapsone is carried out by these same methods.
Because of the importance of the measurement of the susceptibility of strains of M. leprae to dapsone, both to the patient and to the community, the measurement must be carried out with extreme care. This implies meticulous attention to detail in the prepar ation of the dapsone-containing mouse diets, and control of the quality of the diets by assaying samples for their content of dapsone.
Preparation of dapsone-containin& mouse diets. Dapsone is added, either as a crystalline solid or in solution in ethanol, to mouse diet in powder (mash) form and thoroughly mixed in a blender. If one has available a blender suitable for mixing liquids with solids, one
27 Adapted from reference no. 141.
28 Adapted from reference no. 113. WHO/CDS/LEP/86.4 page 72 simply prepares a stock solution by dissolving a weighed quantity of dapsone in 95% etha nol. A convenient stock solution of dapsone is prepared by dissolving 5 g crystalline dapsone in 500 ml 95% ethanol.
In preparing a series of dapsone-containing diets, one should begin with the diet containing the lowest concentration of the drug. Thus, one can prepare diets of progres sively larger concentration without stopping to wash the blender between diets, there being no possibility of contamination of a diet with one of 10-fold higher concentration. The weighed portion - usually 5 kg - of mash is divided between the two shells of the blender, which are then tightly closed. The blender is activated, and a 50 ml aliquot of the dapsone solution is slowly added the attached funnel and tubing. After addition of the solution has been completed, the funnel and tubing are washed through with several 10 ml portions of 95% ethanol, and the blending is continued for one hour. The diet contain ing 0.0001 g dapsone per 100 g diet is prepared from a 1:100 dilution of the stock solu tion; that containing 0.001 g dapsone per 100 g diet is prepared by adding to 5 kg batches of mash in the blender 50 ml aliquots of a 1:10 dilution of the stock solution. A 50 ml aliquot of the stock solution is added to each 5 kg batch of mash in the blender, in order to prepare the diet with the highest concentration of dapsone - 0.01 g per 100 g diet.
If no liquid-solid blender is available, but only one suitable for mixing solids with solids, one begins by diluting a weighed proportion of crystalline dapsone with a small quantity of some inert solid, such as lactose or mash diet. The solid drug, thus diluted, is further diluted by adding weighed portions to weighed amounts of diet in the blender. Although one may be tempted to prepare diets of higher dapsone concentration first, and to dilute portions of these in order to prepare the diets of lower concentration, one runs the risk of contamination, without thorough washing of the blender between diets. As was the case when adding dapsone in solution, one should begin with the diet containing the lowest concentration of dapsone.
Thus, 2.5 g crystalline dapsone are added to 250 g lactose or mash and mixed thor oughly by hand, yielding a stock with a dapsone concentration of 10 g per kg diet. Two serial ten-fold dilutions are prepared by adding a 25 g portion of the stock to 225 g mash, mixing thoroughly, adding a 25 g portion of this 1:10 dilution to another 225 g batch of mash, and mixing thoroughly once again. This procedure yields additional stocks with dapsone concentrations of 1 g per kg and 0.1 g per kg, respectively. Fifty gram portions of each stock, beginning with that of the lowest dapsone concentration, are then added to 5 kg batches of mash in the blender, and the blender is activated for one hour. (It is assumed that one will wish to prepare 20 kg of each diet at once. This is suffic ient for 20 mice for 200 days, the quantity needed for one measurement of dapsone suscept ibility.)
Preparation of mouse diets containing other drugs. Because of the great convenience of administering drugs to mice by incorporating them into the mouse diet, this is the most desirable mode of drug-administration. It may be used for all drugs except those that are not absorbed from the gastrointestinal tract and those drugs that are not stable when incorporated into the mouse diet (rifampicin appears to be an example of such a drug). Drugs that are not absorbed from the gastrointestinal tract of the mouse must be adminis tered by injection. Those drugs that are not stable when incorporated into the diet may be administered through a cannula into the oesophagus - i.e., by "gavage" - or by injec tion. Clofazimine, ethionamide and protionamide may be incorporated into the mouse diet in the manner described for incorporation of dapsone. Because of the limited solubility of these compounds in ethanol, it is necessary to add these drugs to the mouse diets as solids. WHO/CDS/LEP/86.4 page 73
Assaying dapsone in mouse diets. 29 Simply m~x~ng the required amounts of dapsone in the diet mash does not ensure that the dapsone will be present in the diet in the desired concentration. It is necessary to verify that the weighings have been carried out cor rectly and the mixing thorough. This is done by analysing samples of the prepared diets for dapsone by the following procedure.
Two gram portions of each of the dapsone-containing diets are extracted by shaking them vigorously for at least one minute with 20 ml ethyl acetate in a stoppered centrifuge tube on a vortex mixer. After centrifugation and filtration of the ethyl acetate (upper) layer, 10 ml of the ethyl acetate extract is back-extracted by shaking with 2 ml 2N HC1. Prior to extraction with HC1, ethyl acetate extracts from the diets containing the highest concentrations of dapsone are first diluted with ethyl acetate to give a calculated con centration of about 1 mg dapsone per ml -i.e., a 10-fold dilution for diets prepared to contain 0.01 g dapsone per 100 g diet. One ml aliquots of the 2N HCl extracts are then diluted with equal volumes of ethanol and reacted by the successive addition at 5 min intervals of 2 drops (0.01 ml) of freshly prepared 1% (w/v) aqueous sodium nitrite, 10% (w/v) aqueous ammonium sulfamate, and 1% (w/v) N-l-naphthylethylenediamine dihydrochloride in 1:1 acetone/water (v/v). Fifteen minutes later, the extinction (absorbance) of the reaction product is measured at 570 urn by means of a spectrophotometer. The concentration of dapsone in the mouse diet is calculated by comparison with the results obtained by extracting 10 ml ethyl acetate containing 1 ~g dapsone per ml with 2N HCl and reacting it in the same way. The results should be corrected for any Bratton-Marshall positive compounds extracted from diet to which no dapsone had been added.
Assay of mouse diets for other drugs. No methods have yet been described for the assay in drug-containing mouse diets of drugs other than dapsone. Clofazimine may be assayed in mouse diets by the following method, which has been applied to blood (101).
Two-gram portions of each of the clofazimine-containing diets are suspended in 5 ml IN acetic acid, and extracted by shaking them vigorously for at least one minute with 15 ml benzene in a stoppered centrifuge tube on a vortex mixer. After centrifugation and filtration of the benzene (upper) layer, 10 ml of the benzene extract is back-extracted by shaking with 2 ml 25% (v/v) H2S04' The concentration of clofazimine in the 25% H2S04 extract is measured spectrophotometrically by means of the native absorbance of the drug at 530 urn.
To assay rifampicin in rifampicin-containing mouse diets, the following method,30 adapted from that of Sunahara and Nakagawa (202) is appropriate. One g diet is suspen ded in 3 ml distilled water, and the suspension is extracted with 6 ml amyl alcohol (2 methylbutanol). The extract is filtered, and its absorbance at 475 nm is then measured in a spectrophotometer.
Detection of Persisting M. 1eprae
Microbial persistence may be defined as the survival in the host of a small fraction of the bacterial population despite treatment with bactericidal concentrations of drugs, to which the organisms are fully susceptible (204). A poorly understood phenomenon, microbial persistence is a property of many bacteria in addition to M. 1eprae.
The first demonstration of persisting M. 1eprae was reported by Waters and his colleagues (212). Seven of 12 lepromatous patients who had completed at least 10 years of continuous dapsone monotherapy were found to harbour small numbers of M. 1eprae cap able of infecting mice; three strains were passaged into new mice and found to be fully susceptible to dapsone. These findings were in contrast to those of Shepard and his
29 Taken from reference no. 48.
30 Contribuced by G.A. Ellard WHO/CDS/LEP/86.4 page 74 colleagues, who reported (185) that H. 1eprae became non-infective for mice after an average of 100 days of treatment with dapsone.
An explanation of this paradox is attempted in Table 10. As shown in this table, a heavilr infected patient with lepromatous leprosy begins treatment with a total population of 101 H. 1eprae, which is consistent with a BI of 4+. [Shepard has estimated (173, 174). that a 60 kg man has a skin surface-area of 1.70 m2 , and that the skin is involved to a depth of 2 mm. Assuming the skin to be 30% involved, that 1 cm3 of skin weighs 1 g, and that a BI of 4+ is equivalent to a concentration of 10 8 AFB per g skin, one may calcu late that the total population of H. 1eprae of such a patient is of the order of lOll.] The MI is 10%, indicating that 10% of this patient's organisms are viable, and the H. leprae recovered from a pre-treatment biopsy specimen infect mice. [A biopsy speci men from this patient might contain 10 7 - 10 8 H. leprae, of which 10 6 - 10 7 are viable (28)] .
Table 10. Course of events during dapsone monotherapy of multibacillary leprosy 31
Results Interpretation
Duration of Treatment BI MI Mouse Total Viable (months) Inoculation H. leprae H. 1eprae
0 4+ 10% + lOll 1010
1 4+ 1% + 1011 10 9
2 4+ <1% + lOll 10 8
3 4+ <1% lOll <108
12 3+ <1% 1010 <107
24 2+ <1% 10 9 <106
36 1+ <1% Not 10 8 <105 possible
120 0-1+ Not + <10 7 ? 32 possible
At the end of the first month of dapsone therapy, 90% of the viable H. leprae have been killed; accordingly, the MI has decreased to 1%. As the inoculum of 10 3. 7 to 104 organisms contains about 50 to 100 viable H. 1eprae, this patient's organisms remain infective for mice. By the end of the second month of treatment, 90% of the remaining viable H. 1eprae have been killed, and no solidly staining organisms are seen when 100 are examined in the course of measuring the MI. By this time, the inoculum contains only
31 Adapted from reference no. 103. 32 This number is certainly no greater than the total number of H. 1eprae «107) at 120 months, but can be no smaller than 1/1000 of the total number. WHO/CDS/LEP/86.4 page 75 the minimal number of organisms required to infect mice, and some mice escape infection, rendering the results of mouse inoculation irregular.
By the end of the third month of treatment, the patient's M. 1eprae are no longer infective for mice, simply because the proportion of viable organisms has become so small. The total bacterial population, the great majority of which is dead, has not decreased appreciably until the end of the first year of treatment, by which time it has decreased by 90%. Throughout the remaining years of this patient's treatment, so long as his organ isms do not infect mice, one may conclude only that the viable M. 1eprae comprise less than 1:1000 of the total.
On the other hand, the demonstration by Waters et al., (212) that M. 1eprae multiplied in normal mice inoculated with no more than 104 AFB per foot pad indicates that the proportion of viable organisms had increased to at least 1:1000. This could not have resulted from multiplication of M. 1eprae in the patient, because the organisms were fully susceptible to dapsone, and the patient was continuously treated with dapsone in full dosage (100 mg by mouth daily, or 350 mg by intramuscular injection twice weekly). Therefore, the appearance late in therapy of organisms infective for mice could only have resulted from clearing of the dead organisms, whereas the viable organisms remained. That is, the number of viable organisms fails to decrease in proportion to the decrease of the total bacterial population, and the decrease of the total population is accomplished primarily at the expense of the dead organisms.
Detection of persisting M. 1eprae was reported subsequently in patients with lepro matous leprosy after treatment with acedapsone, 225 mg intramuscularly every 75 days for three to four years (166), after treatment with rifampicin as monotherapy in a daily dose of 600 mg for from two to five years (156), and after treatment with the combina tion 600 mg rifampicin plus 100 mg dapsone, each drug administered in a full daily dose for six months (64).
In Table 11, an explanation is attempted of the results of therapy with daily rifam picin for two years reported by Rees and his co-workers (156). Here, as in the previous example, at the end of two years of treatment, a small inoculum (103. 7 to 104 M. 1eprae) fails to infect normal mice; however, a larger inoculum (105 organisms) infects immuno suppressed mice. Because the minimal number of viable M. leprae required to infect T900R mice is the same (about five) as that required to infect normal mice, the proportion of viable M. leprae may be estimated to be about 1:104. As was stated previously, the proportion of viable M. leprae had decreased to less than 1:1000 after just a few days of this same treatment; the data of Rees and his colleagues suggest that, despite contin ued chemotherapy, M. leprae do not continue to be killed at the same rate as that meas ured initially. It is the remaining viable organisms that are identified as persisters.
The identification of persisting M. leprae appears to have been best established by Russell and his co-workers, in their report (166) of the results of a trial of acedap sone monotherapy. In this trial, it had been planned to measure the response to chemo therapy only in terms of the proportion of solidly staining organisms detected in smears. By the end of the first year of therapy, solid M. leprae had disappeared from the smears of all patients. However, solidly staining organisms were seen in the smears of one patient after three years of treatment, and in those of additional patients after treat ment for four years. After the solid organisms had been seen, skin lesions were biopsied and normal mice were inoculated, with the result that mice were infected with M. leprae that proved to be susceptible to dapsone. WHO/CDS/LEP/86.4 page 76
Table 11. Course of events during rifampicin monotherapy of multibacillary leprosy33
Results Interpretation
Duration of BI MI Mouse Total Viable Treatment Inoculation /1. 1eprae /1. 1eprae
0 4+ 10% + lOll 1010
3 days 4+ 10% 1011 <108
1 week 4+ 10% 1011 <108
2 weeks 4+ 1% 1011 <108
1 month 4+ <1% 1011 <108
3 months 4+ <1% 1011 <108
1 year 3+ <1% 1010 <107
2 years 2+ <1% 10 9 <106 (small inoculum)
+ 10 9 10 5 (large inoculum)
Although mice had not been inoculated earlier in this study, Shepard and his co workers had reported (187) that, as the result of an earlier trial of acedapsone, /1. 1eprae became non-infective for mice after about six months of acedapsone treatment. As shown in Table 12, in which a reconciliation of these findings is attempted, the abso lute number of viable organisms appears to have reached a minimum after two years of treatment; thereafter, the total number of /1. 1eprae decreased still further, unmasking the persisting organisms, the proportion of which actually increased. In this case, it appears even more justified to term the surviving /1. 1eprae "persisters".
In summary, it is clear that immunologically intact mice inoculated in the hind foot pad with 5 000 - 10 000 /1. 1eprae are not useful for the detection of persisting /1. 1eprae, at least early in the therapy of the lepromatous patient. The proportion of viable organisms (persisters) is so small that a large inoculum (2 10 5) would be required to detect them. So large an inoculum might well serve as a vaccine, immunizing the immun ologically intact animal so that viable /1. 1eprae contained in the inoculum are unable to multiply. On the other hand, animals that are sufficiently immune-deficient are not immunized by the large inocula required for the demonstration of persisters, and should permit multiplication of viable /1. 1eprae, even when these represent only a minute proportion of the inoculum. Later in therapy, when the overburden of dead /1. 1eprae has decreased, and the proportion of viable organisms is larger, it may certainly be possible readily to detect persisting /1. 1eprae by inoculation of intact mice. Even in this situation, however, the use of immune-deficient mice appears preferable.
33 Adapted from reference no. 103. WHOjCDSjLEPj86.4 page 77
In the study carried out by Waters and his co-workers (212), immunologically intact and T900R mice were inoculated in parallel. Multiplication of M. 1eprae occurred more frequently in T900R mice - i.e., persisting organisms were detected by inoculation of immune-deficient mice in a larger proportion of the specimens studied. At first glance, this is puzzling, especially because only small numbers of AFB were recovered from the large majority of the specimens, and, in some cases, M. 1eprae multiplied in mice inocu lated with suspensions that contained so few AFB that none could be counted (this is not illogical; if one organism is counted, the total number of organisms in the suspension
Table l2.Course of events during acedapsone monotherapy of multibacillary leprosy34
Results Interpretation
Duration of Treatment BI MI Mouse Total Viable (months) Inoculation M. 1eprae M. 1eprae
0 4+ 10% Not Done 1011 1010
12 3+ <1% Not Done 1010 <10 8
24 2+ <1% Not Done 10 9 <10 7
36 1+ >1% + 108 _106
is approximately 104). As has already been stated (see p. 66), immune-deficient mice are capable of detecting smaller proportions of viable M. 1eprae than are immune-competent mice, because the former are not so readily immunized by the large inocula that must be employed, if one is to detect small proportions «1:10 000) of viable organisms. It appears that, under certain conditions, immune-deficient mice may be more suitable for the detection of viable M. 1eprae, even when they are present in larger proportions, or when it is not possible to recover sufficient M. 1eprae for the preparation of large inocula. It will be recalled (see pp. 66 - 67) that, for the purpose of detecting persisting M. 1eprae, mice should be inoculated with minimally diluted suspensions of organisms. As a consequence, not only are the M. 1eprae minimally diluted; also, the human-tissue components inevitably present in suspensions of M. 1eprae prepared from biopsy specimens are also minimally diluted. It may well be that immune-competent mice are non-specific ally "immunized" by the foreign tissue present in the bacterial suspensions. Some evidence that such a phenomenon may occur, with resulting inhibition of multiplication of M. 1ep rae, has been found in data from clinical trials (21), from a study of the action of mouse interferon, which had been prepared in murine cell cultures maintained on medium containing foetal calf serum (108), and from an attempt to employ rabbit serum as a medium in which to freeze M. 1eprae (98).
Procedure. To demonstrate persisting M. 1eprae, a lesion is biopsied, a bacterial suspension is prepared with minimal dilution, so as to obtain the largest possible inocu lum, and large numbers (105 - 10 7) of M. 1eprae are inoculated into the hind footpads of immune-deficient rodents - i.e., T900R mice, nude mice, or neonatal1y thymectomized rats. A one-hundred-mi1lion-fo1d increase - i.e., from 1 to 10 8 - represents 27 doub 1ings; at an average rate of one doubling per two weeks, 54 weeks (about one year)
34 Taken from reference no. 103. WHO/CDS/LEP/86.4 page 78 should be a time sufficient for unequivocal multiplication of any viable H. leprae pres ent in the inoculum. Thus, one year or more after inoculation, harvests are performed and the H. leprae counted.
Work in progress in two laboratories suggests that viable H. leprae present in small proportions may indeed multiply in immunocompetent mice administered large inocula. The total number of H. leprae does not increase above the normal ceiling of 10 6 - 10 6. 3, however, and the presence of an increased proportion of viable organisms can be demon strated only by the passage of small numbers (103. 7 - 104) of H. leprae to new intact mice.
EXPERIMENTAL APPLICATIONS
Drug Screening
Inoculation of H. leprae into the footpads of immunologically intact mice has also been applied to the screening of drugs for antimicrobial activity. Three basic techniques are employed in drug-screening: (1) the "continuous" technique; (2) the "kinetic" technique; and (3) the technique of "proportional bacteriocide".
Continuous Technique. The continuous technique, the first technique employed for demon strating activity of a drug against H. leprae in mice (181), involves continuous administration of a drug beginning from the day of inoculation. Multiplication of H. leprae in untreated control mice is followed by interval harvests. When the evidence of multiplication is unequivocal - i.e., the organisms have multiplied in the footpads of control mice from an inoculum of 10 3. 7 organisms to a level of at least 10 5. 7 (500 000) organisms per footpad, harvests of H. leprae are performed from the footpads of both control and treated mice. An active drug is one that inhibits multiplication of the H. leprae. If on two successive occasions, approximately one month apart, the number of H. leprae harvested from treated mice does not fall within the range of the results of four simultaneous harvests from control mice, the drug can be stated to have been active with a probability of 0.04 (180) (see pp. 83 - 84).
In screening a drug, one ordinarily administers first the largest dosage tolerated by the mice; to have administered the drug in a smaller dosage without demonstrating anti microbial activity is inconclusive, and leaves open the possibility that, had the drug been administered in a larger dosage, activity against H. leprae would have been evi dent. Having established by this technique the antimicrobial activity of a drug, one may, by the same technique, proceed to establish the minimal effective dosage (MED) - the smallest dosage of the drug that produces an antimicrobial effect. One administers frac tions of the largest tolerated dosage until one finds the dosage that is without an anti microbial effect; the next larger dosage is the MED. An example of an application of this technique is shown in Table 13. In this experiment, even the smallest dosage of dapsone 0.00001 g% - inhibited multiplication of the strain of H. leprae employed; therefore, the MED is ~ 0.00001 g%. [The minimal inhibitory concentration (MIC) of the drug is the concentration found in the plasma and tissues of mice administered the drug in the MED. The MEDs and MICs of a number of antileprosy drugs are shown in Table 14.]
An important disadvantage of the continuous technique is that it requires large quantities of drug. Assuming that 20 mice will be administered the drug in a concentra tion of 0.1 g per 100 g mouse diet, that each mouse eats 5 g diet per day, and that it will be necessary to administer the drug for six months, a minimum of 18 g of the drug will be needed for the screen. A second disadvantage of the continuous technique is that it does not distinguish between bacteriostatic and bactericidal activity. WHO/CDS/LEP/86.4 page 79
Table 13. Effect of dapsone on multiplication of H. 1eprae in mice35
Harvest (AFB/mouse) ( x 104)
Mouse g% dapsone 220-221 320-336 447-449 no. in diet days days days
1 - 20 Nil 138 60 94 21- 40 0.03 <2 <2 2 41 - 60 0.01 <2 <2 <2 61 - 80 Nil 8 29 29 81 - 100 0.003 <2 <2 <2 101 - 120 0.001 <2 <2 <2 121 - 140 Nil 19 2 7 141 - 160 0.0003 <2 <2 4 161 - 180 0.0001 <2 <2 <4 181 - 200 Nil 131 29 24 201 - 220 0.00003 <2 2 <2 221 - 240 0.00001 <2 2 3 241 - 260 Nil 54 42
Table 14. Minimal effective dosages (MEDs) to mice and minimal inhibitory concentrations (MICs) for H. 1eprae of drugs employed in chemotherapy of leprosy
Drug MED MIC (g% in diet) (ng/ml)
Dapsone 0.0001 3 Rifampicin 0.003 300 Clofazimine 0.0001-0.001 Cannot be measured Ethionamide/protionamide 0.01 50
Kinetic Technigue. To distinguish between bacteriostatic and bactericidal effects, Shepard developed the kinetic technique (177, 178, 196). This technique is identical to the continuous technique, except in one particular - the period during which the drug is administered. Drug administration is usually begun 60 days after inoculation, when the organisms are in early or mid-logarithmic phase, and is continued for 60 - 90 days. Multiplication of H. 1eprae in untreated mice is followed by means of interval harvests. Just before drug administration is stopped, harvests of H. 1eprae are made from control and treated groups of mice. And following cessation of drug administration, additional harvests are performed from both control and treated mice .- usually at intervals of 28 56 days. Statistical considerations are those described for the continuous method.
A purely bacteriostatic drug will inhibit multiplication of H. 1eprae for as long as the drug is administered; after administration has been stopped, slowly eliminated drugs will continue to inhibit multiplication as long as active concentrations of the drug
35 Adapted from reference no. 194. WHO/CDS/LEP/86.4 page 80 remain. Evidence of a bacteriostatic effect is shown in Figure 19, as a growth curve of H. leprae in treated mice that is parallel to that in contr0l mice, but which lags behind the control growth curve by a length of time no greater than that during which the drug was administered. A growth curve of H. leprae that is parallel to that for untreated mice, but delayed by a time longer than that during which drug was administered, also shown in Figure 19, may be taken as evidence of incomplete bactericide (killing of a portion of the organisms) or prolonged bacteriostasis ("bacteriopause"). Finally, the failure of resumed bacterial growth to reach the maximum following withdrawal of treatment (see Fig. 20) is evidence that some of the H. leprae were killed.
Figure 19. Bacteriostatic and bacteriopausal effects of dapsone on H. leprae in mice. Mice were inoculated in the hind footpad with 10 3. 7 H. leprae, and dapsone was adminis tered for 155 days from the day of inoculation, as indicated by the shaded bar. Dapsone in a concentration of 0.00003 g per 100 g mouse diet (x) produced only a bacteriostatic effect, as shown by the prompt resumption of multiplication when drug administration was stopped. Administered in a concentration of 0.0001 g per 100 g diet (+), the drug pro duced a bacteriopausal effect; inhibition of multiplication persisted longer than can be accounted for by presence of the drug in an effective concentration. Multiplication of H. leprae in mice administered 0.00001 g dapsone per 100 g diet (0) was not different from that in control mice. 36
+
10' o • •
.'• /llI :...__•+ II .. I
'.))::.:} DOS ::::':::,, i o 100 200
NUMBER OF DAYS AFTER INOCULATION
The kinetic technique is less sensitive than the continuous technique to the antimi crobial activity of less potent drugs. However, it distinguishes between bacteriostasis on one hand, and bacteriopause or bactericide on the other. Another advantage of the kinetic technique is that the drug is administered for a shorter period of time than in the continuous technique; therefore, smaller quantities are required.
36 Taken from reference no. 112. WHO/CDS/LEP/86.4 page 81
Figure 20. Bactericidal effect of polyinosinic:polycytidylic acid (poly 1:C) on H. lep rae in mice. Mice were inoculated in the hind footpad with 10 3. 7 H. leprae, and drugs were administered locally in the inoculated hind footpads at intervals of 12 hours for 15 doses during the interval indicated by the stippled bar. Results of harvests of H. lep rae show that saline ~O) may have had a small effect, whereas poly 1:C administered intraperitoneally (A)' was not different from no treatment (.). Both polyinosinic acid (poly I) (0) and poly 1:C (A) administered into the footpads had a definite effect; that of poly 1:C is more clearly bactericidal than is that of poly 1. 37
0 et I Q. ,I .... 6.0 0 I .'I 0 I lL. I
"•0 ~ 0- ~ :E 0: , LlJ I m ,, :E 5.0 , ::> I Z ,I I 2 I C) 0 -'
rf'i.---r--r---,.---y----r-..--.....-I I .....I o 70 130 190 250 310 TIME AFTER INOCULATION (days) Proportional Bactericide Technique. Colston and his co-workers (24) devised this tech ni for measuring the bactericidal activity of a drug. An inoculum containing 10 3. 7 or 104ueAFB per 0.03 ml is prepared in the usual way. This is serially diluted in 10-fold steps by adding to a measured volume of each bacterial suspension 9 times the volume of HBSS. The first dilution provides an inoculum containing 10 2 . 7 (500) or 10 3 (1 000) AFB per 0.03 ml; the further dilution of this suspension provides an inoculum containing 101. 7 (50) or 10 2 (100) AFB per 0.03 ml; the final dilution should be that calculated to contain, on average, 0.5 or 1 AFB per 0.03 ml. Each dilution is used to inoculate 10 - 20 mice. Except for the control group, the mice are treated for a period of time that may vary, depending on the drug, from 1 - 60 days, after which treatment is stopped. The mice are then held for at least one year, a period of time theoretically sufficient for one surviving H. leprae to multiply to 10 6. After this time, harvests of H. leprae are performed from individual footpads, and the proportion of organisms killed is calculated by a "most probable number" (MPN) technique; alternatively, one may calculate the median infectious dose ("1D50), the number of organisms required to infect half of the number of mice (see pp. 86 - 106).
37 Taken from reference no. 108. WHO/CDS/LEP/86.4 page 82
An example of the use of the technique of proportional bactericide is given in Table 15, which shows that 10 organisms did not give rise to multiplication in any of five untreated mice, whereas inoculation of 100 AFB was followed by multiplication in all of the five untreated mice inoculated. In contrast, only the largest inoculum - 104 M. lep rae - gave rise to multiplication in the mice that had been treated continuously with ethionamide, 0.1 g% in the diet. Thus, as a first approximation, one could conclude that the treatment had killed 99% of the viable M. leprae in the inoculum. Similarly, ethi onamide three times weekly by gavage permitted multiplication in four of the five mice inoculated with 10 3 M. leprae per footpad; thus, this treatment killed approximately 90% of the viable organisms inoculated. Finally, ethionamide administered once weekly was virtually without bactericidal effect, whereas dapsone, administered continuously in a dosage of 0.01 g%, killed about 95% of the viable M. leprae initially present.
Because of the requirements for inoculating groups of mice with multiple dilutions of the inoculum, and for holding the mice for at least one year before harvesting M. leprae, the technique of proportional bactericide requires more mice and more time than either of the other techniques. On the other hand, this technique offers the most accurate means for assessing the bactericidal effects of drugs on M. leprae.
Table 15. Measurement of the bactericidal effects of drugs by means of the "proportional bactericide" technique38
No. AFB inoculated per mouse
10 Proportion of Drug viable used No. mice showing multiplication/no. inoculated M. leprae
None 5/5 5/5 5/5 0/5 0.024
Ethionamide 0.1% 5/5 0/5 0/5 0/5 0.0002 continuously
Ethionamide 500 mg/kg 5/5 4/5 1/5 0/5 0.0017 three times weekly
Ethionamide 500 mg/kg 5/5 5/5 3/4 0/5 0.013 once weekly
Dapsone 0.01% 4/4 3/5 1/5 0/5 0.0011 continuously
Experimental Chemotherapy
Immunologically intact mice are suitable for drug-screening, the purpose of which is to determine the ability of a certain concentration of a drug to inhibit multiplication of M. leprae or to kill them. However, the population of M. leprae in the intact mouse, even after maximal multiplication, is very much smaller than that of the untreated lepro matous patient (approximately 10 6, compared to 1010 or more). Therefore, one could not use the M. leprae-infected, immune-competent mouse as a model of the lepromatous patient
38 Adapted from reference no. 25. WHO/CDS/LEP/86.4 page 83 undergoing chemotherapy. So small a population of H. leprae is unlikely to include drug-resistant organisms (the maximal frequency with which drug-resistant mutations occur is probably not greater than 1:106, based on analogies with drug-resistant H. tuberculo sis). Moreover, the immunologically intact mouse does not tolerate even this small population (106) of H. leprae, but rapidly kills the organisms. In intact mice, one could not allow the organisms to multiply, then administer treatment for some duration, and, finally, withdraw treatment and study the subpopulation of persisting H. leprae. Nor could one study in intact mice the development of drug resistance, which occurs only in large populations (~ 10 6) of H. leprae, although one could demonstrate the presence of drug-resistant organisms (which requires only 10 3. 7 - 104 H. leprae, with the poten tial to multiply only to 10 6) in intact mice. Thus, because of the small bacterial popu lation, one could not study in immune-competent mice either of the two phenomena - drug resistance and microbial persistence, both phenomena characteristic of much larger popu1a tions of H. leprae - that appear to determine the outcome of chemotherapy of a patient with lepromatous leprosy. In short, one could not study the intact mouse as if it were the patient. On the other hand, the H. leprae-infected immune-deficient rodent, which tolerates a much larger bacterial population, may serve as a model of the lepromatous patient for studies of chemotherapy.
Studies of experimental chemotherapy have been carried out on H. leprae-infected, neonata11y thymectomized rats (54, 56). Animals at the age of weaning are inoculated, either into the footpad or intravenously, with large numbers of H. leprae, and the organisms are permitted to multiply. After about one year, the animals are sampled, to make certain that multiplication to a good level (at least 10 7 per footpad) has occurred. The animals are then divided among control and treated groups, drugs are administered for a short or a more prolonged period, animals are sacrificed both during and after the period of drug administration, and H. leprae are harvested - from the hind footpads only of animals originally inoculated in the hind footpad, and from hind and fore-footpads, ears, tail and testes of animals that had been inoculated intravenously. Organisms are passaged - in small numbers (103. 7 - 104) to intact mice, and in large numbers (~105) to thymectomized rats - in order to measure the proportions of viable H. leprae. In short, the neonata11y thymectomized rat is employed as a model of the lepromatous patient. Although chemotherapeutic regimens must eventually be tested in patients, there are many important questions about regimens, questions relating to dosage variables and duration, for example, that could be answered much more cheaply and quickly by studies in experimen tal animals than by clinical trial.
Calculations and Statistics
The calculations involved in micrometry and in determining the number of H. leprae in a given volume of suspension have been described earlier in this chapter. Remaining to be presented are the statistical techniques to be employed in assessing the significance of the results of mouse inoculation in a variety of applications, and the calculations involved in the proportional bactericide technique.
RESULTS OF MULTIPLE HARVESTS OF H. LEPRAE39
In comparing the results of harvests of H. leprae from the footpads of experimental mice - e.g., mice treated with a drug or vaccine - with those from the footpads of untreated, control mice, one wishes to learn if the observed differences are statistically significant. To phrase the question more precisely, one wishes to learn the likelihood (probability) that the observed difference might have arisen by chance from the same group of mice - i.e., from the same population of harvest results.
The most direct approach is to perform multiple harvests from one of the two groups of mice, or to perform harvests from multiple groups of mice treated (or untreated) in the same way. In the usual experiment performed for the purpose of screening drugs, one
39 Adapted from reference no. 180. WHO/CDS/LEP/86.4 page 84 should include four groups of untreated controls among 8 - 12 groups of drug-treated mice. Harvests of M. leprae from pools of four mice from each of ~he four groups of control mice are begun between 100 and 130 days after inoculation (one learns by trial-and-error how soon after passage M. leprae of a particular strain are likely to have multiplied to a readily detectable level), and are repeated at intervals of 28 - 30 days. Simultaneous harvests, one harvest per group, from treated mice are begun as soon as multiplication has been detected in all four control groups, and are repeated at the same 28- to 30-day intervals. The likelihood that the numbers of M. leprae harvested from all four control groups will exceed the number harvested from a given experimental group is 1/5 (0.20) [there are five possible arrangements (ascending order is one possible arrangement) of the five results, and any given arrangement may be encountered with equal probability]. That the same arrangement may be encountered at 100 and again at 130 days is less likely; in fact, the probability of such an occurrence is simply the product of the two individual probabilities (0.20 x 0.20 0.04). At this point, one may conclude that the drug is active, and has produced a delay in multiplication of M. leprae of at least 30 days. Should the same arrangement of five results be encountered on yet a third occasion, 151 160 days after inoculation, the probability that such an arrangement of three sets of harvests could have occurred by chance after repeated harvests from the same (or identi cal) group(s) of mice is even smaller (0.23 = 0.008), and one may conclude that the drug has produced a delay of multiplication of M. leprae of at least 51 - 60 days.
If one wishes also to examine the difference between two treatments, one must perform more than a single harvest from each group at each interval. No overlapping of values between two sets of three harvests yields a probability of 0.05, and no overlapping of values between two sets of two harvests on each of two occasions yields a probability of 0.028. In order to examine the difference between two effective treatments, it may be necessary to perform additional harvests after an interval shorter than 28 - 30 days, so that the M. leprae will not have multiplied to the maximal level of approximately 10 6 organisms per footpad in both groups before all the required harvests had been performed.
FISHER'S EXACT PROBABILITY CALCULATION
This extraordinarily useful statistical technique involves calculation of the exact probability with which a given arrangement of two sets of results may occur. In fact, the last two probability values given in the preceding section were obtained by means of this calculation.
The probability of any particular arrangement of data in the 2 x 2 "contingency" table,
a b
c d is given by the equation:
(a + b)! x (c + d)' x (a + c)! x (b + d)! P
(a + b + c + d)! x a! x b! x c! x d! in which the symbol, "''', following a number indicates the "factorial" of the number i. e., WHO/CDS/LEP/86.4 page 85
n! n x (n - 1) x (n - 2) x (n - 3) x ... x [n - (n - 1)].
Thus, in the first example of two sets of three harvests, one sets up the following 2 x 2 table, choosing some value, x, intermediate between the two sets of harvests results:
Result >x Treatment 1 o 3 Treatment 2 3 o Employing the convention that both l! and O! 1, 3! x 3! x 3! x 3! (3!) 2 P 0.05. 6! x 3! x 3! x O! x O! 6! In the second example of two sets of two harvests, repeated once, the following 2 x 2 table is appropriate: Result >x Treatment 1 o 2 Treatment 2 2 o P 0.167; 0.167 x 0.167 0.0278. 4! x (2!)2 Of course, the difference between the two sets of results being compared may not always be so obvious. Suppose that, among eight patients being treated by regimen A, all but one show decided improvement, whereas, among an equal number of patients under treat ment by regimen B, only two show improvement. To determine the likelihood that the two regimens are equally effective (the "null" hypothesis, that the difference between the two treatments is not significant, and that two such different results are not unlikely when successive eight-patient samples are selected from among a much larger number of patients treated by either of the two regimens), one analyses the following 2 x 2 contin gency table: Improvement + 0 Regimen A 7 1 Regimen B 2 6 WHO/CDS/LEP/86.4 page 86 For this particular distribution of 16 results, (8!)2 x 9! x 7! P 0.0196 . l6! x 7! x 6! x 2! To this value of P must be added other values, the probabilities that even more favourable distributions of two sets of eight results, yielding the same overall totals of improved and unimproved patients, could have occurred. In this case, there is only one more favourable distribution possible: Improvement + 0 Regimen A 8 o Regimen B 1 7 (8!)2 x 9! x 7! For this distribution, P 0.0007 . l6! x 8! x 7! The sum of these two probabilities, 0.0203, is smaller than 0.05. Therefore, one should reject the null hypothesis that both groups of patients were drawn from the same popula tion - i.e., that the observed difference could have occurred by chance, and conclude that regimen A was more effective than regimen B. One problem sometimes encountered in using Fisher's exact probability calculation is that calculation of the factorials of numbers larger than those employed in these two examples can be very tedious. Calculation of factorials as large as that of 69 (69! = 1.71 x 10 98) is possible on many electronic calculators, including those of pocket-size. Moreover, one may find tables of factorials and (even more valuable) of loglO factorials in many books of mathematical tables (Table 16, for example) (one must remember that logarithms are to be added and subtracted, rather than multiplied and divided). For easy use of Table 16, a table of antilogarithms, Table 17, is provided. Some collections of statistical tables include tables of exact probabilities - e.g., references no. 137 and 197. Shepard has also supplied some values in reference no. 180. A simple table from Shepard's paper, and a more complete table from reference 197 appear as Tables 18 and 19. RESULTS OF THE PROPORTIONAL BACTERICIDE TECHNIQUE Two methods of calculation have been proposed for the results of the proportional bactericide technique - the traditional MPN calculation, that has been widely used in sanitary surveillance of water and milk supplies, and calculation of the IDsO. Calculation of the MPN The calculation assumes that the organisms are randomly distributed in the suspension (i.e., they are not clumped), and that one viable organism is capable of multiplying in a tube of culture medium, or, in the case of H. leprae, in the footpad of a mouse. If, then, series of culture tubes (usually five or ten) or of mouse footpads have been inocu lated with serially diluted (usually in 10-fold steps) suspensions of organisms, and if the dilutions have been chosen well, multiplication will be found to have occurred in all of the tubes or feet inoculated from one dilution, in a proportion of the tubes or foot pads inoculated from the next lower dilution, and in none of the tubes or footpads inocu lated from all of the lower dilutions. One then calculates the number of viable organisms WHO/CDS/LEP/86.4 page 87 most likely to have given rise to the observed distribution of tubes or footpads showing multiplication and of those showing no multiplication. Examples of the results of a study of the proportion of viable H. 1eprae in a number of different suspensions (214) is shown in Table 20. In most public health laboratories, the MPN is determined by reference to a table which presents an array of many if not all possible distributions and the MPN associated with each distribution. However, the use of such a table will frequently require that approximations be made, because the standard number (five or ten) of footpads may not be available for each dilution of the original suspension of H. 1eprae. More difficult to use, but more satisfactory, in that it makes use of all of the information available and requires no approximations, is the general equation of Halvorson and Ziegler (71): 0.1 P3 + + 1 - e- l Ox 1 - e-O. l x in which nI, n2 and n3 are the numbers of the footpads harvested of mice that had been inoculated with the three consecutive serial dilutions; PI, P2 and P3 are the numbers of corresponding footpads showing multiplication; and x is the absolute number (not the proportion) of viable H. 1eprae actually administered to the mice inoculated from the intermediate of the three serial dilutions. The value of the right side of the equation is first calculated (this is III for a series of 10 footpads, 88.8 for a series of 8 footpads, etc). Then, the value of x is varied by trial-and-error, until the left side of the equation equals the right. This calculation is greatly facilitated by a programmable hand calculator. ~ ~ OQ 0 40 (1) <, Table 16. Common Logarithms of Factorials of the Integers 1-999 () 00t::l 00t/) <, t"' t"1 If o 2 3 4 5 6 7 8 9 ...... "d I 00 C1' o 0.00000 I 0.00000 0.30103 0.77815 1.38021 2.07918 2.85733 3.70243 4.60552 5.55976 10 6.55976 7.60116 8.68034 9.79428 10.94041 12.11650 13.32062 14.55107 15.80634 17.08509 .t::'- 20 18.38612 I 19.70834 21.05077 22.41249 23.79271 25.19065 26.60562 28.03698 29.48414 30.94654 30 32.42366 33.91502 35.42017 36.93869 38.47016 40.01423 41.57054 43.13874 44.71852 46.30959 40 47.91165 49.52443 51.14768 52.78115 54.42460 56.07781 57.74057 59.41267 61.09391 62.78410 so 64.48307 66.19064 67.90665 69.63092 71.36332 73.10368 74.85187 76.60774 78.37117 80.14202 60 81.92017 83.70550 85.49790 87.29724 89.10342 90.91633 92.73587 94.56195 96.39446 98.23331 70 100.07841 101.92966 103.78700 105.65032 107.51955 109.39461 111.27543 113.16192 115.05401 116.95164 80 118.85473 120.76321 122.67703 124.59610 126.52038 128.44980 130.38430 132.32382 134.26830 136.21769 90 138.17194 140.13098 142.09476 144.06325 146.03638 148.01410 149.99637 151.98314 153.97437 155.97000 100 157.97000 159.97433 161.98293 163.99576 166.01280 168.03398 170.05929 172.08867 174.12210 176.15952 110 178.20092 180.24624 182.29546 184.34854 186.40544 188.46614 190.53060 192.59878 194.67067 196.74621 120 198.82539 200.90818 202.99454 205.08444 207.17787 209.27478 211.37515 213.47895 215.58616 217.69675 130 219.81069 221.92797 224.04854 226.17239 228.29950 230.42983 232.56337 234.70009 236.83997 238.98298 140 241.12911 243.27833 245.43062 247.58595 249.74432 251.90568 254.07004 256.23735 258.40762 260.58080 150 262.75689 264.93587 267.11771 269.30240 271.48993 273.68026 275.87338 278.06928 280.26794 282.46933 160 284.67345 286.88028 289.08980 291.30198 293.51683 295.73431 297.95442 300.17713 302.40244 304.63033 170 306.86078 309.09378 311.32930 313.56735 315.80790 318.05094 320.29645 322.54442 324.79484 327.04770 180 329.30297 331.56065 333.82072 336.08317 338.34799 340.61516 342.88467 345.15651 347.43067 349.70713 190 351.98589 354.26692 356.55022 358.83578 361.12358 363.41362 365.70587 368.00034 370.29700 372.59586 200 374.89689 377.20008 379.50544 381.81293 384.12256 386.43432 I 388.74818 391.06415 393.38222 395.70236 210 398.02458 400.34887 402.67520 405.00358 407.33400 409.66643 412.00089 414.33735 416.67580 419.01625 220 421.35867 423.70306 426.04942 428.39772 430.74797 433.10015 435.45426 437.81029 440.16822 442.52806 230 444.88978 447.25340 449.61888 451.98624 454.35546 456.72652 459.09944 461.47418 463.85076 466.22916 240 468.60937 470.99139 473.37520 475.76081 478.14820 480.53737 482.92830 485.32100 487.71545 490.11165 2SO 492.50959 494.90926 497.31066 499.71378 502.11862 504.52516 506.93340 509.34333 511.75495 514.16825 260 516.58322 518.99986 521.41816 523.83812 526.25972 528.68297 531.10785 533.53436 535.96250 538.39225 270 540.82361 543.25658 545.69115 548.12731 550.56506 553.00439 555.44530 557.88778 560.33183 562.77743 280 565.22459 567.67330 570.12354 572.57533 575.02865 577.48349 579.93986 582.39774 584.85713 587.31803 290 589.78043 592.24432 594.70971 597.17657 599.64492 602.11474 604.58603 607.05879 609.53301 612.00868 300 614.48580 616.96436 619.44437 621.92581 624.40869 626.89299 I 629.37871 631.86585 634.35440 636.84436 310 639.33572 641.82848 644.32263 646.81818 649.31511 651.81342 654.31310 656.81416 659.31659 661.82038 320 664.32553 666.83204 669.33989 671.84910 674.35964 676.87152 679.38474 681.89929 684.41516 686.93236 330 689.45087 691.97070 694.49184 697.01428 699.53803 702.06307 704.58941 707.11704 709.64596 712.17616 340 714.70764 717.24039 719.77442 722.30971 724.84627 727.38409 729.92317 732.46350 735.00508 737.54790 350 740.09197 742.63728 745.18382 747.73160 750.28060 752.83083 755.38228 757.93495 760.48883 763.04392 360 765.60023 768.15773 770.71644 773.27635 775.83745 778.39974 780.96323 783.52789 786.09374 788.66077 370 791.22897 793.79834 796.36888 798.94059 801.51347 804.08750 806.66268 809.23903 811.81652 814.39516 380 816.97494 819.55587 822.13793 824.72113 827.30546 829.89092 832.47751 835.06522 837.65405 840.24400 390 842.83507 845.42724 848.02053 850.61492 853.21042 855.80701 858.40471 861.00350 863.60338 866.20436 400 868.80642 871.40956 874.01379 876.61909 879.22547 881.83293 884.44146 887.05105 889.66171 892.27343 410 894.88622 897.50006 900.11496 902.73091 905.34791 907.96595 910.58505 913.20518 915.82636 918.44857 420 921.07182 923.69611 926.32142 928.94776 , 931.57512 934.20351 936.83292 939.46335 942.09480 944.72725 430 947.36072 949.99520 952.63068 955.26717 957.90466 960.54315 963.18263 965.82312 968.46459 971.10705 440 973.75051 976.39495 979.04037 981.68677 984.33415 986.98251 989.63185 992.28216 994.93344 997.58568 4SO 1000.23889 1002.89307 1005.54821 1008.20431 1010.86136 1013.51937 1016.17834 1018.83825 1021.49912 1024.16093 460 1026.82369 I 1029.48739 1032.15203 1034.81761 1037.48413 1040.15158 1042.81997 1045.48929 1048.1595311050.83071 470 1053.50280 11056.17582 1058.84977 1061.52463 1064.20041 1066.87710 1069.55471 1072.23322 1074.91265 1077.59299 480 1080.27423 1082.95637 1085.63942 1088.32337 1091.00821 1093.69395 1096.38059 1099.06812 1101.75654 1104.44585 490 1107.13604 1109.82712 1112.51909 1115.21194 1117.90566 1120.60027 1123.29575 1125.99211 1128.68934 1131.38744 40 Taken from reference no. 38, and reproduced by permission of the publishers of the Geigy Scientific Tables. Table 16 (continued) 500 1134.08641 11136.78624 1139.48695 11142.18851 11144.89094 1147.59424 i 1150.29839 1153.00339 1155.70926 1158.41598 510 1161.12355 1163.83197 1166.54124 1169.25135 1171.96232 1174.67412 1177.38677 1180.10026 1182.81459 1185.52976 520 1188.24576 1190.96260 1193.68027 1196.39877 1199.11810 1201.83826 1204.55925 1207.28106 1210.00369 1212.72715 530 1215.45142 , 1218.17652 1220.90243 I 1223.62916 I 1226.35670 1229.08505 1231.81422 1234.54419 1237.27497 1240.00656 540 1242.73896 1245.47215 1248.20615 I 1250.94095 1253.67655 1256.41295 1259.15014 1261.88813 1264.62691 1267.36648 550 1270.10684 ! 1272.84799 1275.58993 1278.33266 1281.07617 1283.82046 1286.56554 1289.31139 1292.05803 1294.80544 560 1297.55363 1300.30259 1303.05232 1305.80283 1308.55411 1311.30616 1314.05898 1316.81256 1319.56691 1322.32202 570 1325.07790 1327.83453 1330.59193 1333.35008 1336.10899 1338.86866 134\.62908 1344.39026 1347.15219 1349.91487 580 1352.67829 i 1355.44247 1358.20739 1360.97306 1363.73948 1366.50663 1369.27453 1372.04317 1374.81254 1377.58266 590 1380.35351 . 1383.12510 1385.89742 1388.67048 1391.44426 1394.21878 1396.99403 1399.77000 1402.54670 1405.32413 600 1408.10228 1410.88115 1413.66075 1416.44107 1419.22210 1422.00386 1424.78633 1427.56952 1430.35343 1433.13804 610 1435.92337 1438.70941 144\.49617 1444.28363 1447.07179 1449.86067 1452.65025 1455.44054 1458.23152 1461.02322 620 1463.81561 1466.60870 1469.40249 1472.19698 1474.99216 1477.78804 1480.58462 1483.38188 1486.1798.4 1488.97849 630 1491.77784 1494.57787 1497.37858 1500.17999 1502.98208 1505.78485 1508.58831 1511.39245 1514.19727 1517.00277 640 1519.80895 1522.61581 1525.42334 1528.23155 1531.04044 1533.85000 1536.66023 1539.47114 1542.28271 1545.09496 650 1547.90787 1550.72145 1553.53570 1556.35061 1559.16619 1561.98243 1564.79933 1567.61690 1570.43513 1573.25401 660 1576.07356 1578.89376 1581.71461 1584.53613 1587.35830 1590.18112 1593.00459 1595.82872 1598.65350 1601.47892 670 1604.30500 1607.13172 1609.95909 1612.78710 1615.61576 1618.44507 1621.27501 1624.10560 1626.93683 1629.76870 680 1632.60121 1635.43436 1638.26814 1641.10256 1643.93762 1646.77331 1649.60964 1652.44659 1655.28418 1658.12240 690 1660.96125 1663.80073 1666.64083 1669.48157 1672.32293 1675.16491 1678.00752 1680.85075 1683.69461 1686.53909 700 1689.38418 1692.22990 1695.07624 1697.92320 1700.77077 1703.61896 1706.46776 1709.31718 1712.16721 1715.01786 710 1717.86912 1720.72099 1723.57347 1726.42656 1729.28026 1732.13456 1734.98948 1737.84500 1740.70112 1743.55785 720 1746.41518 1749.27312 1752.13165 1754.99079 1757.85053 1760.71087 1763.57181 1766.43334 1769.29547 1772.15820 730 1775.02152 1777.88544 1780.74995 1783.61505 1786.48075 1789.34704 1792.21391 1795.08138 1797.94944 1800.81808 740 1803.68731 1806.55713 1809.42754 1812.29853 1815.17010 1818.04225 1820.91499 1823.78831 1826.66222 1829.53670 750 1832.41176 1835.28740 1838.16362 1841.04041 1843.91778 1846.79573 11849.67425 1852.55335 1855.43302 1858.31326 760 1861.19407 1864.07546 1866.95741 1869.83994 1872.72303 1875.60669 1878.49092 1881.37571 1884.26108 1887.14700 770 1890.03349 1892.92055 1895.80816 1898.69634 1901.58508 1904.47439 ; 1907.36425 1910.25467 1913.14565 1916.03718 780 1918.92928 1921.82193 1924.71514 1927.60890 1930.50321 1933.39808 1936.29351 1939.18948 1942.08601 1944.98308 790 1947.88071 1950.77889 1953.67761 1956.57689 1959.47671 1%2.37707 1965.27799 1968.17944 1971.08145 1973.911399 800 1976.88708 1979.79072 1982.69489 1985.59961 1988.5048(, 19 41Taken from reference no. 37, and reproduced by permission of the publishers of the Geigy Scientific Tables. Table 17 (continued) .so 3162 3170 3177 31&4 3192 3199 3206 3214 3221 3228 1 I 1 2 3 4 4 5 6 7 .51 3236 3243 3251 3258 3266 3273 3281 3289 3296 3304 1 2 2 3 4 5 5 6 7 .52 3311 3319 3327 3334 3342 3350 3357 3365 3373 3381 1 2 2 3 4 5 5 6 7 .53 3388 3396 3404 3412 3420 3428 3436 3443 3451 3459 1 2 2 3 4 5 6 6 7 .54 3467 3475 3483 3491 3499 3508 3516 3524 3532 3540 1 2 2 3 4 5 6 6 7 .55 3548 3556 3565 3573 3581 3589 3597 3606 3614 3622 1 2 2 3 4 5 6 7 7 .56 3631 3639 3648 3656 3664 3673 3681 3690 3698 3707 1 2 3 3 4 5 6 7 8 .57 3715 3724 3733 3741 3750 3758 3767 3776 3784 3793 1 2 3 3 4 5 6 7 8 .58 3802 3811 3819 3828 3837 3846 3855 3864 3873 3882 1 2 3 4 4 5 6 7 8 .59 3890 3899 3908 3917 3926 3936 3945 3954 3963 3972 1 2 3 4 5 5 6 7 8 .60 3981 3990 3999 4009 4018 4027 4036 4046 4055 4064 1 2 3 4 5 6 6 7 8 .61 4074 4083 4093 4102 4111 4121 4130 4140 4150 4159 1 2 3 4 5 6 7 8 9 .62 4169 4178 4188 4198 4207 4217 4227 4236 4246 4256 1 2 3 4 5 6 7 8 9 .63 4266 4276 4285 4295 4305 4315 4325 4335 4345 4355 1 2 3 4 5 6 7 8 9 .64 4365 4375 4385 4395 4406 4416 4426 4436 4446 4457 1 2 3 4 5 6 7 8 9 .65 4467 4477 4487 4498 4508 4519 4529 4539 4550 4560 1 2 3 4 5 6 7 8 9 .66 4571 4581 4592 4603 4613 4624 4634 4645 4656 4667 1 2 3 4 5 6 7 9 10 .67 4677 4688 4699 4710 4721 4732 4742 4753 4764 4775 1 2 3 4 5 7 8 9 10 .68 4786 4797 4808 4819 4831 4842 4853 4864 4875 4887 1 2 3 4 6 7 8 9 10 .69 4898 4909 4920 4932 4943 4955 4966 4977 4989 5000 1 2 3 5 6 7 8 9 10 .70 5012 5023 5035 5047 5058 5070 5082 5093 5105 5117 1 2 4 5 6 7 8 9 11 .71 5129 5140 5152 5164 5176 5188 5200 5212 5224 5236 1 2 4 5 6 7 8 10 11 .72 5248 5260 5272 5284 5297 5309 5321 5333 5346 5358 1 2 4 5 6 7 9 10 11 .73 5370 5383 5395 5408 5420 5433 5445 5458 5470 5483 1 3 4 5 6 8 9 10 11 .74 5495 5508 5521 5534 5546 5559 5572 5585 5598 5610 1 3 4 5 6 8 9 10 12 .75 5623 5636 5649 5662 5675 5689 5702 5715 5728 5741 1 3 4 5 7 8 9 10 12 .76 5754 5768 5781 5794 5808 5821 5834 5848 5861 5875 1 3 4 5 7 8 9 11 12 .77 5888 5902 5916 5929 5943 5957 5970 5984 5998 6012 1 3 4 5 7 8 10 11 12 .78 6026 6039 6053 6067 6081 6095 6109 6124 6138 6152 1 3 4 6 7 8 10 11 13 .79 6166 6180 6194 6209 6223 6237 6252 6266 6281 6295 1 3 4 6 7 9 10 11 13 .80 6310 6324 6339 6353 6368 6383 6397 6412 6427 6442 1 3 4 6 7 9 10 12 13 .81 6457 6471 6486 6501 6516 6531 6546 6561 6577 6592 2 3 5 6 8 9 11 12 14 .82 6607 6622 6637 6653 6668 6683 6699 6714 6730 6745 2 3 5 6 8 9 11 12 14 .83 6761 6776 6792 6808 6823 6839 6855 6871 6887 6902 2 3 5 6 8 9 11 13 14 .&4 6918 6934 6950 6966 6982 6998 7015 7031 7047 7063 2 3 5 6 8 10 11 13 15 .85 7079 7096 7112 7129 7145 7161 7178 7194 7211 7228 2 3 5 7 8 10 12 13 15 .86 7244 7261 1278 1295 7311 7328 7345 7362 7379 7396 2 3 5 7 8 10 12 13 15 .87 7413 7430 7447 7464 7482 7499 7516 7534 7551 7568 2 3 5 7 9 10 12 14 16 .88 7586 7603 7621 7638 7656 7674 7691 7709 7727 7745 2 4 5 7 9 11 12 14 16 .89 7762 7780 7798 7816 7834 7852 7870 7889 7907 7925 2 4 5 7 9 11 13 14 16 .90 7943 7962 7980 7998 8017 8035 8054 8072 8091 8110 2 4 6 7 9 11 13 15 17 .91 8128 8147 8166 8185 8204 8222 8241 8260 8279 8299 2 4 6 8 9 11 13 15 17 .92 8318 8337 8356 8375 8395 8414 &433 8453 8472 &492 2 4 6 8 10 12 14 15 17 .93 8511 8531 8551 8570 8590 8610 8630 8650 8670 8690 2 4 6 8 10 12 14 16 18 .94 8710 8730 8750 8770 8790 8810 8831 8851 8872 8892 2 4 6 8 10 12 14 16 18 .95 8913 8933 8954 8974 8995 9016 9036 9057 9078 9099 2 4 6 8 10 12 15 17 19 .96 9120 9141 9162 9183 9204 9226 9247 9268 9290 9311 2 4 6 8 11 13 15 17 19 .97 9333 9354 9376 9397 9419 9441 9462 9484 9506 9528 2 4 7 9 11 13 15 17 20 .98 9550 9572 9594 9616 9638 9661 9683 9705 9727 9750 2 4 7 9 11 13 16 18 20 ~ 9908 9931 9954 9977 2 5 7 9 11 14 16 18 20 'g .99 9772 9795 9817 9840 9863 9886 ()Q 0 n> ..... ("') 1.Ot:;;l ...... Ul e;; ...... '"d 00 0- ~ WHO/CDS/LEP/86.4 page 92 Table 18. Critica142 P values by Fisher's exact test in four-fold tables43,44 a + b c + cl a c P a + b c + cl a c P 3 3 0 3 0.050 10 10 0 10 0.000005 4 4 0 4 0.014 1 10 0.00006 2 10 0.0004 5 5 0 5 0.004 3 10 0.001 0 4 0.024 4 10 0.005 6 6 0 6 0.001 5 10 0.016 5 0.008 6 10 0.043 0 9 0.00006 1 9 U.005 2 9 0.U03 1 5 0.040 3 9 0.010 0 4 0.030 4 9 0.029 0 8 0.0004 7 7 0 7 0.003 1 8 0.003 1 7 0.002 2 8 0.012 0 6 0.002 3 8 0.035 0 7 0.002 1 6 0.015 1 7 0.010 0 5 0.010 2 7 0.035 8 8 0 8 0.00008 0 6 0.005 0 7 0.0007 1 6 0.029 1 7 0.005 0 5 0.016 2 7 0.020 0 4 0.043 0 6 0.003 1 6 0.020 0 5 0.013 0 4 0.038 42 Includes all instances in which P < 0.05 by the one-tailed test. 43 Tables are constructed as follows: Positive Negative Regimen 1 a b a + b Regimen 2 c d c + d Interchange the "positive" and "negative" columns as necessary, to provide a fit to the table. 44 Taken from reference no. 180. 45 Table 19. Table of critical values of D (or C) in the Fisher test Level of significance Totals in right margin B (or A)f Level of significance .05 .025 .01 .005 Totals in right margin B (or A)f A +B = 3 C+D =3 3 0 - - - .05 .025 .01 .005 , , ~ ~ - . 8 - A +B = 4 C +D = 4 4 0 0 - - 7 C+D =3 4 0 - - - 6 5 A +B = 5 C +D = 5 5 1 1 . 0 0 4 4 0 0 - - 8 C+D = 4 5 1 0 0 - 7 4 0 - - - 6 C+D = 3 5 0 0 - - 5 C +D = 2 5 0 - - - 8 7 A +B = 6 C +D = 6 6 2 1 1 0 6 5 1 0 0 - 5 4 0 - - - 8 C +D = 5 6 1 0 0 0 7 5 0 0 - - 6 4 0 - - - 5 I C+D = 4 6 1 0 0 0 8 5 0 0 - - 7 C +D = 3 6 0 0 - - 6 5 0 - - - 8 C +D = 2 6 0 - - - 7 ----- C+D=2 8" o o A +B = 7 C +D = 7 7 3 2 1 1 --1-·------6 1 1 0 0 A +B = 9 C+D=9 9 5 4 3 3 5 0 0 - - 8 3 3 2 1 4 0 - - - 7 2 1 1 0 C+D=6 7 2 2 1 1 6 1 1 0 0 6 1 0 0 0 5 0 0 5 0 0 - - 4 0 4 0 - - - C+D=8 I 9 4 3 3 2 5 C +D = 7 2 1 0 0 8 3 2 1 1 6 1 0 0 - 7 2 1 0 0 5 0 - - - 6 1 0 0 C+D = 4 7 1 1 0 0 5 0 0 6 0 0 - - C +D = 7 I 9 3 3 2 2 5 0 - - - 8 2 2 1· 0 C+D = 3 7 0 0 0 - 7 1 1 0 0 &~ 6 0 - - - 6 0 0 Ib_ C+D = 2 7 0 (") - - - 5 0 \Ct:;j woo to" t"1 45Taken from reference no. 197, which was adapted from Finney, D.J. 1948. The Fisher-Yates test of significance -"'d ...... in 2x2 contingency tables. Biometrika, 35, 149-154. 00 Table 19 is reproduced by permission of the publishers, McGraw-Hi11 , New York. . .j::-."" 'g ~ 0Cl 0 lD_ Table 19, continued n \Ct:::l ~cn Level of significance Level of significance -ij Totals in right margin B (or A)t Totals in right margin B (or A)t ...... 05 .025 .01 .005 .05 .025 .01 .005 00 . 0- A +B = 9 C +D = 6 9 3 2 1 1 A+B=lO C+D=6 10 3 2 2 1 . 8 2 1 0 0 9 2 1 1 0 ~ 7. 1 0 0 - 8 1 1 0 0 6 0 0 - - 7 0 0 - - 5 0 - - - 6 0 - - - C +D = 5 9 2 1 1 1 C +D = 5 10 2 2 1 1 8 1 1 0 0 9 1 1 0 0 7 0 0 ,- - 8 1 0 0 - 6 0 - - - 7 0 0 - - C+D=-1 9 1 1 0 0 6 0 -- - 8 0 0 0 - C+D=4 10 1 1 0 0 7 0 0 - - 9 1 0 0 0 6 0 - - - 8 0 0 - - C+D=3 9 1 0 0 0 7 0 -- - 8 0 0 - - C+])=3 10 1 0 0 0 7 0 - - - 9 0 0 - - C+J)=2 9 0 0 - - 8 0 - - - _.- --- - (j = 2 10 0 0 - .I + B = 10 C+D=lO 10 5 -1 3 C +D - 9 4 3 3 2 9 0 - - - 8 3 2 1 1 A+B-=l1 C+D=l1 11 7 6 5 4 I 1 1 0 10 7 i 2 5 4 3 3 6 1 0 0 - 9 4 3 2 2 - 5 i 0 0 - 8 3 2 1 1 4 0 - - - 7 2 1 0 0 C +D = 9 10 5 -1 3 3 6 1 0 0 - !J 4 3 2 2 5 0 0 - - 8 2 2 1 1 4 0 - - - 7 1 1 0 0 = 10 11 6 5 4 4 I C + D 6 I 1 0 0 - 10 4 4 3 2 5 0 0 - - 9 3 3 2 1 C + D = 8 10 -1 4 3 2 8 2 2 1 0 !J I 3 2 2 1 7 1 1 0 0 8 I 2 1 1 0 6 1 0 0 - 7 1 1 0 0 5 0 - -- 6 0 0 - - C+D=9 11 5 4 4 3 5 0 - - - 10 4 3 2 2 C +D = 7 10 3 3 2 2 9 3 2 1 1 9 2 2 1 1 8 2 1 1 0 8 1 1 0 0 7 1 1 0 0 7 1 0 0 - 6 0 0 -- 6 0 0 - - 5 0 - -- 5 I 0 Table 19, continued Level of significance Level of significance Totals in right margin B (or A)t .05 .025 .01 .005 Totals in right margin H (or A)t A+B=11 C+D=8 11 4 4 3 3 .05 .025 .01 .005 10 3 3 2 1 9 2 2 1 1 A + B = 12 C+D=11 12 7 6 5 5 8 1 1 0 0 11 5 5 -1 3 7 1 0 0 - 10 -1 3 2 2 6 0 O· - - 9 3 2 2 1 5 0 - - - 8 2 1 1 0 C +D = 7 11 4 3 2 2 7 1 1 0 0 10 3 2 1 1 6 1 0 0 - 9 2 1 1 0 5 0 0 -- 8 1 1 0 0 C + D = 10 12 6 5 5 -1 7 0 0 - - 11 5 -1 3 3 6 0 0 - - 10 -t 3 2 2 C +D = 6 11 3 2 2 1 9 3 2 1 1 10 2 1 1 0 8 2 1 0 0 9 1 1 0 0 t 1 0 0 0 8 1 0 0 - 6 0 0 -- 7 0 0 - - 5 0 - -- 6 0 - - - C +D = 9 12 5 5 -1 3 C+D=5 11 2 2· 1 1 11 -1 3 3 2 10 1 1 0 0 10 3 2 2 1 9 1 0 0 0 9 2 2 1 0 8 0 0 - - 8 1 1 0 0 7 0 - - - 7 1 0 0 - C +D = 4 11 1 1 1 0 6 0 0 -- 10 1 0 0 0 5 0 - -- 9 0 0 - - C+D=8 12 5 -1 3 3 8 0 - - - 11 3 3 2 2 C +D = 3 11 1 0 0 0 10 2 2 1 1 10 0 0 - - 9 2 1 1 0 9 0 -- - 8 1 1 0 0 C +D = 2 11 0 0 - - 7 0 0 -- 10 0 -- - 6 0 0 - - A + B = 12 C + D = 12 12 8 7 6 5 C+D=7 12 -1 3 3 2 11 6 5 -1 4 11 3 2 2 1 10 5 4 3 2 10 2 1 1 0 9 1 1 0 9 4 3 2 1 0 ~ 3 2 1 1 8 1 0 0 - 'g 8 OQ 0 7 2 1 0 0 7 0 0 - - (1)_ C") 6 1 0 0 - 6 0 - - - \Ot:;l 5 0 0 - - - V'Itn 4 0 - - - ~ -'"d ...... 00 .0' ~ 'g ~ (IQ 0 Table 19, continued (D- C') IDt:::I 0'00 Level of significance i ~ Totals in right margin B (or A)t Level of signifieunee -'"d .05 .025 .01 .005 Totals in right margin B (or A)t - .05 .025 .01 .005 00 A + B = 12 C +D = 6 12 3 3 2 2 0'1 A +B = 13 C+D=l1 13 7 6 5 5 -. 11 2 2 1 1 12 6 5 4 3 ~ 10 1 1 0 0 11 4 4 3 2 0, 9 1 0 0 10 3 3 2 1 8 0 o· - - 9 3 2 1 1 7 0 0 - - 8 2 1 0 0 6 0 - - - 7 .1 0 0 0 C+D=5 12 2 2 1 1 6 0 0 -- 11 1 1 1 0 5 0 - - - 10 1 0 0 0 C + D = 10 ]3 6 6 5 4 9 0 0 0 - ]2 5 4 3 3 8 0 0 - - 11 4 3 2 2 7 0 - - - 10 3 2 1 1 C +D ... 4 12 2 1 1 0 9 2 1 ] 0 11 1 0 0 0 8 1 1 0 0 10 0 0 0 - 7 1 0 0 - 9 0 - - 0 6 0 () -- 8 0 - - - 5 0 - - - C+D=3 12 1 0 0 0 C+D=9 ]3 5 5 4 4 11 0 0 0 - 12 4 4 3 2 10 0 0 - - 11 3 3 2 1 9 0 - - - ]0 2 2 1 1 (,'+])=2 () 12 0 - - 9 2 1 0 0 11 0 - - - - " "------"" 1------8 1 1 0 0 A + B = 13 C + D = 13 1:~ U 8 7 6 7 ! 0 0 - - 12 7 6 5 4 G 0 0 -- 11 6 5 4 3 5 0 - - - 10 4 4 3 2 C+D=8 ]3 5 4 :~ 3 9 3 3 2 1 12 4 3 2 2 8 2 2 1 0 11 3 2 1 1 7 2 1 0 0 10 2 1 1 0 6 1 0 0 - 9 1 1 0 0 5 0 0 - - 8 1 0 0 - 4 0 - - - 7 0 0 - - C + J) = 12 13 8 7 6 5 6 0 - - - 12 6 5 5 4 C +D = 7 13 3 3 2 11 5 4 3 3 12 3'"" 2 2 1 10 4 3 2 2 11 2 2 1 1 9 3 2 1 1 10 1 1 0 0 8 2 1 1 0 9 1 0 0 0 7 1 1 0 0 8 0 0 -- G 1 0 0 - 7 0 0 - - 5 0 0 - - 6 0 - -- L"" Table 19, continued Level of significance Level of significance Totals in right margin B (or A)t Totals in right margin B (or A)t .05 .025 .01 .005 .05 .025 .01 .005 A + B= 14 C + D = 13 14 9 8 7 6 13 7 6 5 5 12 6 5 4 3 13 3 3· 2 2 A + B = 13 C +D = 6 11 5 -1 3 2 12 2 2 1 1 10 4 3 2 2 2 1 1 0 11 9 3 2 1 1 1 1 0 0 10 8 2 1 1 0 9 1 0 0 - 7 1 1 0 o 8 0 0 - - 6 1 0 - - 7 0 - - - 5 0 0 - - 13 2 2 1 1 C+D =5 C + D = 12 14 8 7 6 6 12 2 1 1 0 13 6 6 5 4 11 1 1 0 0 12 5 4 4 3 10 1 0 0 - 11 4 3 3 2 I 9 0 0 - - I 10 3 3 2 1 8 0 - - - I 9 2 2 1 1 13 2 1 1 0 C+D = 4 8 2 1 0 0 12 1 1 0 0 I 7 1 0 0 - 11 0 0 o 6 0 0 - - 10 0 0 5 0 - - - 9 0 C+D=l1 14 i 6 6 5 13 1 1 0 0 C+D = 3 I 13 6 5 4 4 12 0 0 0 12 5 4 3 3 11 0 0 11 4 3 2 2 10 0 10 3 2 1 1 13 0 0 0 C+D =2 I 9 2 1 1 0 12 I 0 8 1 1 0 0 ! 7 1 0 0 - 14 9 8 7 A+B=14 C + D = 14 I I 10 6 0 0 - - 13 7 6 5 I 8 5 0 - - 12 6 5 4 - C + D = 10 14 6 6 5 4 11 -1 3 3 I : 13 5 4 4 3 10 4 3 2 2 12 4 3 3 2 9 3 2 2 1 11 3 3 2 1 8 I 2 2 1 0 10 2 2 1 1 I 1 1 0 0 ~ 7 9 2 1 0 0 'g 6 1 0 0 ()Q 0 8 1 1 0 0 l1) ...... 5 0 0 n 7 0 0 0 - 4 0 \Ot:::l 6 0 0 - - ..... Cl) 5 0 - - - t""' trJ "'0 00 .0'1 .l::- ~~ (1)_ Table 19, continued n \Ct::t 0000 ~ . - '"C Level of significance Level of significanee Totals in right margin B (or A)t ~ .05 .025 .01 .005 Totals ill right margin B (or A)t -. ~ A + B = 14 C +D = 9 14 6 5 4 4 .05 .025 .01 .005 13 4 4 3 3 . 12 3 3 2 2 11 3 Z 1 1 .4 + II ... 14 C + D ... 4: 14 2 1 1 1 10 2 1 1 0 13 1 1 0 0 9 1 1 0 0 12 1 0 0 0 8 1 0 0 - 11 0 0 - - 7 0 0 - - 10 0 0 - - 6 0 - - - 9 0 - - - C+D=8 14 5 -I ·1 3 C+D .. 3 14 1 1 0 0 13 4 3 2 2 13 0 0 0 - 12 3 2 2 1 12 0 0 - - 11 2 2 1 1 11 0 - - - 10 2 1 0 0 C+D ... 2 1,1 0 0 0 - 9 1 0 0 0 13 0 0 - - 8 0 0 0 - 12 0 - - - 7 0 0 - - - 15 15 11 10 9 8 6 I 0 - - A + B ... 15 C + D ... C+D=7 14 i 4 3 3 2 14 9 8 7 6 13 3 2 2 1 13 7 6 5 5 12 2 2 1 1 12 6 5 4 4 11 2 1 1 0 11 5 4 3 3 10 1 1 0 0 10 4 3 2 2 9 1 0 0 - 9 3 2 1 1 8 0 0 - - 8 2 1 1 0 7 0 - - - 7 1 1 0 0 C+D=6 14 3 3 2 2 6 1 0 0 - 13 2 2 1 1 5 0 0 - - 12 2 1 1 0 4 0 - - - 11 1 1 0 0 C +D ... 14 15 10 9 8 7 10 1 0 0 - 14 8 7 6 6 9 0 0 - - 13 7 6 5 4 8 0 0 - - 12 6 5 4 3 7 0 - - - 11 5 4 3 2 C+D=5 14 2 2 1 1 10 4 3 2 1 13 2 1 1 0 9 3 2 1 1 12 1 1 0 0 8 2 1 1 0 11 . 1 0 0 0 7 1 1 0 0 10 0 0 - - 6 1 0 -- 9 0 0 - - 5 0 - -- 8 0 - - - Table 19, continued Level of significance Level of significance Totals in right margin H (or A)t .05 .025 .01 .005 Totals in right margin IH (or A)t .05 .025 .01 .005 15 6 5 4 4 15 9 8 7 7 A + B == 15 C+D = 9 A + B = 15 C + D = 13 14 5 4 3 3 14 7 7 6 5 13 4 3 2 2 13 6 5 4 4 12 3 2 2 1 12 5 4 3 3 11 2 2 1 1 11 4 3- 2 2 10 2 1 0 0 10 3 2 2 1 9 1 1 0 0 9 2. 2 1 0 8 1 0 0 8 2 1 0 0 7 0 0 7 1 0 0 - 6 0 6 I 0 0 - - I 15 5 4 4 3 5 0 - - - C+D=8 14 4 3 3 2 15 8 7 i 6 C + D = 12 I 13 3 2 2 1 14 7 6 5 4 I 12 2 2 1 1 13 6 5 4 3 I 11 2 1 1 0 12 5 4 3 2 10 1 1 0 0 11 4 3 2 2 9 1 0 0 10 3 2 1 1 8 0 0 9 2 1 1 0 7 0 8 1 1 0 0 6 0 i 1 0 0 - 15 4 4 3 3 6 0 0 - - C+D=7 14 3 3 2 2 5 0 - - - 13 2 2 1 1 15 t 7 6 5 C+D=l1 I 12 2 1 1 0 14 6 5 4 -1 11 1 1 0 0 13 5 -1 3 3 I 10 1 0 0 0 12 -1 3 2 2 9 0 0 11 3 2 2 1 8 0 0 10 2 2 1 1 t 0 9 2 1 0 0 15 3 3 2 2 8 1 1 0 0 C+D=6 14 2 2 1 1 7 1 0 0 - 13 2 1 1 0 6 0 0 - - 12 1 1 0 0 5 0 - - - 11 1 0 0 0 15 6 6 5 5 C + D = 10 I 10 0 0 0 14 5 5 4 3 9 0 0 - 13 4 4 3 2 8 0 - - - 'g~ 12 3 3 2 2 15 2 2 2 1 ()Q 0 11 3 2 1 1 C+-P=5 tll ...... 14 2 1 1 1 (") 10 2 1 1 0 13 1 1 0 0 \Ot::I 9 1 1 0 0 \Coo 12 1 0 0 0 ...... 8 1 0 0 - t-I 11 0 0 0 - t:<:l 7 0 0 - - 10 0 0 - - "'d 6 0 - - - ...... 9 0 - - - 00 .0'\ ,J::oo WHO/CDS/LEP/86.4 page 100 Table 19, continued Level of significnncc Totals in right margin B (or ..1)t .05 .025 .01 .005 :1 + n = 15 C + J) = ·1 15 2 1 1 1 1·1 1 I 0 0 1:1 1 0 0 0 12 0 0 0 11 0 0 10 0 C+D=3 15 1 1 0 0 H 0 0 0 0 13 0 0 12 0 0 11 0 C+D=2 15 0 0 0 1-l 0 0 1:\ 0 Table 20. Proportion of viable M. 1eprae in several suspensions46 No. AFB inoculated 50,000 5,000 500 50 5 0.5 Exper- Proportion 95% 0.69 iment of viable confidence no. No. mice showing multiplication/no. inoculated M. 1eprae limits !D50 1 10/10* 7/10 2/10 0/10 0/10* 0.0003 0.0002-0.0001 0.00035 2 10/10* 10/10 6/10 1/10 0/10* 0.0018 0.0009-0.004 0.0022 3 10/10* 10/10 7/10 0/10 0/10* 0.0024 0.0010-0.0044 0.0022 4 10/10* 10/10 2/2 1/10 0/10* 0.0036 0.0019-0.0093 0.0055 5 10/10* 10/10 7/10 1/10 0/10* 0.024 0.012-0.05 0.028 6 10/10* 10/10 7/10 0/10* 0.24 0.12-0.54 0.22 7 10/10* 10/10 9/10 0/10* 0.46 0.22-1.0 0.35 * Assumed values. The MPN calculation may produce highly improbable results. This problem has been approached by de Man (119), who has devised tables in which one may find the "normal" results obtained in 95% of cases, the "less-likely" results obtained in 4% of cases, and the 95 and 99% confidence limits for each value of the MPN falling into either of these two categories. (The even less likely results are not shown in de Man's tables, and are stated to be unacceptable.) The most useful of de Man's tables appears as Table 21. 46 Adapted from reference no. 214. WHO/CDS/LEP/86.4 page 101 Median Infectious Dose (IDSO) Because the assumption that M. leprae are randomly distributed within a suspension appears invalid, determination of the median infectious dose (IDSO)' the number of M. leprae required to infect half of the animals inoculated, has been proposed as an alternative to calculation of the MPN. A suitable method for determining the IDSO has been described by Shepard (180). As shown in Table 22 for the first two examples in Table 20, the 10glO numbers of M. leprae inoculated per mouse (termed log F) are calculated and transformed by the appropriate addition or subtraction so that the smallest value (termed x) is 1 (in this case, 1.3 is added to each value of log F). The values for F and, therefore, for log F and for x, must be evenly spaced. One then calculates the proportion (Pi) of mice showing multiplication at each dilution, and sums these to calculate rn, which is defined by the equation: m xk + 1/2 in which xk is the largest value of x employed, d is the difference between successive values of x, and SPi is the sum of the individual values of Pi. The value of m is then decreased (or increased) by the quantity originally added to (or subtracted from) log F, yielding log IDSO' the estimate of the logarithm of the number of M. leprae needed to infect half of the mice. Dividing 0.69, the number of viable M. leprae theoretically required to infect half the mice, by the IDSO (antilog of log IDSO) yields the proportion of viable M. leprae in the inoculum. The values calculated for the seven experiments of Table 20, shown in the left-hand-most column of the table, compare very well with those in the third column from the left, which had been calculated from the MPN. To calculate the significance of the difference between two values for log IDSO' one first calculates the variance of rn, Vm, according to the equation: in which d is the difference between successive values of log F or x; S indicates the sum; Pi is the proportion of infected mice; qi 1 - Pi, the proportion of uninfected mice; and ni is the total number of mice inoculated from each dilution. The square root of the sum of two values of Vm represents the standard deviation of the difference between the corresponding values of log IDSO. In the case of experiments 1 and 2 of Table 22, with log IDSO values of 3.3 and 2.S, respectively, + 0.28 . Then, one divides the difference between the two values of log IDSO by the standard devia tion of the difference: IDSO IDSO 3.3 - 2.S 2 1 2.9 , + 0.28 and finds, in Table 23, the fraction of the area under the normal curve represented by such a "deviation" from 0 - the mid-point of the curve. From this table, one finds that· a deviation of 2.9 includes 99.63% of the area under the curve - i.e., all but 0.27% of the area is included; thus, the probability that the two values of log IDSO were drawn from the same population is only 0.0027, less than 0.01. ~~ (IQ 0 Confidence limits v~lues. fo~ the ~N47 (D- Table 21. Qf C") I-'t::l Ot/) N ...... t'" MPN/g(ml) 10 x 0.1000 ; 10 x 0.0100 g(ml) t:<:I MPN/g(ml) 10 x 1 ; t'tl 10 x 1 ; 10 x 0.1000 ; 10 x 0.0100 g(ml) category confidence limits 00 result MPN 95% 0- result MPN category confidence limits 1 2 99% -. 95% 1 2 99% 0.6 2.5 ~ 1.2 x 0.4 2.9 0.51 ? 1 0 3.2 O.? 2.? 0 1 0.09 x <0.01 0.6? 0.01 1 1 1.3 x 0.5 0 0.68 0.01 0.51 ? x 0.6 3.5 0.8 3.0 0 1 0 0.09 x <0.01 ? 1 2 1.5 o.? 2.? x 0.02 0.85 0.05 ,0.6? 1.3 x 0.5 3.3 0 2 0 0.18 ? 2 0 3.5 0.8 3.0 0 0.09 x <0.01 0.?1 0.01 0.54 2 1 1.5 x 0.6 1 0 0.05 0.69 ? x O.? 3.8 0.9 3.2 1 0 1 0.19 x 0.02 0.89 ? 2 2 1.? 0.8 3.0 0.02 0.89 0.05 o.?o 1.5 x 0.6 3.6 1 1 0 0.19 x ? 3 0 3.9 0.9 3.3 0.29 x 0.06 1.06 0.09 0.85 1 1.? x o.? 1 2 0 0.06 0.?3 ? 3 o.? 3.9 0.9· 3.3 2 0 0 0.20 x 0.02 0.93 ? 4 0 1.? x 1.0 3.6 x 0.1 1.1 0.1 0.9 1 1.9 x 0.8 4.2 2 0 1 0.3 ? 4 4.3 1.0 3.6 1 0.3 x 0.1 1.1 0.1 0.9 0 1.9 x 0.8 2 0 0.1 1.0 ? 5 0.5 3.3 0.6 2.8 2 1 1 0.4 x 0.1 1.3 8 0 0 1.3 x O.? 3.1 0.1 1.3 0.2 1.0 1 1.5 x 0.6 3.? 2 2 0 0.4 x 1.2 8 0 4.1 0.8 3.4 0 0.5 x 0.1 1.5 0.2 8 0 2 1.? x o.? 2 3 1.2 0.1 0.9 0.6 3.? O.? 3.1 3 0 0 0.3 x 0.1 8 1 0 1.5 x 0.9 3.4 x 0.1 1.4 0.2 1.1 1 1.? x O.? 4.1 3 0 1 0.4 1.1 8 1 4.5 1.0 3.? 1 0 0.4 x 0.1 1.4 0.2 8 1 2 1.9 x 0.8 3 x 0.2 1.5 0.2 1.3 x O.? 4.1 0.9 3.5 3 1 1 0.5 8 2 0 1.~ 4.5 1.0 3.8 0 0.5 x 0.2 1.5 0.2 1.3 2 1 1. x 0.8 3 2 0.3 1.4 8 0.9 4.9 1.2 4.2 3 2 1 0.6 x 0.2 1.? 8 2 2 2.1 x 1.0 3.9 x 0.2 1.7 0.3 1.4 1.9 x 0.8 4.6 3 3 0 0.6 1.2 8 3 0 5.0 1.2 4.2 0 0 0.4 x 0.1 1.4 0.2 8 1 2.1 x 0.9 4 1.6 0.2 1.3 3 x 1.0 5.4 1.3 4.6 4 0 1 0.6 x 0.2 8 3 2 2.4 1.2 4.3 0.6 x 0.2 1.7 0.2 1.4 2.2 x 0.9 5.1 4 1 0 1.5 8 4 0 1.3 4.6 4 1 1 0.7 x 0.2 1.8 0.3 8 4 1 2.4 x 1.0 5·5 1.8 0.3 1.5 1.1 5.5 1.3 4.7 4 2 0 0.7 x 0.2 8 5 0 2.4 x 1.5 5.1 x 0.3 2.0 0.4 1.7 x 1.2 6.0 4 2 1 0.8 8 5 1 2.7 6.1 1.5 5.2 4 0 0.8 x 0.3 2.1 0.4 1.7 6 0 2.7 x 1.2 3 0.5 1.9 8 0.6 4.5 0.8 3.? 4 4 0 0.9 x 0.3 2.2 9 0 0 1.7 x 1.0 4.1 0 0 0.6 x 0.2 1.8 0.2 1.5 0 1 1.9 x 0.7 5.0 5 2.0 0.3 1.6 9 x 0.9 5.6 1.1 4.6 5 0 1 0.7 x 0.2 9 0 2 2.2 1.0 4.2 1 0 0.7 x 0.2 2.0 0.3 1.7 1 0 1.9 x 0.7 5.1 5 2.2 0.4 1.8 9 0.9 5.6 1.1 4.7 5 1 1 0.9 x 0.3 9 1 1 2.2 x 1.3 5.2 x 0.3 2.2 0.4 1.8 2.5 x 1.0 6.2 5 2 0 0.9 2.0 9 1 2 5.8 1.2 4.7 2 1 1.0 x 0.4 2.4 0.5 9 2 0 2.2 x 0.9 5 0.4 2.4 0.5 2.0 1.0 6.4 1.' 5.3 5 3 0 1.0 x 9 2 1 2.5 x 1.5 5.8 1 1.1 x 0.4 2.6 0.6 2.2 2 2 2.8 x 1.2 7.0 5 3 2.6 0.6 2.2 9 1.1 6.5 1.3 5.4 5 4 0 1.1 x 0.4 9 3 0 2.5 x 1.5 6.0 0.8 x 0.3 2.2 0.3 1.8 1 2.9 x 1.2 7.2 6 0 0 2.0 9 3 1.4 ?8 1.8 6.6 6 0 1 0.9 x 0.3 2.4 0.4 3 2 3.2 x 2.4 0.4 2.0 9 1.2 7.3 1.6 6.1 6 1 0 0.9 x 0.3 9 4 0 2.9 x 1.8 6.7 x 0.4 2.6 0.5 2.2 x 1.4 8.0 6 1 1 1.1 9 4 1 3.3 8.8 2.0 7.4 1.1 x 0.4 2.6 0.5 2.2 4 2 3.? x 1.6 6 2 0 0.6 2.4 9 1.4 8.2 1.8 6.9 6 2 1 1.2 x 0.5 2.9 9 5 0 3.3 x 2.9 0.6 2.5 x 1.6 9·0 2.0 7.6 6 3 0 1.2 x 0.5 9 5 1 3.7 2.3 8.3 1 1.4 x 0.6 3.2 0.7 2.7 4.2 x 1.9 9.8 6 3 2.7 9 5 2 9.2 2.1 7.7 6 4 0 1.4 x 0.6 3.2 0.7 6 0 3.8 x 1.7 3.4 0.8 2.9 9 x 1.9 10.0 2.4 8.5 6 5 0 1.5 x 0.7 9 6 1 4.3 2.4 8.7 x 0.3 2.6 0.5 2.2 4.4 x 1.9 10.3 ? 0 0 1.0 9 ? 0 7.2 1.2 5.8 1 1.2 x 0.4 2.9 0.6 2.4 0 0 2.3 x 0.9 ? 0 0.7 2.? 10 ? 0 2 1.3 x 0.5 3.2 47 Taken from reference no. 119 Table 21 1 con.tinued MPN/g(ml) MPN/g(ml) 10 x 1; 10 x 0,,1000 ; 10 x 0.0100 g(ml) 10 x 1 ; 10 'x 0.1000 ; 10 x 0.0100 g(ml) result MPN category confidence limits result MPN category confidence limits 1 2 99% 95% 1 2 99% 95% 1.1 8.3 1.4 6.7 90 x 30 260 40 210 10 0 1 2.7 x 10 10 6 60 270 10 0 2 3.1 x 1.3 9.5 1.6 7.7 10 10 7 120 x 40 330 1.1 8.6 1.4 6.9 160 x 60 450 80 370 10 1 0 2.7 x 10 10 8 110 600 10 1 1 3.2 x 1.3 9.9 1.7 7.9 10 10 9 230 x 80 760 10 1 2 3.8 x 1.5 11.3 '2.0 9.2 10 2 0 3.3 x 1.3 10.3 1.7 8.3 10 2 1 3.9 x 1.6 11.7 2.0 9.6 10 2 2 5 x 2 13 2 11 Category 1: Normal results, obtained in 95% of cases x 2 15 3 12 Category 2: Less likely results, obtained only in 4% of case. 10 2 3 5 These are not to be used for important decisions 10 3 0 4.0 x 1.6 12.4 2.0 10.0 1 x 2 14 2 12 Results that are even less likely than those of category 2 10 3 5 are not mentioned in the table and are always unacceptable. 10 3 2 6 x 2 16 3 13 10 3 3 7 x 3 18 3 15 10 4 0 5 x 2 15 3 12 ~o 4 1 6 x 2 17 3 14 10 4 2 7 x 3 19 3 16 10 4 3 8 x 3 21 4 17 10 5 0 6 x 2 18 3 15 10 5 1 7 x 3 20 4 17 x 3 22 4 19 10 5 2 9 21 10 5 3 10 x 4 25 5 10 6 0 8 x 3 22 4 18 10 6 1 9 x 4 24 5 20 10 6 2 11 x 4 27 5 23 10 6 3 12 x 5 30 6 25 10 6 4 14 x 6 32 7 27 10 7,0 10 x 4 27 5 22 10 7 1 12 x 4 30 6 25 10 7 2 14 x 5 33 7 28 10 7 3 15 x 6 36 8 31 10 7 4 17 x 7 40 9 34 10 8 0 13 x 5 34 6 28 10 8 1 15 x 6 38 8 32 10 8 2 17 x 7 42 9 36 10 8 3 20 x 8 47 10 40 10 B 4 22 x 10 52 12 44 10 8 5 25 x 11 57 14 48 10 9 0 17 x 6 47 9 38 10 9 1 20 x 8 53 10 43 10 9 2 23 x 9 60 12 49 10 9 3 26 x 11 68 14 56 10 9 4 30 x 13 77 16 64 10 x 15 86 18 72 .~ ~ 9 5 35 21 82 10 9 6 40 x 17 97 ()Q 0 10 10 0 24 x 9 77 12 61 Cl> <, x 11 94 15 75 n 10 10 1 29 .... t:::l 10 10 2 35 x .13 114 17 91 OCf.l 10 10 3 40 x 20 140 20 110 l".J <, 20 170 30 140 t'"" 10 10 4 50 x tr.l 10 10 5 70 x 30 210 30 170 "tl 00 -.0'1 ~ WHO/CDS/LEP/86.4 page 104 Table 22. Proportion of viable M. leprae calculated by the rOsO method No. M. leprae No. mice Experiment inoculated log10F X hiuvested "positive" q/ni Pi 1 0.5 -0.3 1 10 o * 0 0 5.0 0.7 2 10 o * 0 0 50 1.7 3 10 o * 0 0 500 2.7 4 10 2 0.2 0.0178 5,000 3.7 5 10 7 0.7 0.0233 50,000 4.7 6 10 10* 1.0 0 0.0411 Spi = 1.9; SpiCll = __ n i-I m1 = 6 + 1/2 - 1 x 1.9. = 4.6; 4.6 - 1.3 3.3; antilog 3.3 1,995; 0.69/1,995 = 0.00035; v~ = 0.0411. No. M. 1eprae No. mice Experiment inoculated log10F X hiuvested "positive" q/ni Pi 2 0.5 -0.3 1 10 o * 0 0 5.0 0.7 2 10 o * 0 0 50 1.7 3 10 1 0.1 0.01 500 2.7 4 10 6 0.6 0.0267 5,000 3.7 5 10 10 1.0 0 50,000 4.7 6 10 10* 1.0 0 0.0367 Spi = 2.7; SpiCll = ni - 1 m2 "= 6 + 1/2 - 1 x 2.7 = 3.8; 3.8 - 1.3 2.5; antilog 2.5 316; 0.69/316 = 0.0022; v~ = 0.0367 • * Assumed values WHO/CDS/LEP/86.4 page 105 Table 23. Fractional areas under the normal curve48 Deviation c -~ Integral '------1I.lllKI 11.1101 1I.111I! 0.11113 O.lll'" 1I.IM15 O. 48 Taken from reference no. 39, and reproduced by permission of the publishers of the Geigy Scientific Tables. WHO/CDS/LEP/86.4 ·page 106 Table 23 continued Deviation c -~ Integral O.IXKI ().OOl 0.002 O.lKI3 O.IXI" O.n05 0.006 0.1107 0.008 0.(109 ~o~ 0.1 0.1 0.1 0.1 o.! O. 0.50 38292:38363'38433\38504 38574 38644 38714138785\38855,'38925 0.51 38995'39065'139135 39205 39275 39345 39415 39484 39554 39624 0.52 39694 39763 39833 39903 39972 40042 40111 40181 402SO,40319 0.53 40389 40458 40527 40597 40666 40735 40804 40873 40942 41011 0.54 41080 41149 41218 41287 41356 41425 41493 41562 41631 41699 0.55 417(~ 41837 41905 41974 42042 42111 42179 42247 42316 42384 0.56 42452 42520 42588 42657 42725 42793 42861 42929 42997 4J064 0.57 43132 43200 432t»8 43336 43403 43471 43538 43606 43674 43741 0.58 43809 43876 43943 44011 44078 44145 44212 44280 44347144414 0.59 44481 44548 44615 44682 44749 44816 44882 44949 45016 45083 0.60 45149 45216 45283 45349 45416 45482 45549 45615 45681 45748 0.61 45814 45880 45946 46012 46078 46145 4621I 46277 46342 46408 0.62 46474 46540 46606 46672 46737 46803 46869 46934' 47000 47065 0.63 47131 47196 47261 47327 47392 47457 47522 47588 47653 47718 0.64 47783 47848 47913 47978 48042 48107 48172 48237 48302 48366 0.65 48431 48495 48560 48624 48689 48753 48818 48882 48946'49010 0.66 49075 49139 49203 49267 49331 49395 49459 49523 49587'496SO 0.67 49714 49778 49842 49905 49969 50032 50096 S0159 S0223 S0286 0.68 S03SO 50413 50476 S0539 50602 50666 50729 50792 50855 50918 0.69 50981 51043 51106 51169 51232 51294 51357 51420 51482 51545 0.7 51607 52230 52848 53461 54070 54675 55275 55870 56461 57047 0.8 57629 58206 58778 59346 59909 60467 61021 61570 62114 62653 0.9 63188 63718 64243 64763 65278 65789 66294 66795 67291 67783 1.0 68269 687SO 69227 69699 70166 70628 71086 71538 71986 72429 1.1 72867 73300 73729 74152 74571 74986 75395 75800 76200 76595 1.2 76986 77372 77754 78130 78S02 78870 79233 79592 79945 80295 1.3 80640 80980 81316 81648 81975 82298 82617 82931 83241 83547 1.4 83849 84146 84439 84728 8S013 85294 85571 85844 86113 86378 1.5 86639 86896 87149 87398 87644 87886 88124 88358 88589 88817 1.6 89040 89260 89477 89690 89899 90106 90309 90508 90704 90897 1.7 91087 91273 91457 91637 91814 91988 92159 92327 92492 92655 1.8 92814 92970 93124 93275 93423 93569 93711 93852 93989 94124 1.9 94257 94387 94514 94639 94762 94882 95000 95116 95230 95341 2.0 954SO 95557 95662 95764 95865 95964 96060 96155 96247 96338 2.1 96427 96514 96599 96683 96765 96844 96923 96999 97074 97148 2.2 97219 97289 97358 97425 97491 97555 97618 97679 97739 97798 . 2.3 97855 97911 97966 98019 98072 98123 98173 98221 98269 98315 2.4 98360 98405 98448 98490 98531 98571 98611 98649 98686 98723 2.5 98758 98793 98826 98859 98891 98923 98953 98983 99012 99040 2.6 99068 99095 99121 99146 99171 99195 99219 99241 99264 99285 2.7 99307 99327 99347 99367 99386 99404 99422 99439 99456 99473 2.8 99489 99505 99520 99535 99549 99563 99576 99590 99602 99615 2.9 99627 99639 996SO 99661 99672 99682 99692 99702 99712 99721 3.0 99730 99739 99747 99755 99763 99771 99779 99786 99793 99800 3.1 99806 99813 99819 99825 99831 99837 99842 99848 99853199858 3.2 99863 99867 99872 99876 99880 99885 99889 99892 99896 99900 3.3 .99903 99907 99910 99913 9991699919 99922 99925 99928199930 3.4: 99933; 3.5: 99953; 3.6: 99968; 3.7: 99978; 3.8: 99986; 3.891: 99990 WHO/CDS/LEP/86.4 page 107 CHAPTER 7 - IDENTIFICATION OF MYCOBACTERIUM LEPRAE Cultivation of M. leprae in cell-free culture has been reported not infrequently. Generally, the isolates have differed from one another in one or more properties; some have not even been acid-fast. They all possess one property in common, however; most of the organisms claimed to be M. leprae have been isolated from a tissue or body fluid of a leprosy patient. One researcher has pointed out that, because the isolates possess such a wide variety of properties, and differ so widely, one from another, they cannot all be M. leprae (175). Indeed, not one of these claims has met with general acceptance. It is difficult to prove that an organism is M. leprae, because, until recently, it has not been possible to fulfil Koch's postulates49, and because most all of the gener ally accepted taxonomic criteria for identification of M. leprae are negative - that is, M. leprae lacks a given property. Nevertheless, as difficult as it may be to identify an organism as M. leprae, or to exclude M. leprae as a possible identity, it appears incumbent upon each claimant to successful cultivation to examine his putative M. leprae according to the following established criteria: (1) M. leprae is unable to multiply in culture media appropriate to other Myco bacteria and organisms of other genera; (2) M. leprae is almost universally agreed to belong to the genus Mycobacterium, with the morphological features, including acid-fastness, and biochemical char acteristics common to all members of the genus; (3) M. leprae multiplies in the footpad of the immunologically normal mouse in a characteristfc fashion; (4) M. leprae is usually inhibited from multiplying in mice by dapsone adminis tered in a very small concentration; (5) a "lepromin" prepared from M. leprae provokes a typical reaction when applied as a skin test to patients with tuberculoid leprosy, and no reaction in patients with polar lepromatous leprosy. In addition, properties with respect to oxidation of dihydroxyphenylalanine (DOPA), and loss of acid-fastness upon extraction with pyridine, have been claimed to be charac teristic. Also the cell walls of M. leprae contain both glycine and meso diaminopimelic acid, a rare situation. Finally, means will soon be available to identify antigens and fragments of deoxyribonucleic acid (DNA) that are specific to the species M. leprae - that is, it will soon be possible to identify M. leprae according to what it is, rather than what it does (or does not do). These criteria are examined more fully in the remainder of this chapter. A general word of caution appears necessary. One must be careful to carry out the tests for identifying an isolate as M. leprae on subcultures, so as to avoid "carry over". Certainly, one wishes to demonstrate the properties, not of the inoculum itself, which may well include M. leprae capable of surviving for some time, but rather of the progeny of the inoculated organisms. Morphology M. leprae is an acid-alcohol-fast bacillus with mean width 0.3 ~m and mean length 2.1 ~m. Compared to other members of the genus Mycobacterium, M. leprae is less able to withstand heating during fixation and staining. 49 Koch's postulates, enunciated by Robert Koch, 1843 - 1910, discoverer of the tubercle bacillus, described the kinds of experimental evidence required to establish the etiologic relationship of a given micro-organism to a given disease. The requirements are: (1) the organism must be observed in every case of the disease; (2) it must be isolated and grown in pure culture; (3) the isolated organism must reproduce the disease upon inoculation into an experimental animal; and (4) the organism must be observed in and recovered from the diseased experimental animal (14). WHOjCDS/LEPj86.4 page 108 H. 1eprae is commonly described as pleomorphic. This characteristic had been described by many workers (34, 117, 129), who found large proportions of poorly-stained organisms in skin-scrapings and biopsy specimens of patients undergoing chemotherapy, and interpreted these morphological changes as evidence of bacterial degeneration and death (see pp. 25 - 29). Careful study of individual H. 1eprae by both light and electron microscopy appeared to confirm this hypothesis (154, 155), as has a study of the rela tionship of the morphological appearance of H. 1eprae under light microscopy to infec tivity of the organisms for mice (121, 191). H. 1eprae may be further characterized as occurring in bunches, termed "globi" , in human lepromatous lesions. Study of H. 1eprae by electron microscopy has revealed a cell wall composed of two layers; a three-layer plasma membrane; mesosomes, which appear to be invaginations of the plasma membrane, with which they are sometimes seen to be in continuity; a nucleoid; and cytoplasmic granules (78, 79). The cytoplasm of H. avium was reported to be much more uniformly granular than that of H. 1eprae, particularly in ultrathin sections, suggesting that the disorganized appearance of the cytoplasm of H. 1eprae might have resulted from degeneration of the organisms (12). Studies of the fine structure of the cell wall have shown two structural features. Paired fibrous structures, similar to those described in the cell walls of a number of cultivable mycobacterial species (203), have been seen in shadow-casted and negatively stained preparations of the cell walls of H. 1eprae (86). These structures varied in diameter from 3 to 30 nm, and appeared to cover the surface of cellular "ghosts". Transverse-band structures, originally described in studies of H. 1epraemurium (138), have been seen in shadow-casted preparations of H. 1eprae (132). Varying in diameter from 10 to 30 nm, the band structures appeared to be interwoven with the paired fibrous structures, and even to be continuous with them. Virtually all reports of electron microscopic observations of H. 1eprae suggest that the ultrastructure of this organism is very much like that of other members of the genus. Multiplication of an Organism Certainly, before cultivation of H. 1eprae can be considered to have been success ful, the putative isolate must be shown to have multiplied in culture. The most direct means by which multiplication may be demonstrated is to count the number of organisms inoculated, and show that the number harvested is larger than that inoculated. There are two considerations of the greatest importance: the absolute number of the AFB counted; and the magnitude of the counting error. One must actually count the organisms present in a measured volume of sample. It is not sufficient to show, for example, that the average number of organisms per macrophage has increased; in this example, one must also show that the number of macrophages has not decreased, and that the apparent increase of AFB has not resulted merely from their redis tribution. Even assuming that the absolute number of AFB can be shown to have increased, it is also necessary to show that the increase is greater than can be expected from chance variation. As long as the particles may be assumed to be distributed both randomly and indepen dently, one from another the distribution of particles in a volume of suspension is described by the Poissons0 distribution. This assumption is probably valid for the distribution of red blood cells in a blood-cell counting chamber. However, although red blood cells are distributed randomly, they are not distributed uniformly. Thus, as long as one counts only a sample of the total population of red blood cells (the number seen to overlie a given area of the counting chamber), and wishes to generalize to the entire population of cells, one must estimate the counting error - i.e.,the error that results 50 S.D, Poisson, 1781 - 1840, French mathematician. WHO/CDS/LEP/86.4 page 109 because the sample counted is not identical with every other possible sample of the popu lation of red blood cells. The estimate of the counting error is then used to adjust the observed value, in order to provide an estimate of the range of values within which the "true" value lies. An important property of the Poisson distribution is that the standard deviation of the mean, A, the number counted in the sample, and taken to represent all possible samples, is simply Al / 2 . Thus, if one counts A red blood cells per mm3, the 95% confidence limits around the value of A - i.e., the range that may be expected to include the value of A obtained from 95% of all possible samples - is simply: k + to.95 x Al / 2 , where to.95 is the "Student's t" value. This is approximately 2.0 for any value of k > 30; therefore, the 95% confidence limits are: k + 2 x Al / 2 , and A - 2 x Al / 2 . The second important property of the Poisson distribution is a direct consequence of the first: the larger the actual number of particles counted, the smaller the counting error, in relation to the value of A, and the narrower the 95% confidence limits. Thus, if in a given area of the counting chamber one counted 100 red blood cells, then the standard deviation is 1001/2 = 10, and the 95% confidence limits are 100 - 2 x 10 = 80 and 100 + 2 x 10 = 120 - i.e., A ± 20% A. If, on the other hand, one were willing to continue counting until he had counted 10 000 cells, the 95% confidence limits would be much narrower - 10 000 + 2 x 10 000 1/2 = 10 000 - 200 and, 10 000 + 200 - i.e., + 2% A - and one could have mu-;;h greater security that the observed value represented the "true" value. Thus, one may increase greatly the precision of the estimate of the number of red blood cells by counting a larger fraction of the ruled surface of the counting chamber. The same considerations apply to the counting of radioactive disintegrations - events that occur randomly and independently in time. Of course, because of their tendency to occur in "clumps", H. leprae are not ran domly distributed in a bacterial suspension or on the surface of a smear, and their dis tribution is not that of the Poisson distribution (97). Nevertheless, it is useful to apply the properties of the Poisson distribution to the counting of H. leprae, and then to make some adjustment for non-randomness. If, in a smear prepared according to Shepard's technique, one counted a single AFB, the total number of organisms per footpad would be calculated as 10 3. 7 - 104 - about the same as that inoculated. A count of 10 organisms would be calculated as 104. 7 - 10 5 AFB per footpad - the result of lO-fold multiplication. But the confidence limits would be at least: 10 ± to.95 (for n = 10) x 101/2 = 10 ± 2.63 x 3.16 = 10 + 8.3. Thus, the range of values within which the true value should lie 95% of the time includes 2, a number of organisms that might have been counted if there had been no multiplication. If, on the other hand, one had counted 100 organisms, then the confidence limits will probably be farther apart than 100 ± 20% x 100, because of a non-random distribution, but one could conclude with some security that the H. leprae had multiplied. To summarize briefly a very complex subject: (1) if H. leprae have been successfully cultured, one should be able to show, by actual counts of the numbers of AFB, that the organisms have multiplied; (2) "multiplication" means that the number of AFB harvested is at least 10-fold greater than that inoculated. This is the criterion of multiplication generally accep~ed for work in the mouse-footpad system; WHO/CDS/LEP/86.4 page 110 (3) at least as important as the "fold-multiplication" are the numbers of AFB actu ally counted. The larger these are, the greater ~he confidence one may have in the values calculated from them, and in the conclusion that M. leprae have truly multiplied. Non-Cultivability of H. 1eprae With respect to M. leprae, "non-cultivability" implies failure of the organisms to multiply in standard bacteriological culture media employed for cultivation of M. tuber culosis and other, cultivable mycobacterial species. In fact, in many laboratories in which mice are inoculated with M. leprae, it is standard practice to inoculate media such as Lowenstein-Jensen or Dubos-Middlebrook with a portion of every suspension of organisms, to exclude the possibility that the organisms counted in the suspension are cultivable and, therefore, not M. leprae. Moreover, it appears likely that M. leprae have failed to grow in many other media - both standard and specialized - used by bacteri ologists in cultivating other organisms, including organisms of other genera. Growth of H. 1eprae in the Mouse Footpad This subject is considered in detail in the sections of Chapter 6 dealing with inocu lation of mice. In brief, when immunologically normal mice of virtually any strain are inoculated in the hind footpad with a number of M. leprae (obtained from tissues of man, mouse or armadillo) not exceeding 104, of which no more than 10 need be viable, the organ isms multiply with a doubling time during logarithmic multiplication of 11 - 13 days, to a maximum that varies with the strain of mouse. The maximum of bacterial multiplication in the most suitable strains - BALB/c, CBA, CFW - is usually 10 6. 0 - 10 6. 3,. and may be lower in other, less suitable mouse strains. After the number of M. 1eprae has reached its maximum, multiplication stops, and the number of viable organisms appears to decrease exponentially. The rate at which the organisms are killed may also vary among strains of mice. At no time does the mouse foot reveal gross evidence of disease. Histopatholog ically, one finds little change until bacterial multiplication is maximal or near-maximal. At this time, one sees evidence of a chronic inflammatory infiltrate characterized by lymphocytes and macrophages, the intensity of which may vary both with the strain of mice and the strain of organisms (193). In the footpad of the nude mouse, M. leprae multiplies to much higher levels - ~ 1010 per footpad, and gross enlargement of the inoculated foot is seen almost always, providing the mice survive for a sufficiently long time after inoculation. Susceptibility of H. 1eprae to Dapsone Until recently, virtually all strains of M. leprae recovered from previously untreated patients with lepromatous leprosy were inhibited from multiplication in mice administered dapsone in a concentration in the diet of 0.0001 g per 100 g; many strains were also inhibited by dapsone administered in a concentration of 0.00003 g per 100 g, and a few by dapsone in a concentration of 0.00001 g per 100 g diet (112). The concentra tion of dapsone in the plasma of the mouse that results from the administration of 0.00003 g dapsone per 100 g diet, about 3 ng (0.003 mg) per ml, may be considered the MIC of dapsone for M. leprae. This exquisite susceptibility of M. leprae to the antimi crobial effects of dapsone is distinctly unusual, among both organisms and antimicrobial drugs. Recently, primary resistance of M. leprae to dapsone has been recognized with increasing frequency (221). Presumably, some patients had been infected ab initio with M. leprae that were resistant to dapsone, the infection having resulted from con tact with a patient who had relapsed with secondary dapsone resistance and was infectious. However, most strains of M. leprae isolated from patients with primary resistance have been quite susceptible to dapsone, multiplying in mice administered 0.0001 g but not in those administered 0.001 g dapsone per 100 g mouse diet - i.e., the MIC has increased from 3 to about 100 ng per ml. WHO/CDS/LEP/86.4 page 111 As the result of intensive screening, a few cultivable mycobacterial strains have been found to be susceptible to dapsone. For example, "M. 1ufu" [the temporary name of one environmental strain recently isolated in Zaire (146)) is about lO-fold less suscep tible than M. 1eprae [i.e., the MIC of dapsone for M. 1eprae is about one-tenth that for M. 1ufu (170)), and a few strains of M. kansasii have been found to be about lOOcfold less susceptible (140). With these few exceptions, the cultivable Mycobac teria that have been screened were not inhibited by dapsone in a concentration of 1 ~g (1000 ng) per ml. Thus, exquisite susceptibility to dapsone does not appear to be unique to M. 1eprae, nor does it characterize all M. 1eprae, but its presence would strongly support any claim to have cultivated M. 1eprae. "Lepromin" - Testing One of the properties of M. 1eprae generally accepted as unique is the failure of a standard suspension of killed organisms to provoke a late (3-4 weeks) "Mitsuda" reaction after intradermal inoculation of polar lepromatous patients, and the ability to stimulate a significant (~ la mm in diameter) Mitsuda reaction in patients with polar or near-polar tuberculoid leprosy. "Lepromins" prepared from M. 1eprae recovered both from mouse (182) and from armadillo (122) have behaved in this fashion. That a lepromin prepared from M. 1eprae-like organisms recovered from ferally infected armadillos also behaved in this fashion (123) may be taken as evidence that these organisms were indeed M. 1eprae, or may suggest only that the specificity of the criterion is limited. Oxidation of Dihydroxyphenylalanine In a series of studies, Prabhakaran has found activity of the enzyme o-diphenoloxi dase in preparations of M. 1eprae, but not in any of a large number of cultivable myco bacterial species studied. He concluded that the enzyme is a constitutive enzyme unique to M. 1eprae (147 - 149). Although o-diphenoloxidase occurs in mammalian cells as well as in plants, the mammalian enzyme oxidizes L-DOPA (3,4-dihydroxydiphenylalanine) to DOPAchrome with an absorbance maximum at 475 nm, and shows little activity toward D-DOPA or derivatives of DOPA, such as epinephrine and norepinephrine, whereas the enzyme of M. 1eprae converted both D- and L-DOPA to indole-5,6-quinone, and also oxidized a vari ety of phenolic substrates to quinones. Indole-5,6-quinone is distinguished from DOPA chrome by its absorbance maximum at 540 nm. Not all workers have succeeded in reproducing Prabhakaran's results, nor is there universal agreement that the enzyme activity does not represent the activity of host-tissue enzymes adsorbed onto the surface of the M. 1eprae. PROCEDURES Prabhakaran has described several procedures by which the o-diphenoloxidase activ ity of a suspension of M. 1eprae may be measured: spectrophotometric, radioisotopic, and a "spot" test. Spectrophotometric Procedure A suspension of M. 1eprae containing 1.5 to 2 mg bacterial protein is incubated in 0.1 M phosphate buffer adjusted to pH 6.8 with L-DOPA in a final concentration of 0.002 M and a total volume of 3 ml at 370C for 30 minutes. After incubation, the reaction mixture is centrifuged for 45 minutes at 15 000 x g, and the absorbance of the supernatant is measured at 540 nm, the absorption maximum of indole-5,6-quinone. Controls include the entire reaction mixture except the bacterial suspension, to exclude auto-oxidation, and the reaction mixture including organisms that had been heated at 1000C for 15 minutes, to exclude non-enzymatic oxidation. WHO/CDSjLEP/86.4 page 112 Radioisotopic Procedure One ~Ci of 3H-L-DOPA is incubated in a reaction mixture that includes M. 1eprae, representing 1-2 mg bacterial protein, and 40 ~moles KH2P04-Na2HP04 buffer, pH 6.8, in a volume of 2 ml. After incubation for 60 minutes at 370C, the reaction is stopped by adding 0.2 ml 20% trichloracetic acid, and the mixture is centrifuged at 10 000 x g for 10 minutes. The supernate is passed over a 0.5 x 4.0 cm column of Dowex 50 (H+) resin to remove phenolic substrates, after which the column is eluted with three 0.5 ml portions of deionized glass-distilled water. A portion of the eluate, which contains 3H-H20 formed by oxidation of 3H-DOPA, is diluted with scintillation fluid, and radioactivity is assayed in a liquid scintillation spectrometer. The controls are the same as those for the spectro photometric procedure. Spot Test A drop (approximately 0.05 ml) of M. 1eprae suspension, containing approximately 2 x 1010 organisms per ml, is placed on a porcelain tile; to this are added a drop each of 0.5 M KH2P04-Na2P04 buffer, pH 6.8, and 0.01 M L-DOPA solution. After 16 - 24 hours at room temperature in a moist atmosphere, a purple-black colour is seen. The drop of bacterial suspension may also be spotted on a filter paper that has previously been dipped in a buffered solution of DOPA, air-dried, and stored in a desiccator. Pyridine Extraction The claim has been made that the acid-fastness of M. 1eprae is extractable by relatively brief exposure to pyridine, and that this property is unique to M. 1eprae. The technique of pyridine extraction of Mycobacteria was first applied to M. 1ep- rae by Campo-Aasen and Convit (15), who wished to distinguish M. 1eprae from other non-cultivable Mycobacteria, especially M. 1epraemurium. These authors claimed that sections and smears prepared from biopsied tissues of leprosy patients contained organ isms, of which a surface-component was phospholipid, as shown by the Baker test for phos pholipids (31); moreover, the phospholipids were removed by the Baker technique (31), which consists primarily of extraction with the weakly basic organic solvent, pyridine, for two hours at room temperature. On the other hand, tissue sections and smears prepared from lesions of rats infected with M. 1epraemurium contained organisms which were stained by the Baker technique for phospholipids even after extraction with pyridine. Fisher and Barksdale (59) subsequently demonstrated that pyridine extraction of M. 1ep rae caused loss not only of surface phospholipids but also of acid-fastness of the M. 1eprae recovered from 15 of 19 skin biopsy specimens, and argued that these proper ties differentiated M. 1eprae from true Mycobacteria. In subsequent publications, Convit (29, 30), Fisher and Barksdale (60), and McCormick and Sanchez (120) reported that M. 1eprae recovered from human and armadillo tissues lost their acid-fastness upon extraction with pyridine, whereas cultivable Mycobacteria did not. However, Skinsnes et al., (198, 199) and Slosarek and his co-workers (200, 201) reported that, under some circumstances, M. 1eprae do not lose their acid-fastness upon pyridine extraction, whereas some cultivable Mycobacteria do. Skinsnes proposed that loss of acid-fastness upon pyridine extraction is a property of aged or dead Mycobacteria. PROCEDURE The specimen - paraffin section, cryostat section or smear - is fixed in Bouin's fluid for from 5 to 16 hours, after which it is immersed first in 70% ethanol for 5 min utes, and then in 50% ethanol for 5 minutes. After rinsing in tap water for 2 minutes, the specimen is immersed in fresh, analytical-grade pyridine for 2 hours. Then, the specimen is rinsed in tap water for 2 minutes, and fixed in formol-calcium for 1 hour. Finally, it is stained by means of the standard acid-fast stain, together with a duplicate specimen not subjected to extraction. WHO/CDS/LEP/86.4 page 113 Characterization and Identification of Bacteria by Study of their Genome5l GENOME SIZE The size of a bacterial genome, expressed as length, number of purine and pyrimidine base-pairs, or molecular weight, can be determined by methods based on electron microscopy or on the physical properties of the bacterial deoxyribonucleic acid (DNA) (5). The lack of precision of the measurements made by these methods, and the limited range of known molecular weights of bacterial genomes (108. 60 - 10 9. 60 daltons52) limit the appli cation of these measurements to the classification of bacteria. The molecular weight of mycobacterial DNA has been reported to vary from 10 9. 11 - 10 9. 48 daltons (87). BASE COMPOSITION OF THE BACTERIAL GENOME Base composition is expressed as the proportion of bacterial DNA represented by the content of adenine + thymine (A + T) or guanine + cytosine (G + C) in moles per cent or in per cent by weight. Usually, one wishes to know the G + C content - actually the propor tion (G + C)/(G + C + A + T). Three methods are routinely used. Chromatography of DNA The DNA is hydrolyzed, and the free bases are separated either by filter paper chrom atography or by high performance liquid chromatography. The quantity of each base is determined, and the content of G + C calculated in moles per cent. This technique, which involves the identification and quantification of each base directly, is the reference technique; it also permits the measurement of methylated bases. Buoyant Density Using an analytical ultracentrifuge, the buoyant density, P, of a DNA molecule is determined in a cesium chloride gradient, and used to calcclate the G + C content by means of an experimentally derived formula: % (G + C) = 1038.47(P- 1.6616). Thermal Denaturation The hyperchromicity (greater absorbance of light with a wave-length of 260 nm) of single-stranded DNA is employed to determine the temperature, Tm, at which 50% of the bacterial DNA, initially double-stranded, has been denatured (converted to single-stranded DNA). The Tm is determined by gradually heating the DNA, and measuring the increase of absorbance at 260 nm as a function of temperature. The G + C content of the DNA may be calculated from the value of the Tm by experimentally derived formulae, which vary accord ing to the composition of the buffer in which the DNA is dissolved (139). The G + C content of bacterial DNA ranges from 25 - 75 moles per cent. This broad range of values permits exclusion of an unknown organism from a species, if the respective values of G + C content vary greatly. Strains within recognized species show a narrow range of values, differing by only ± 2%, whereas species within a given genus show a somewhat wider range of values of G + C content (± 8%). G + C content of mycobacterial species has been found to range from 56 - 69% (G + C) (76, 139). The G + C content of the DNA of M. 1eprae was found to be 56% by both the thermal denaturation (17, 87), and the buoyant density techniques (17). 51 Contributed by H. Bercovier. 52 A dalton is an atomic mass unit - 1.66041 x 10-24 (lO-23.78) g. WHO/CDS/LEP/86.4 page 114 HOMOLOGIES BETWEEN BACTERIAL GENOMES Homology and divergence between sequences of DNA bases of two organisms may be studied and measured by DNA-DNA hybridization. The proportion of DNA sequences common to two organisms is measured by permitting dissociated (denatured; single-stranded) DNA from one organism to reassociate with single-stranded DNA from the second organism. The resulting dup1exes (hybrids) are a measure of the homology between the DNAs of the two organisms. The degree of re1atedness (homology) between the DNAs of two organisms is usually determined by permitting fragments of the proper size (about 500 bases) of radioactively labelled single-stranded DNA of one organism to react with similarly sized fragments of unlabelled single-stranded DNA of the second organism. Under suitable conditions - e.g., large excess of the unlabelled DNA; optimal time and temperature of incubation; appropriate ionic strength, random collisions between molecules, such as occur between molecules of any solute in solution, permit the complementary bases of homologous DNA sequences to reassociate, forming new, hybrid double-stranded DNA molecules. The degree of re1atedness between the DNAs of the two organisms is determined by comparing the quan tity of DNA hybrids formed in the homologous reaction (labelled DNA reacting with non labelled DNA from the same organism) with that formed in the heterologous reaction (label led DNA of one organism reacting with non-labelled DNA of the second organism). Three methods are commonly used for DNA-DNA hybridization. Hybridization on a Membrane Filter A solution of unlabelled single-stranded DNA fragments of high molecular weight is filtered through a nitrocellulose filter, to which the DNA binds. Radioactively labelled (with 32p or 3H) DNA that has been mechanically sheared and denatured is added to the filters, which are then incubated under conditions that ensure maximal reassociation between the two DNAs. After washing, the radioactivity of the DNA dup1exes that remain bound to the filter is determined in a liquid scintillation spectrometer. The degree of re1atedness between the two DNA molecules is calculated from the ratio of dup1exes formed in the heterologous reaction to that of dup1exes formed in the homologous reaction. This method can be improved by using a competition assay, in which unlabelled DNA of one (unknown) organism is first incubated in solution with labelled DNA of the second organism. This solution is then incubated with a nitrocellulose membrane, to which unla belled DNA of the second organism had previously been bound. The quantity of labelled DNA bound to the filter is inversely proportional to the degree of homology between the two DNAs. This indirect method yields more reproducible results than does the first, more direct method. Hybridization in Solution Unlabelled DNA of one organism, that has been sheared and denatured, is incubated in 1,sOO-fold excess with labelled and sheared single-stranded DNA of the second organism under the conditions of ionic strength, temperature and time (these conditions vary according to the G + C content of the DNA molecules) that permit optimal formation of dup1exes. The small concentration of labelled DNA makes it unlikely that reassociation of labelled DNA will occur with itself during the short period of incubation; the large excess of unlabelled DNA forces the reaction to completion. The labelled hybrid duplexes that result are then separated from the remaining labelled single-stranded DNA by chroma tography on a hydroxyapatite column, or by digestion of the single-stranded DNA by "Sl nuclease", an enzyme that acts only on single-stranded DNA, followed by purification of the remaining double-stranded DNA, which may be accomplished by various methods. These two methods - hydroxyapatite chromatography and SI nuclease digestion - have been care fully evaluated (63). The degree of relatedness between the two DNA molecules is meas ured by comparing the heterologous with the homologous reactions. WHO/CDS/LEP/86.4 page 115 Hybridization in solution also permits the study of the quality of the hybrid dup lexes, in terms of their thermal stability. One may carry out the hybridization at a stringent (higher) temperature, at which only closely related base-sequences reassociate. Alternatively, one may estimate the number of mismatched base-sequences apparently matched under non-stringent conditions from the quantities of labelled single-stranded DNA eluted (washed) from the hydroxyapatite column as the temperature of the column is increased in increments of SoC. Again, one compares the temperature at which 50% of labelled DNA from the heterologous reaction is eluted with that at which 50% of labelled DNA from the homol ogous reaction is eluted (the ~Tme)' By these methods, it has been shown, especially for Enterobacteriaceae species, that a genetically well-defined species includes strains that are at least 70% related under "optimal reassociation conditions", with a ~Tme < SoC. When using the technique of digestion by Sl nuclease, the value of ~Tme does not change, but the homology between strains of the same species is at least 50%. Spectrophotometric Method Using unlabelled DNA, it is possible to measure the homology between DNAs of various bacterial strains by measuring the initial rate of reassociation of heterologous DNAs in solution, and comparing this to the initial reassociation rate of the homologous DNAs. Two DNAs, A and B, previously sheared, are mixed in spectrophotometer cuvettes, denatured by heating, and allowed to reassociate by lowering the temperature rapidly. The rate of reassociation is measured in the rate of decrease of absorbance at 260 urn. The degree of homology is calculated from a comparison of the initial reassociation rates of the mix tures A + A, A + B, and B + B. This method does not permit a study of the quality of the hybrid duplexes, nor can it provide data on mismatched sequences. Moreover, the influence of genome size on measurement of DNA-DNA homology by this technique is difficult to evalu ate, whereas study of the reciprocal reactions by the labelled DNA techniques already described make this estimation easy. If two bacteria belong to different genera or families, DNA-DNA hybridization cannot be used for taxonomy, but hybridization is a useful and powerful technique both for class ification and for the study of divergence among related organisms. Few data on DNA-relatedness among Mycobacteria have been published (4, 68, 87). By means of a spectrophotometric method, M. leprae DNA has been shown to be related to DNAs of both Mycobacteria and Corynebacteria species, but at a level of relatedness that does not permit further conclusions (87). More data are required to permit the classification of M. leprae among other bacterial species. Two good general sources of information on this topic are the articles by Johnson (90) and Brenner (11). Cell-Wall Compositions3 As in other members of the family Actinomycetales, which includes the genus Myco bacteria (see Table 24), the cell-wall of M. leprae contains mycolic acids (high mole cular weight, branched-chain hydroxy-fatty acids), arabinogalactan and peptidoglycan. The mycolic acids, which resemble two of the three types found in M. tuberculosis, account for 50% of the weight of the lipid of the cell-wall of M. leprae. Minor amounts of other mycolic acids may also be present. Some of these mycolic acids may be unique to M. leprae. Peptidoglycan, a polymer composed of alternating units of N-acetylglucosamine and N acylmuramic acid, forms the basis of the cell-wall of most bacteria. Chains of these units are cross-linked by small peptides, the structure of which varies among bacterial species. In the cell-walls of all Mycobacteria thus far studied, with the exception of 53 Adapted from references no. 3, 41 - 43. WHO/CDS/LEP/86.4 page 116 M. leprae, the peptide is L-alanyl-D-isoglutaminyl-meso-diaminopimelyl-D-alanine. In the peptide of M. leprae, L-alanine has been completely replaced by glycine. Table 24: Taxonomy of the genus Mycobacterium54 Order: Actinomycetales Families: Actinomycetaceae Mycobacteriaceae Frankiaceae Actinoplanaceae Dermatophilaceae Nocardiaceae Streptomycetaceae Micromonosporaceae Genus in Family Mycobacteriaceae: Mycobacterium Species in Genus Mycobacterium: Slowgrowers: M. tuberculosis M. terrae M. microti M. triviale M. bovis M. gordonae M. africanum M. scrofulaceum M. kansasii M. paraffinicum M. marinum M. intracellulare M. simiae M. avium M. gastri M. xenopi M. nonchromogenicum M. ulcerans Rapid growers M. phlei M. flavescens M. vaccae M. fortuitum M. diemhoferi M. peregrinum M. smegmatis M. chelonei M. thamnopheos Having special growth requirements: M. paratuberculosis M. leprae M. lepraemurium Both the mycolic acids and the peptidoglycan appear to possess unique features that could be used to identify M. leprae. Like other Mycobacteria, M. leprae produces typical complex lipids. Two are of special interest, because they may be recovered from the supernatants of homogenates of infected human or armadillo tissue after sedimentation of the organisms by centrifugation. One of these is an antigenically active phenolic glycolipid containing a unique trisac charide (84); both leprosy patients and armadillos form antibodies against this glyco lipid. 54 Adapted from reference no. 165. WHO/CDS/LEP/86.4 page 117 Antigenic Constitution55 A number of antigens of M. leprae have been partially characterized, and more will be characterized in the near future, aided by the increasing availability of specific monoclonal antibodies. It should soon be possible to identify an isolate as M. leprae by demonstrating that it possesses antigens hitherto detected only in preparations of M. leprae recovered from human, armadillo or murine tissues. M. leprae synthesizes an envelope of glycosylated diacylphthiocerol compounds. Three of these phenolic glycolipids (PG) have been characterized structurally. The major PG contains a terminal 3,6-di-0-methylglucose linked through a ~1-4 linkage to 2,3 dimethylrhamnose, which is in turn linked through an al-2 linkage to 3-methylrhamnose. The terminal dimethylglucose is the dominant antigenic and immunogenic portion of the molecule, as shown by loss of antigenicity following partial hydrolysis, which removes this portion of the molecule, or by inhibition of the binding of leprosy patients' sera (antibody) to the PG by synthetic oligosaccharides containing a terminal 3,6-di-0-methyl glucose linked through a ~1-4 linkage to rhamnose (85). Molecules of this structure have not yet been detected in other Mycobacteria; thus, this antigen provides a means for identifying M. leprae. Monoclonal antibodies dir ected against PG react specifically with the surface envelope of the organism to produce blue-green fluorescence in indirect immunofluorescence assay (226). PG is degraded slowly within infected tissues, and may accumulate to achieve a mass twice that of the M. leprae in some human lepromatous lesions (224). The cell-wall of M. leprae contains arabinomannan, a carbohydrate antigen shared with most other Mycobacteria. Monoclonal antibodies directed against this antigen, which may be useful in the serodiagnosis of infection with M. leprae, have been prepared (125). Proteins with subunit molecular weights of 53 000, 43 000 and 36 000 daltons are exposed on the surface of M. leprae, as shown by labelling intact organisms with 1251 using either lactoperoxidase or "Iodogen" (13). Indirect evidence suggests that the 36 OOO-dalton protein contains antigenic determinants or epitopes specific for M. leprae (65). No monoclonal antibodies directed against any of these proteins have yet been identified. "Antigen 7", of unknown subunit molecular weight and chemical composition (74), may also reside within the cell-wall of M. leprae. A recently reported monoclo nal antibody may aid in characterizing this antigen (18). Internal to the cell-wall, M. leprae contains proteins with subunit molecular weights> 94 000, 68 000 (66), 59 000, and 12-14 000 daltons. Monoclonal antibodies have been prepared, which indicate M. leprae-specific epitopes on the 68 000- (13) and 12-14 OOO-dalton (88) proteins. Antibodies present in the sera of patients with leprosy can inhibit the binding of monoclonal antibodies to the 68 OOO-dalton protein (13, 88), indicating that the M. leprae-specific determinants are recognized by these patients. Identification of an Isolate as M. leprae For confirmation of its identity as M. leprae, an isolated organism should exhibit the following properties (of course, not all of these properties are unique to M. lep rae): (1) acid-fastness, with the morphological characteristics, in terms of size, shape, and staining quality, of M. leprae originally recovered from untreated patients with lepromatous leprosy; 55 Contributed by T.M. Buchanan. WHO/CDS/LEP/86.4 page 118 (2) definite multiplication under the cultural conditions for which the claim has been made (see pp. 107 - 109); (3) inability to grow on generally employed culture media and on media used for culturing Mycobacteria - e.g., Lowenstein-Jensen medium; (4) multiplication in the footpads of normal mice, with doubling time during logar ithmic multiplication of 10 - 15 days; (5) inhibition of multiplication in the mouse when dapsone is administered contin uously in the diet in a concentration of 0.0001 g per 100 g diet; of course, if the strain of M. leprae is resistant to dapsone, an increasingly frequent occurrence, the isolate will not be inhibited by the drug; (6) an intracutaneous dose of 10 6. 60 - 10 7. 20 organisms in 0.1 ml provokes a Mitsuda reaction with ~ 10 mm in duration in patients with TT leprosy, and no reaction in patients with LL leprosy; (7) multiplication in the nude mouse, with the appearance of a characteristic lesion in the inoculated hind footpad 12 - 18 months after inoculation; (8) G + C content 56%; (9) DNA homology with authentic M. leprae of at least 70% under optimal conditions of reassociation; (10) contains the mycolic acids and peptidoglycans characteristic of M. leprae; (11) contains M. leprae-specific peptidoglycan and other antigens or epitopes, demonstrated by reaction with specific monoclonal antibodies. Demonstration of these last five properties requires very large numbers of organisms. These should be readily obtainable from a successful culture system, but could be obtained in any case from the infected nude mice, or from a heavily infected armadillo. Further, if he is to apply criteria nos. 7 - 11, the investigator will probably need to seek the collaboration of an established laboratory with the required expertise, because the required techniques are so demanding. He should not hesitate to do so, because cultivation of M. leprae in vitro is so intensely desired a goal. If he has applied carefully criteria nos. 1 - 6, and has made available complete records of his work, he should experience no difficulty in securing the needed collaboration. In fact, recent advances in our knowledge and skill in the molecular biology have been so rapid that, having satisfied criteria nos. 1 - 6, the worker who believes he has cultivated M. leprae should immediately undertake to have the identity of his isolate established by measurement of the G + C content and DNA hybridization. WHO/CDS/LEP/86.4 page 119 CHAPTER 8 - IMMUNOLOGICAL STUDIES This chapter on immunological methods for leprosy is a brief one for several reasons. First, except for lepromin-testing, few immunological techniques have been assigned the same importance and even indispensability that has been assumed by such techniques as the Ridley-Jopling classification and the mouse foot-pad technique. Second, some immunologi cal techniques cannot be performed without equipment costing thousands of dollars; an example is the lymphocyte-transformation test, which cannot be performed without a liquid scintillation spectrometer. Finally, the present period is one of very rapid advance in immunological techniques, many of which are being exploited in leprosy research, and a technique much in vogue today may have been rendered obsolete by further advances one or two years hence. The immunological techniques described are those most likely to be employed in leprosy-endemic countries. These include: lepromin-testing; skin-testing with antigens other than lepromin; Abe's fluorescent leprosy-antibody-absorption (FLA-ABS) test for antibodies directed against antigens of H. leprae; and Buchanan's ELISA assay for antibodies directed against the species-specific phenolic glycolipid. Skin-Testing APPLYING THE SKIN-TEST56 Skin-testing with soluble antigens is performed by the technique employed for tuber culin testing. Equipment Syringes and needles for skin-testing are kept separate from those used for other purposes. Leak-proof graduated glass syringes are used, with platinum or steel 25- or 26 gauge, 10 mm needles. Platinum needles can be flamed to red-heat without damage, and remain sharp. On the other hand, although steel needles quickly become corroded when treated in this manner, making injection painful, they are far cheaper, and may be replaced more frequently, for example daily. Before each skin-testing session, syringes and needles are disassembled, washed and brushed with water, and sterilized in an autoclave. If an autoclave is not available, the disassembled syringes are boiled for at least 10 minutes in distilled or demineralized water, to avoid mineral deposits. During a single session, providing that the needle is flamed between tests, and that the needle remains on the syringe, the same syringe and needle may be used for many injections. If the needle and syringe become detached, they must be resterilized. A metal box in which to keep the syringes and needles, a small spirit lamp for flam ing, and suitable forceps for mounting the needle on the syringe are also needed. The syringe box and forceps should be cleaned and sterilized together with the syringes and needles. A small transparent ruler graduated in millimetres is required to measure the reac tions. Administering the Test The skin-test is usually applied to the dorsal surface of the forearm, but the volar surface may also be used. If two different tests are to be administered simultaneously, they are applied one in each arm. To avoid bias in reading, the different tests should not be administered systematically, one in the left arm and the other in the right, but 56 Prepared by J. Guld and H.G. ten Dam. WHO/CDS/LEP/86.4 page 120 should be allocated according to some random procedure; the allocation must be recorded accurately for each person. The tip of the syringe is flamed; the sterilized needle is taken up with forceps, the hub of the needle is heated, and the needle is mounted on the syringe in such a way that the bevel faces the side of the graduated scale. Heating the hub of the needle causes slight expansion and consequently a tight fit on the syringe when the needle has cooled. The whole needle is then flamed, and the syringe is filled from the vial or ampoule con taining the skin-test antigen. Air remaining in the syringe after filling is expelled. Before each injection, the tip of the needle is flamed to a dull red; then, a drop of antigen is expelled to cool the needle, and to make certain it is not obstructed. Norm ally, a clean skin is not sterilized before injection. Holding the syringe by the barrel, the tip of the needle (with the bevel upwards) is inserted superficially into the skin, parallel to the long axis of the arm, the skin kept slightly stretched in the direction perpendicular to the long axis. When the needle has been inserted satisfactorily, the position of the piston is observed, the syringe is held firmly in place, and the piston is advanced gently until it has reached a point 0.1 ml ahead of the original position. Then, with the syringe held parallel to the arm, the needle is withdrawn. The injection should be administered slowly, so that tissue damage is minimized. A well made intradermal injection of 0.1 ml will produce a raised but flat anaemic wheal with an "orange-peel" appearance and a diameter of some 7 mm. If the injection has been administered too deeply, the wheal will be smaller, dome-shaped, and less anaemic. This makes the reaction more difficult to read. When the test has been administered, details are recorded. The intradermal administration of the skin-test antigen usually poses no problem, but from time to time, even the most experienced tester will experience a technical incident, such as inadvertent separation of syringe from needle, with loss of a portion of the antigen, that results in inadequate administration of the dose, In such cases, no attempt at correction should be made; only if the incident happens before any antigen has been administered may the test be administered again at a different site. Although an occas ional mishap is unavoidable, it will usually be of little consequence, providing that the mishap has been recorded clearly, so that the test result will not be interpreted as if the test had been administered correctly. Testing should be carried out in as uniform a manner as possible. Reading the Reaction A reaction exhibits attributes such as induration, surrounding oedema, erythema, density ("firmness"), and the presence of bullae or vesicles, lymphangitis, necrosis, and fever. All of these attributes reflect the degree of sensitivity, and depend upon the dose of antigen administered. For routine testing, the measurement of the reaction is restricted to only one of these attributes; the most practical are induration and ery thema. Because it is difficult to measure erythema on a very dark skin, measurement of the induration is preferred. The test is read 48 or 72 hours (preferably at both intervals) after it has been applied. The test-site is carefully palpated, and, if induration is present, its diameter is measured with the transparent ruler in a uniform way -e.g., perpendicular to the long axis of the arm - and recorded in millimetres. If there is no induration, this is recor ded. The readings should be made by trained observers, so that they are both accurate and consistent. If two or more readers work in the same programme, small, systematic differ ences in the readings may occur, even if the readers are equally accurate and consistent. Therefore, the differences between readers should be measured. For example, if the reac tions must be read by two readers, it would be appropriate to allocate half of the popula tion to each of them, employing some random procedure. Double readings can be done in a population sample, and the results compared in a correlation table. WHO/CDS/LEP/86.4 page 121 To avoid bias, the reader should not be informed of the possible sensitivity of the person tested; for example, he should not know the result of a previous test, nor should he have access to records that contain information regarding previous skin-test results. In general, these records should be maintained by a clerk, to whom the reader dictates his findings. LEPROMIN-TESTING The lepromin test is not a diagnostic test, but rather a means of classifying patients. First described by Mitsuda (128) and Hayashi (75), the lepromin test involves the intradermal injection of an autoclaved, emulsified preparation of lepromatous tissue standardized according to its content of H. leprae. Preparation of standard lepromin is described in Appendix 3. However, because this is so demanding a procedure, the investigator is better advised to request a supply from the Leprosy Unit, World Health Organization, Geneva. Testing with Lepromin One-tenth ml is injected intradermally into the flexor surface of a forearm, using a 25-gauge hypodermic needle. The Fernandez reaction is read, like a reaction to tubercu lin, after 48 to 72 hours. The longest diameter of the area of induration, and the diam eter perpendicular to this are measured with a millimetre rule, and the measurements are recorded. The Mitsuda reaction is read after four weeks, employing a millimetre rule to measure the longest and perpendicular diameters, and recording the results. If ulceration is observed, this is also recorded. For purposes of classification, the Fernandez reac tion is not so well standardized as the Mitsuda reaction; patients with LLp and LLs leprosy should have a Mitsuda reaction no larger than 3 mm. COMPARING SKIN-TEST ANTIGENS57 The need sometimes arises to compare two or more skin-test antigens, in terms of the sizes of the reactions they provoke in defined populations. For example, one may wish to compare (standardize or calibrate) "lepromin A" - a lepromin prepared from the tissues of H. leprae-infected armadillos - with "lepromin H", the lepromin prepared from the tissues of patients with lepromatous leprosy that has been diluted to contain 4 million (106. 60) or 16 million (107. 20) AFB per 0.1 ml. Such a comparison is carried out among patients with polar tuberculoid and polar lepromatous leprosy, with the objective of ascertaining the quantity of lepromin A that produces in tuberculoid patients reactions of the same size as those produced by a standard amount of lepromin H, and at the same time produces in lepromatous patients reactions no larger than those produced by lepromin H. The same need arises when one wishes to compare with lepromin H a "lepromin" prepared from a putative isolate of H. leprae. Range-Finding One begins the process of calibrating a new skin-test antigen - in this case lepr omin A- against the standard lepromin H by selecting three concentrations of each of the two antigens to be compared; it is desirable that the lowest of the three concentrations produce some reaction in at least some subjects, and that the highest concentration not produce a maximal reaction in all of the subjects. This is accomplished by randomly assigning subjects in groups of 10 - either BT-TT patients or normal volunteers - to testing by each of the concentrations of each of the antigens; each subject would be administered only one skin-test. 57 Prepared with the assistance of T.K. Sundaresan. WHOjCDSjLEPj86.4 page 122 For lepromins, the initial selection of antigen concentrations is easy; the M. lep rae present in each preparation can be counted, and one already knows which concentra tions of lepromin H produce reactions in virtually all TT patients. One might begin, therefore, with dilutions of both standard lepromin H and the new lepromin A containing 10 6. 60, 10 6. 00 and 10 5. 40 AFB per 0.1 ml. Each of these is administered to 10 subjects selected at random; 21 days later, the skin-test sites are examined and the reactions measured. Assuming that these three concentrations of both antigens meet the stated criteria i.e. that some subjects have at least a small reaction to the dilutions containing 10 5. 40 AFB per 0.1 ml, and that the dilutions containing 10 6. 60 have not produced large necrotic reactions in all of the subjects tested, one goes on to carry out a "parallel line assay". To testing by each concentration of each antigen, one assigns 50 patients with BT-TT leprosy, who are selected at random from a pool of 300 such patients. Skin-tests are administered with materials identified only by a code number or letter; three weeks later, the skin-test sites are examined, and the reactions measured and recorded without knowledge of which concentrations of which antigens have been employed. After the meas urements of all of the reactions have been recorded, the code is broken, the mean reaction size is computed for each concentration of each antigen, and dose-response curves are plotted for each antigen. If the two lepromins behave in identical fashion - i.e., if mean reaction size depends only upon the number of AFB injected, and is the same for AFB obtained from armadillo as for those obtained from human tissues, then the two dose response curves should be superimposable, one on the other. It appears equally likely that the two antigens will not behave in identical fashion, in which case one expects the dose-response curves to be parallel, but not superimposable. From the parallel curves, one may readily compute the ratio of potency of the two antigens, and adjust the dose of the new antigen so as to produce the reaction expected from a standard dose of the stan dard antigen. Of course, one also expects that a lepromin will provoke no or very small reactions in patients with BL-LL leprosy. To test the new lepromin in this regard, one need employ only one concentration - the highest - of each antigen to test 50 BL-LL patients selected randomly from a pool of 100. In this case, one computes the proportion of patients react ing to each antigen, and compares them by means of Fisher's exact probability calculation. The new lepromin should provoke reactions in such patients with no greater frequency than does the standard lepromin. SKIN-TESTING WITH SOLUBLE ANTIGENS FROM M. LEPRAE The Mitsuda type of lepromin is useful for clinical assessment and for predicting to a certain extent the prognosis of leprosy, but it is of little value in epidemiological studies, in which the purpose is to determine whether an individual has been exposed to M. leprae or not, and for monitoring delayed-type hypersensitivity (DTH) reactions to M. leprae after vaccination or immunotherapy. For this reason, soluble skin-test anti gens prepared from M. leprae have been developed, the use of which is based mainly on the model of tuberculin-testing. Tuberculin has been extensively studied, and has been of great value in unravelling the epidemiology of tuberculosis. Some believe that the early "Fernandez" reaction, read 48 - 72 hours after injection of integral (prepared from whole M. leprae) lepromin, reflects a DTH reaction similar to that elicited by tuberculin, but the literature on this subject is not clear. There are two types of soluble antigens have been prepared from M. leprae, often referred to as the Rees and Convit antigens. The Rees antigen is prepared from armadillo derived M. leprae that are sonicated, centrifuged at high speed, filtered through a membrane of 0.22 ~m pore-size, and standardized at 1 ~g protein per dose. The Convit antigen is also prepared from armadillo-derived M. leprae that are passed through a French press and centrifuged. The low-molecular-weight fraction is separated, autoclaved, WHO/CDS/LEP/86.4 page 123 and standardized at 5 or 10 ~g protein per dose. Reactions to both soluble antigens are read at 48 - 72 hours. Preliminary results from studies in man indicate that patients with lepromatous leprosy uniformly fail to react to soluble antigen, whereas large proportions of patients with tuberculoid leprosy and significant numbers of healthy contacts of leprosy patients react. The soluble antigens have been successfully used to study the sensitizing poten tial of vaccine preparations containing killed M. leprae among human volunteers. However, in humans and experimental animals, both antigens show some cross reactivity with PPD, indicating that these antigens lack specificity, and limiting their usefulness for epidemiological studies. Furthermore, because of a lack of information regarding the active antigen(s), the preparations may be standardized only by protein concentration; therefore, batch-to-batch variation is quite common. In summary, skin-testing with sol uble antigens prepared from M. leprae appears promising, but requires further develop ment. Serodiagnosis A sensitive method for detecting antibodies that are specific for antigens or anti genic determinants of M. leprae would be extremely useful, both for recognizing subclin ical infection - e.g., in epidemiological studies, and to support the diagnosis of leprosy in patients whose clinical picture is not characteristic. Two methods have been described; however, their sensitivity and specificity have not yet been established. SERODIAGNOSIS BY INDIRECT IMMUNOFLUORESCENCE58 Abe and his co-workers have described a fluorescent antibody procedure - the FLA-ABS test - said to recognize antibodies directed against antigens specific for M. leprae. In brief, a standard suspension of M. leprae is smeared on a microscope slide. The serum sample is first mixed - "absorbed" - with cardiolipin and lecithin, to remove non specific proteins of normal human serum that adhere to M. leprae, and subsequently absorbed with suspensions of BCG and M. vaccae, to remove antibodies directed against antigens common to M. leprae and other Mycobacteria. The absorbed serum is placed on the dried smear of M. leprae and incubated, permitting the anti-M. leprae antibodies that might be present in the serum to be bound by the organisms, after which the smear is washed by immersion in phosphate-buffered saline (PBS) , to remove any unbound serum com ponents. Finally, rabbit anti-human-IgG labelled with fluorescein isothiocyanate is placed on the smear; this fluorescent antibody is bound by antibody-coated M. leprae, rendering the organisms fluorescent. Reagents Suspension of M. leprae. M. leprae are separated from a lesion of an untreated lep rosy patient or from M. leprae-infected armadillo tissues as follows. The tissue is homogenized in a blender with 10 ml of physiological saline per 1 g of tissue, while cooling with ice water. The homogenized suspension is centrifuged at 130 x g for 10 minutes. The pellet is again homogenized and centrifuged in the same manner. Both supernatants are pooled and centrifuged at 9000 x g for 20 minutes at 0 - 4oC. After removing the supernatant, pelleted bacilli are suspended in a small volume of saline. AFB are enumerated, and the suspension is diluted with saline to contain 10 8. 00 - 10 8. 10 AFB per ml. The suspension is divided into aliquots, which are frozen and stored at -20oC. Suspension of BCG. BCG is cultivated on Sauton's medium for 4 weeks at 37oC, collected by centrifugation (800 x g, 10 minutes), washed three times with physiological saline and lyophilized. One g of dried organisms is suspended in 20 ml of PBS and sonicated with a 58 Adapted from reference no. 1. WHOjCDSjLEPj86.4 page 124 microtip and minimal power of a cell-disruptor for 15 minutes, to obtain a homogeneous suspension. Sodium azide is added to a final concentration of 0.1%, and the suspension is stored at 0 - 4oC. Suspension of M. vaccae. M. vaccae (ATCC 15483) are cultivated in modified Dubos' medium at 370C for 1 week, collected by centrifugation as above, and washed three times with physiological saline. One volume of pe11eted organisms is suspended in 9 volumes of PBS, sonicated and stored in the same manner as BCG. Fluorescent antibody. Lyophilized anti-human-IgG fluorescent antibody (FA), prepared from rabbit antiserum, with activity against both heavy and light chains, is reconstituted with 0.5 m1 of distilled water per vial at the time of use, and absorbed by adding an equal volume of 5% (wjv) BCG suspension. After incubating at 37 0C for 30 minutes, the mixture is centrifuged at 500 x g for 15 minutes, and the supernatant is again centrifuged at 5500 x g for 5 minutes by means of a microcentrifuge. The clear supernatant is care fully removed from the pe11eted organisms, diluted with PBS to the dilution indicated (usually 1:40), and filtered through a membrane filter (0.2 ~m pore-size). The solution is used immediately after preparation. The Test Smear of M. leprae. One platinum loopfu1 of M. leprae suspension is smeared on each circle of a glass slide used for immunofluorescence tests. The smear is air-dried at room temperature, or, in a humid climate, with warm air (e.g., a hair-dryer). (Because the M. leprae have not been killed, these procedures should be carried out in a biological safety cabinet.) Pretreatment of the smear. The smears are soaked in CC14 (which is renewed after pre treatment of several specimens) at room temperature for 10 minutes, and then dried in air. Each smear is then covered with 2 or 3 drops of 0.1% trypsin solution, and incubated in a moist chamber at 37 0C for 1 hour. The slides are then washed four times by shaking with PBS for 5 minutes. After drying the slides, vertical lines are drawn with nail polish between the circles. This pretreatment is essential to enhance the intensity of the immunofluorescence of the organisms, perhaps because it eliminates tissue 1ipids and proteins that interfere with the antigen-antibody reaction. Absorption and dilution of test serum. The serum (0.05 m1) is mixed with equal volumes of BCG and M. vaccae suspensions, and diluted 10-fo1d by adding 0.35 ml of diluent A (see Appendix 4). The mixture is then incubated at 37 0C for 30 minutes and centrifuged at 500 x g for 15 minutes to sediment the majority of the organisms. The supernatant is again centrifuged at 5500 x g for 5 minutes with a microcentrifuge. The clear supernatant is removed and further diluted with diluent B to prepare serial four-fold dilutions - i.e., 1:40, 1:160, 1:640, 1:2560, etc. In each experiment, there must be positive as well as negative control specimens. Pooled serum collected from active lepromatous patients is used as a positive control, and pooled serum of healthy donors living in an area non-endemic for leprosy as a negative control. Primary reaction. The serum dilutions are placed on the M. leprae smears and incubated in a moist chamber at 370C for 1 hour. After washing with PBS for 5 minutes with shaking, the vertical lines of nail-polish are removed, and the slides are dried with cool air. Secondary reaction. The smear is covered with 1 or 2 drops of FA solution and incubated in a moist chamber for 1 hour at 37oC, or over-night at 4oC. Washing and mounting. After washing with PBS again, the smear is allowed to dry and mounted with 0.05 M Na2C03-buffered glycerol (pH 9.5) and a coverslip. WHO/CDS/LEP/86.4 page 125 Reading. This should be performed with sera coded, so that the reader does not know the origin of the individual serum, employing a fluorescent microscope, and magnification of 400 - 650 x, sufficient for observing the immunofluorescence of H. 1eprae. The BV fil ter system is used in combination with an interference filter system. The intensity of the fluorescence is recorded on a scale of + to 4+, according to the system employed in the FTA-ABS test. The criteria for the reading are as follows: 4+ - very strong fluores cence of almost all organisms; 3+ - strong fluorescence of the majority of the organisms; 2+ - many organisms show definite fluorescence, but weakly fluorescent organisms are also seen; 1+ - definite fluorescence of the minority of organisms, but weak of the majority; ± - weak fluorescence of a few organisms; 0 - organisms recognized, but without observ able specific fluorescence. The specific green colour of fluorescein isothiocyanate is always confirmed by using the interference filter. Clustered organisms or organisms in tissue-fragments are not considered in the readings, because these often emit either exceptionally strong or non-specific fluorescence. 2+ or greater fluorescence of the isolated organisms caused by the 1:40 or greater dilution of serum is considered positive; 1+ or less fluorescence at any dilution, and 2+ or more at the 1:10 dilution are consid ered negative, because such fluorescence is sometimes seen in preparations of sera from non-endemic areas. The antibody titre is expressed by a in the expression 10 x 4a, the maximal dilution of serum giving a positive reaction. DETECTION OF ANTIBODIES AGAINST THE PHENOLIC GLYCOLIPID OF H. LEPRAE59 Tissues infected with H. 1eprae contain large amounts of a phenolic glycolipid (PG), that forms an amorphous capsule around the organisms (84,224,226). This com pound, which can be purified in milligramme amounts from infected armadillo tissues (84), represents the only antigen specific to H. 1eprae that is currently available in amounts sufficient for use in large scale serological screening (225). The specificity of the antigen is conferred by the unique trisaccharide portion of the PG; the terminal sugar -3,6-di-O-methylglucose - appears to be dominant immunologically (226). The humoral immune response to the PG involves primarily the production of IgM antibodies, which are readily detected by an "ELISA" technique (225). Levels of IgM antibody directed at the PG have been reported to be high in patients with lepromatous leprosy, low in patients with tuberculoid leprosy, and to have decreased in the former patients after effective chemotherapy. Household contacts of patients with leprosy have been found to produce antibodies to the PG, presumably indicating that they have been infected with H. 1eprae. Antibodies were detected among 25% of contacts in Mexico, and 35% of contacts in Sri Lanka, compared to only 5 and 9% of individuals in the same communities who were not household contacts of leprosy patients. Studies performed at intervals of 6 or 12 months have indicated that, in many contacts, antibodies to the PG appear (presumably in response to infection with H. 1eprae) and return subsequently to normal levels, whereas in the few contacts who go on to develop clinical leprosy, the antibodies are found to have persisted (16). Procedure Preparation of antigen. The PG may be obtained from infected tissues by extraction by the method of Bligh and Dyer (9), followed by chromatography on a column of silica and celite, in a proportion of 2:1, that has first been washed with CHC13; the PG is eluted from the column with 2% methanol in CHC13 (84). The compound may be further purified by preparative thin-layer chromatography on silica gel G plates using either ether:acetone (9:1) or CHC13:methanol (15:1) as the solvent phase (84, 225). The native PG is extremely hydrophobic, and its use in aqueous systems is facilitated by deacylation, which removes 50% of the lipid portion of the molecule, while leaving the antigenic trisaccharide intact (225). Deacylation is accomplished by incubating the native PG (10 mg) for 16 hours at 1000C in 2 ml 10% NaOH in methanol:benzene (2:1) under 59 Contributed by D.B. Young and T.M. Buchanan. WHO/CDS/LEP/86.4 page 126 N2. After cooling, the pH is brought to about 4 by dropwise addition of HC1, and the lipid is extracted with 3 volumes of CHC13. The deacylated lipid is purified by prepara tive thin-layer chromatography, using as the solvent phase CHC13:methanol (15:1), in which the deacylated compound migrates less rapidly than does the native molecule, because of its decreased hydrophobicity (225). The purified deacylated lipid is stored in a concentration of 5 - 10 mg per ml in CHC13:methanol (2:1) at 0 - 4oC. Its concentration is most conveniently estimated by assaying the carbohydrate content by means of the phenol-sulfuric acid test (44), using rhamnose as the standard. One mg of deacylated PG contains approximately 0.5 mg carbohy drate. Coating the plates. The deacylated PG may readily be prepared as a stable aqueous suspen sion. Fifty ~g of the material is placed in a glass tube, and the organic solvent is permitted to evaporate. One ml of water is then added, and the tube is agitated vigor ously for one minute. The suspension is then incubated at 55 0C for 15 minutes (or over night at room temperature) and agitated again. The suspension may now be diluted for coating plates, or stored for as long as two weeks at room temperature. To coat plates, the suspension of lipid is diluted 10-fold in water, to provide a final concentration of 5 g per ml, and 0.1 ml is pipetted into each of the wells of one half of a polystyrene microtitre plate. The other half of the plate is "coated" with water only, to provide a control. The plate is then covered, incubated overnight at 37oC, and washed four times with 0.2 ml per well of PBS, pH 7.2. The coated plates may be used immediately or covered and stored at 0 - 40C for at least one month. The quality of the water employed may affect the amount of "non-specific" binding measured in the half of the plate that was not coated with antigen. The lowest back grounds are obtained with pyrogen-free water, such as that employed for injections in hospitals, whereas tap water gives very high values. A portion of the background probably results from binding of antibodies in human serum to endotoxin in the water. Polystyrene microtitre plates manufactured by Linbro are usually employed, but those of other manufac turers may also be used, and the PG will also bind to polyvinyl chloride plates. Only part of the PG actually binds to the plastic surface, and it is possible to recover much of the antigen after the over-night coating step. Because the antigen has been prepared in water, recovery of unbound antigen may be conveniently accomplished by lyophilization; the antigen may then be resuspended in a concentration of 5 ~g per ml. As much as 75 per cent of the antigen may be recovered in this manner. Blocking of non-specific binding. Detergents such as Tween 20R will interfere with the binding of the PG to the plastic surface, and should not be used for washing plates. In order to block non-specific binding of antibodies, the coated plate is preincubated with a solution of 5% BSA in PBS (0.1 ml per well) for 2 hours at 37oC. As an additional precau tion to inhibit non-specific binding, sera to be tested are diluted in the presence of 5% foetal calf serum (FCS) (normal goat serum is equally effective). Incubation with test sera. The BSA is removed from the plate and replaced by human serum that has been diluted 1:20 (dilutions as large as 1:200 may be used with good effect if only small quantities of the sera to be tested are available) in PBS containing 5% FCS. Serum samples are added in duplicate to both the antigen-coated and uncoated halves of the plate, in a volume of 0.1 ml per well. The plate is incubated for 2 hours at 37oC, and then washed four times with PBS. Enzyrne-con;ugated secondary antibody. The IgG fraction of goat anti-human IgA + IgG + IgM conjugated with peroxidase is diluted 1:1000 in PBS containing 1% FCS and added to the plate in a volume of 0.1 ml per well. After incubation for one hour at 37oC, the plate is washed four times with PBS. Most of the antibodies to PG are IgM, and it is important to use a secondary antibody that is capable of detecting immunoglobulins of this class. A secondary antibody directed ------ WHO/CDS/LEP/86.4 page 127 toward IgA, IgG and IgM, and a secondary antibody specific for IgM, yielded almost ident ical results in a study of 78 sera from leprosy patients (16). Colour development. A stock solution of the substrate a-phenylenediamine in methanol, 10 mg per ml, is diluted lOO-fold in 0.1 M citrate buffer, pH 5. Hydrogen peroxide is added in a final concentration of 0.003%, and the substrate solution is added to the plate in a volume of 0.1 ml per well. After incubation in the dark for 20 minutes at room temperature, the reaction is stopped by addition of 8 N H2S04 (25 ~l per well), and colour intensity is measured in terms of the absorbance at 492 nm, employing a Titertek Multiscan spectrophotometer. Calculation of Results The means of duplicate samples in the water-coated half of the plate are subtracted from the means of the corresponding duplicates in the antigen-coated half. Sera from normal individuals will show the same amount of binding to both halves of the plate, and yield values of zero, whereas sera from individuals who have been infected with H. 1ep rae yield positive values; a value of at least 0.1 is considered positive. The sera of patients with lepromatous leprosy often yield values greater than 1.0 (225). It is wise to identify a negative serum and a highly positive serum, to be employed as controls each time the assay is performed. Use in Serodiagnosis Those individuals possessing antibodies to the PG clearly include many who will not develop clinically apparent leprosy. However, a persistently positive antibody response may indicate early disease, and should be studied thoroughly. WHO/CDS/LEP/86.4 page 128 CHAPTER 9 - SURVEILLANCE OF DRUG-INTAKE60 Investigations into the regularity of self-medication in the treatment of such chronic diseases as diabetes mellitus, hypertension and pulmonary tuberculosis have indi cated that poor compliance of patients with self-administered treatment is universal. Poor compliance with drugs, primarily dapsone, prescribed for self-administration has also been demonstrated to be common among leprosy patients (49). Furthermore, in leprosy as in tuberculosis, poor compliance is almost certainly one of the most important causes of treatment failure. Because poor compliance is so generally a problem, it could be argued that its exis tence may be assumed, and that an attempt should be made to remedy the situation - by improving the training of paramedical workers and more intensive patient education, or by employing treatment regimens so designed that every dose may be supervised - rather than expend the effort needed to monitor patient compliance. On the other hand, there may well arise situations, such as controlled clinical trials, in which it is clearly important to measure the degree to which patients actually ingest the prescribed treatment. In this chapter, the methods required to monitor compliance of patients with dapsone, rifampicin and the thioamides are presented. Storage and Shipment of Urine Specimens For preservation of urine specimens obtained for the purpose of monitoring patient intake of drugs, a crystal of thymol may be added to each specimen, or thiomersal should be added to the specimens in a final concentration of O.lN. Urine specimens prepared for storage as described may be shipped either frozen on dry ice, cooled to 40C on wet ice, or without any attempt to maintain a lowered temperature. The most important consideration is that the specimens be clearly labelled and so packed as to prevent breaking and spilling. Monitoring Compliance with Dapsone The traditional and most effective means of monitoring intake of dapsone and many other drugs is by employment of a simple laboratory procedure for detection of the drug or its metabolic products in urine. Dapsone and its metabolites are found in higher concen trations in urine than in plasma; patients generally are more willing to provide urine than plasma samples; and if urine is used rather than plasma, the analysis can be carried out on much larger samples, thus enabling detection of lower concentrations. DAPSONE/CREATININE RATIO The most satisfactory colorimetric method for estimating dapsone, in which its aro matic amino groups are diazotised and coupled with N-l-naphthylethylene diamine, gives colours with a wide range of other compounds containing aromatic amino groups. Also, the half-life of dapsone Lnman is long (about 24 hou1;'!') .J:,Or, these reasons, qualitative urine-tests give positive results for several days after the ingestion of a single thera peutic dose of dapsone. One must consider also the effects of urinary concentration and dilution. Thus, one wishes to avoid classifying as positive (for dapsone) concentrated dapsone-free urine samples containing substantial amounts of natural diazotisable com pounds; similarly, one does not wish to characterize dilute dapsone-containing samples as negative. However, the effects of diuresis can be largely overcome by quantitatively determining the ratio of the concentration of dapsone plus its diazotisable metabolites to that of creatinine in the urine, using modifications of the Bratton and Marshall and alkaline picrate colorimetric methods, respectively. 60 Contributed by G.A. Ellard. WHO/CDS/LEP/86.4 page 129 Colorimetric Determination of Dapsone Eight ml 6% (w/v) trichloracetic acid in lN HCl are added to 1 ml of urine. Any samples that contain visible precipitating protein are discarded. The optical density of the solution at 550 nrn is measured in a suitable spectrophotometer, and the reaction is initiated by the addition of two drops (0.04 ml) of 1% aqueous sodium nitrite. Five minutes later, two drops of 10% aqueous ammonium sulfamate are added, and after a further five minutes two drops of 2% N-l-naphthylethylenediamine dihydrochloride in acetone/water (1:1 by volume). Care should be taken to shake the sample thoroughly after the addition of each reagent. The concentration of dapsone plus its diazotisable metabolites is then determined from the increase in the optical density at 550 nrn (read after a further five minutes), and by comparison with a standard containing 30 ~g/ml dapsone in O.lN HCl reacted and read in the same way. Colorimetric Determination of Creatinine One-tenth ml of urine is reacted by adding 0.1 ml N sodium hydroxide and 0.2 ml saturated aqueous picric acid. After standing for 10 minutes, the sample is diluted to 10 ml with distilled water and the optical density measured at 500 nrn. The concentration of creatinine is then calculated by comparison with the optical density obtained by react ing 0.1 ml of a 1 mg/ml solution of creatinine in O.lN HCl in the same way. Among patients who ingest daily 100 mg doses of dapsone regularly, dapsone/creatinine ratios usually fall in the range 50 - 150 ~g dapsone (plus dapsone-metabolites) per mg creatinine. Ratios smaller than 50 probably indicate that the 100 mg dose of dapsone was not taken during the 24 hours preceding collection of the urine specimen. A ratio smaller than 10 probably means that no dapsone has been ingested for at least three days. "ELISA" METHOD The lack of specificity of the Bratton and Marshall procedure for detecting dapsone and its metabolites results in the method being insufficiently sensitive to identify omissions of more than about 4 days of treatment. However, it is probable that signifi cant therapeutic penalties are likely to be incurred only when gaps in dapsone self-admin istration of 6 days or more occur. By this time, urinary dapsone concentrations will have fallen to 1-2% of those encountered when prescribed treatment is regularly ingested. There is therefore a need for more sensitive and specific assay methods to detect dapsone and its metabolites in urine. Huikeshoven and his co-workers (215) have developed an "ELISA" (enzyme-linked immunosorbent assay) method of great sensitivity and specificity. In this method, ali quots of urine and a solution of dapsone coupled to the enzyme, horse-radish peroxidase, are pipetted into the wells of a microtitre plate, the wells having previously been coated with an anti-dapsone antibody raised in rabbits. After standing for 30 minutes at 56oC, the wells are washed free of unbound dapsone and dapsone-peroxidase conjugate, and a colour is developed by the addition to the wells of hydrogen peroxide together with a suitable substrate - e.g., 5-aminosalicylic acid. The intensity of the colour developed is directly proportional to the quantity of dapsone-peroxidase conjugate bound, and, therefore, inversely proportional to the concentration of dapsone and its metabolites in the urine sample tested. Huikeshoven has developed a portable "kit" to facilitate monitoring of dapsone com pliance by the ELISA method in the field. "SPOT" TEST A "spot" test has been described (82, 83) that appears to be more convenient than measurement of the dapsone/creatinine ratio for monitoring intake of dapsone. The spot WHO/CDS/LEP/86.4 page 130 test is more simply performed, although it may be somewhat less sensitive than the latter measurement. Thick strips of filter-paper (e.g., Whatman no. 3) are soaked in dimethyl aminoben zaldehyde-oxalic acid solution, and air-dried in the dark; these impregnated paper strips are stable for a few months if stored in the dark. The test is performed by placing a drop of urine on the impregnated paper strip. After one minute, a yellow ring caused by urea appears at the periphery of the spot of urine, and an inner orange spot appears when dapsone is present. Addition of one drop of 0.5 - 1.0 N HCl to the spot will produce the orange colour if dapsone is present but the urine is excessively alkaline; similarly, if an immediate orange spot (before addition of HC1) has resulted from the presence of sul phonamides, the colour will fade after addition of HC1. Controls are: urine obtained from a subject known not to have taken dapsone that has been acidified by the addition of 10 ml 1 N HCl to 90 ml urine (solution A); and 39 ml solution A, to which has been added 1 ml of a solution of 100 mg dapsone in 10 ml 1 N HCl that has subsequently been diluted 1:50 with distilled water (final concentration 5 ~g dapsone per ml). Monitoring Compliance with Other Drugs The drugs to be used in combination with dapsone for multidrug therapy (MDT) , follow ing the recommendations of the WHO Study Group on Chemotherapy of Leprosy for Control Programmes (221), are rifampicin, clofazimine, ethionamide and protionamide. The regu larity with which these drugs are taken is likely to be a major factor in determining their potential value in curing patients and controlling the disease. RIFAMPICIN The taking of a standard 600 mg dose of rifampicin can often be detected by the characteristic orange-brown colour that it imparts to urine samples collected within 6 to 8 hours of ingestion. Rifampicin together with its desacetyl metabolite may be detected in the urine for at least 12 hours after ingestion by means of a plate diffusion assay, employing Staphylococcus aureus NCTC 10702 and its rifampicin-resistant variant S. aur eus NCTC 10703 (126, 127). Plates containing 15 ml nutrient agar and 1 mg streptomycin per ml are inoculated with one or another of the organisms. Two 6 mm diameter discs of sterile filter paper are dipped into the urine sample with flamed forceps, and the excess urine is removed by touching the discs to the side of the urine container. One of the pair of discs is placed on a quarter of the plate inoculated with the rifampicin susceptible organism, and the other on the plate inoculated with the resistant organism. Solutions containing 1, 10, 100 and 1000 ~g rifampicin per ml are prepared, and dupli cate discs are dipped in these and placed on the plates. The diameters of the zones of inhibition are measured after overnight incubation at 37oC. An assay employing high performance liquid chromatography (HPLC) has also been described (213). CLOFAZIMINE No simple methods have yet been developed for monitoring the compliance of patients with drugs other than dapsone and rifampicin. Clofazimine has a very long half-time of elimination from the body, and only negligible concentrations of the drug or its metabo lites are found in the urine (51, 101). Thus, monitoring compliance with self administered clofazimine presents a very difficult problem. Among light-skinned patients, the degree of skin pigmentation may provide a rough indication of the extent to which the drug is being taken; however, it is in just such patients that clofazimine is least acceptable. ETHIONAMIDE AND PROTIONAMIDE A urine-test method for detecting the ingestion of ethionamide has been described (47), which depends on the extraction and concentration of its yellowish sulphoxide metabolite. The method, which is also applicable to protionamide, gives reliably positive results for at least 12 hours after the ingestion of 250 mg doses of either drug. WHO/CDS/LEP/86.4 page 131 Eight m1 urine are saturated with 3 g sodium citrate and thoroughly extracted with 10 m1 chloroform. The urine is then centrifuged for 10 minutes at 2000 rpm, and the upper aqueous phase is removed by aspiration with a Pasteur pipette and discarded. The chloro form extract (lower phase) is then filtered through Whatman no. 52 filter paper, and re extracted with 0.8 m1 0.1 N HC1 by vigorous shaking. The ch10roform-HC1 mixture is allowed to stand in a 15 mm diameter tube, as the result of which the HC1 separates with out centrifugation, and can be removed from the surface of the chloroform phase with a Pasteur pipette. Ten drops of the HC1 phase are delivered into a semicircular depression of a white porcelain plate, such as is used for blood-group determination. The presence of chromogenic thioamide metabo1ites is most readily detected as a yellow colour in the HCl extract. The test should be read by comparison with a control urine sample that is collected four hours after ingestion of a supervised dose of either ethionamide or pro tionamide. Alternatively, one might administer protionamide in the form of isoprodianR, which includes both dapsone and isoniazid in addition to protionamide. Ingestion of protionamide could then be monitored by analysis of urine for dapsone or acetylisoniazid (46). Because these additional drugs are much more expensive than dapsone, and it is most important to prevent the emergence of M. 1eprae resistant to them, one might wish to ensure that patients comply with MDT by employing the drugs in fully-supervisable regi mens, rather than by attempting to monitor compliance. In fact, the regimen recommended by the WHO Study Group for MDT of multibacillary leprosy was designed to answer the need for supervisability. This regimen consists of four components (221): (1) dapsone, to be self-administered in a daily dose of 100 mg; (2) clofazimine, to be self-administered in a daily dose of 50 mg; (3) clofazimine, to be administered also in a monthly supervised dose of 300 mg; and (4) rifampicin, to be administered in a monthly supervised dose of 600 mg. The monthly 300 mg dose of clofazimine, probably not effective by itself (20), is intended to compensate for any of the self-administered doses that were not taken. On the other hand, the monthly 600 mg dose of rifampicin has been shown to cause killing of M. 1eprae in patients (115). Finally, it is hoped that patients will comply more willingly with treatment that is to be terminated after a finite period of time. There fore, it appears unnecessary to monitor patient-compliance with this regimen in order to ensure a good outcome of the treatment. However, some patients will be unable to tolerate clofazimine because of the skin pigmentation it causes. For them, the Study Group has suggested substitution for clofa zimine of ethionamide or protionamide, to be self-administered in a daily dose of 375 mg (221). In such cases, monitoring of compliance with the thioamide drug is very impor tant. The possibility of dapsone resistance, primary or secondary, makes good compliance with the thioamide mandatory, if secondary resistance to rifampicin is to be prevented. Yet, intolerance to ethionamide and protionamide is so common, even in this relatively small dosage, that the patients may simply not ingest them. WHO/CDS/LEP/86.4 page 132 APPENDIX 1 - PRODUCTION OF If. LEPRAE IN THE ARMADIu.o6l The progressive, lepromatous-leprosy like infection in the nine-banded armadillo, which evolves in a proportion of animals inoculated with M. leprae, has provided for the first time a substantial laboratory source of M. leprae. This encouraged the Immunology of Leprosy (IMMLEP) Scientific Working Group of the UNDPjWorld BankjWHO Special Programme for Research and Training in Tropical Diseases to undertake development of a specific vaccine for leprosy. Substantial progress has been made in standardizing the procedures for obtaining maximal infection in the armadillo and maximal yields of purified organisms freed from armadillo tissue by methods least likely to damage M. leprae. M. leprae are extracted from the livers, spleens, lymph nodes and non-ulcerated skin nodules of heavily infected armadillos, yielding a total per animal of 250-300 g of infected tissue containing 10 9-1010 AFB per g tissue. For extraction and purification of M. leprae from these tissues, IMMLEP has devel oped a method yielding 95% recovery of intact organisms, which appear to have retained their antigenic and immunogenic activities as well as their viability. This has been achieved without the use of proteolytic enzymes for digesting the tissues after homogeni zation, which is carried out at high pH to inhibit the action of tissue proteases. Tissue residues are removed first by means of a Percoll gradient, followed by an aqueous 2-phase system; the purified M. leprae are prevented from clumping by the addition of 0.1% Tween 80 (the sequence of procedures is shown in Table 25). On average, an infected armadillo yields 900 mg dry weight M. leprae by these methods. At autopsy, usually 1-2 years after inoculation, AFB recovered from the tissues of each armadillo are both cultured and inoculated into mice to exclude the possibility of contamination by other species of mycobacteria carried by armadillos. To retain as far as possible the characteristics of M. leprae, armadillos are inoculated initially with M. leprae from man, and armadillo-derived M. leprae are not used to inoculate other armadillos more than once. The IMMLEP protocol for preparing M. leprae from armadillos appears as Appendix 2. 61 Contributed by C. Lowe and R.J.W. Rees. WHO/CDS/LEP/86.4 page 133 Table 25. Purification of M. leprae from armadillo tissues62 Objective Process Effective disintegration of tissue Homogenization mechanically at high pH Inhibition of host-derived lytic Homogenization and washing at enzymes - especially proteases high pH and nucleases Control of physical form of DNA Inhibition of DNAase, presence of Mg ++ , isotonic conditions to preserve nuclei, then hypotonic lysis in presence of DNAase Removal of bulk of insoluble Percoll gradients (non-toxic tissue residues colloidal silica) Removal of traces of tissue Aqueous 2-phase system: poly residue with same density as ethylene glycol 6000 + dextran bacteria T500 Removal of materials used in process Extensive washing with buffered Tween 80 Prevention of clumping of bacteria Use of 0.1% Tween 80, lightly buffered to prevent acidification due to hydrolysis Tween 80. 62 Contributed by C. Lowe and R.J.W. Rees. WHO/CDS/LEP/86.4 page 134 APPENDIX 2 - PURIFICATION OF H. LEP~3 Solutions and Chemicals Homogenization Medium To 9 parts 0.15 M NaCl + 1 part 2 M Tris base, add 1 M MgS04 at the rate of 1 m1 per litre. Washing Buffer 0.15 M NaC1 + 1 part 0.15 M HEPES buffer (adjusted to pH 7.2 with NaOH) , containing 0.1% Tween 80 and 1 mM MgS04. Tween 80 is conveniently added as a 10% (w/w) stock solu tion. DNAase Buffer 0.1% Tween 80 (made up freshly from 10% stock solution) containing 1 ml 1 M MgS04 per litre and 20 ml 0.15 M HEPES buffer, pH 7.2, per litre. Buffered Tween To 0.1% Tween 80, add 0.5 M MES (adjusted to pH 6.8 with NaOH) at the rate of 2 m1 per litre. Perco11R Percoll is stabilized colloidal silica. Aqueous Two-Phase System To 7 g dextran T500, add 4.9 g polyethy1ene glycol (PEG) 6000 and 1 g 10% (w/v) poly ethylene glycol palmitate (the monopalmitate ester of PEG 6000), and water to 70 g. Allow the polymers to dissolve. Add 0.5 ml 2 M NaCl, 2 ml 0.5 M potassium phosphate, pH 6.9, and the H. leprae in a minimal volume of buffered Tween, and make up to a total of 100 m1 with water. DNAase Crystalline deoxyribonuclease I (this is available sterile if required). The sup pliers warn that the enzyme in solution is very easily denatured by shaking. Cruder preparations may contain chymotrypsin, in which case a protease inhibitor might be added the suppliers suggest DFP. Procedure Weigh the tissue. Homogenize in homogenization medium at the rate of 4 ml per 1 g tissue, using an homogenizer. Centrifuge at 10 000 x g for 10 minutes, set aside the supernatant, which contains the phenolic glycolipid (see pp. 125 - 127), re-homogenize in the same volume of homogenization medium, recentrifuge, and discard the supernatant. Resuspend the sediment in washing buffer (volume as for the homogenization medium above), centrifuge as above, and discard supernatant64. Suspend in DNAase buffer (a 63 Adapted by P. Draper from reference no. 220, in which the purification method is referred to as Protocol 1/79. 64 All processing so far is carried out at 10oC. Time of exposure to homogenization medium should be as short as practicable. WHO/CDS/LEP/86.4 page 135 suitable volume is about 100 m1 per 25 g original tissue), add DNAase to give a concentra tion of 4 units per m1, stir at 200C for 1 hour, and filter through a coffee strainer or other suitable netting with approximately 0.5 mm stainless steel mesh. Collect by centrifugation (10 000 x g, 10 min) , suspend in buffered Tween, make 30% (v/v) with respect to Perco11 (a suitable total volume is about 100 m1 per 25 g original tissue), distribute into centrifuge tubes (25 m1 in 27 mm i.d. tubes), and centrifuge at 27 000 x g for 1 hour in an angle rotor. Collect the bacterial band (near bottom of tube; the tissue-derived debris will be in a distinct band near the top of the tube). Dilute the suspension with buffered Tween, and wash with buffered Tween by centrifug ing (8 000 - 10 000 x g, 10 minutes). Four centrifugings and three resuspensions are usually sufficient. Suspend in the minimal volume of buffered Tween, and add to the aqueous 2-phase system. Stir very well, then allow the phases to separate in a separatory funnel. Remove the upper phase, and dilute it with an equal volume of water containing Tween 80 to give a final concentration of 0.1% Tween. Collect and wash with buffered Tween. The interface material and lower phase are discarded. Comments 1. It appears important not to over-homogenize. The manufacturer's recommendation of 3 min at top speed in the Omnimix is sufficient for soft tissues. Emulsifier-type homog enizers are more effective with tough tissue - lymph node and subcutaneous leproma, but too long treatment will give difficulties in the density-gradient step. 2. The conditions of centrifuging are critical in the formation of the Perco11 gradient. The geometry of the gradient is important; the 1ength:width ratio used here (2:1) should be adhered to, if tubes of different sizes are used; long, thin liquid columns do not form gradients properly. Swing-out rotors could probably be used, but these would require longer centrifuging to form the gradient. The bacteria have a buoyant density of about 1.09 in Perco11, but the gradients are not linear, which means that a system must be developed empirically. The concentration of Perco11 is not particularly critical; concentrations between 25 and 50% give satisfactory separation. The bacterial band becomes somewhat diffuse at the higher concentrations, but these may be needed in processing homogenates from tough tissues, in which case there is a lot of fibrous material in the suspension, which is denser than most tissue debris. 3. Washing Perco11 away from recovered bacteria appears to depend on the fact that the bacteria sediment faster than the silica. It is important not to centrifuge too fast or too long at this stage. Speeds giving 8 000 x g will sediment relatively pure bacteria without important losses. 4. Dilute Tween 80 hydrolyses spontaneously and becomes acid. This causes H. leprae to clump, hence the use of a dilute buffer. MES has its best buffering range at about pH 6 and so is a suitable buffer to use, but other buffers with similar pH would probably do as well. 5. The salts added to the 2-phase system are at convenient concentrations, but any con centration might be used that produced the final concentrations of 0.01 M NaC1 and 0.01 M potassium phosphate. 6. The final suspensions are quite well dispersed and stable in Tween. They can be washed once with water, but a second resuspension in water leads to gross clumping and adhesion to glass and plastic surfaces. WHOjCDSjLEPj86.4 page 136 7. To prepare polyethylene glycol monopalmitate, dissolve 100 g PEC65 in 600 ml toluene, and distil off about 100 ml toluene from the solution to relnove traces of moisture. Add 2 g triethylamine previously distilled over phthalic anhydride. Add 5 g palmitoyl chlo ride in 50 ml toluene dropwise with continuous stirring. Reflux mixture gently for 15 minutes, then filter. Cool to 30C and collect precipitated derivative by vacuum fil tration. Recrystallize twice from absolute ethanol. The derivative contains one palmi toyl group per PEG molecule. Fulka AG produce good palmitoyl chloride; many samples from commercial sources have lots of palmitic acid, so that the stoichiometry would come out wrong. The recrystallization may be difficult because PEG-palmitate has quite a low melting point, especially when moist with ethanol or toluene. A refrigerator, cold-room or lots of ice will be required. Absolute ethanol is 100%, not the more readily available recti fied spirit (95%). It appears unwise to use denatured alcohol. 65 In this context PEG means polyethylene glycol 6000, although the method works with other poiymers. It is not difficult to scale up the process. WHO/CDS/LEP/86.4 page 137 APPENDIX 3 - PREPARATION OF LEPROMIN The preparation of lepromin, and the safety requirements in its preparation have been described in a WHO Memorandum (10). The following description is adapted from this publication. Integral (Mitsuda-type) lepromin has been used for many years in millions of patients and healthy individuals, as a skin test for the classification of leprosy and for assess ment of the immune responsiveness to M. 1eprae. The batches used have been made by several laboratories, and attempts at standardization of the batches, based on counts of AFB have been successful. Lepromin is prepared from M. 1eprae obtained from biopsy specimens of skin obtained from heavily infected patients suffering from lepromatous leprosy, or from tissues of armadillos heavily infected with M. 1eprae. The skin or other tissue is removed asept ically, autoclaved at l200C for 15 minutes, and stored at -20oC until required for proces sing. Before beginning the preparations, the following tests are performed on a small portion of each biopsy specimen: (1) the concentration of the AFB is determined by microscopic examination. Only tissues shown to be heavily infected are used; (2) the presence of other bacteria is tested by placing small pieces of the tissue into suitable media. The tests used are those specified in the WHO Requirements for the Sterility of Biological Substances (217). Only those biopsy specimens shown to be sterile are used. Tissues satisfying the requirements of these two tests are subjected to the following procedures, each step being carried out under aseptic conditions as rapidly as possible, and the preparation being completed within one working day: (1) M. 1eprae are released in the following manner. After the epidermis and fat have been removed from the autoclaved tissues, the remaining tissues are weighed, cut into small pieces with sterile scissors, and homogenized mechan ically in sterile, pyrogen-free saline. The homogenate, suitably diluted in saline, is centrifuged at 200 x g for 10 minutes, and the supernate is removed and retained. The sediment is re-homogenized and the process repeated once; (2) the released bacilli are treated in the following manner. The two pooled super nates are passed through nylon gauze, and the number of AFB per millilitre is determined. The sieved supernates are then diluted to give the required concen tration of M. 1eprae, using a solution of analytical-grade phenol, such that the final concentration of phenol in the diluted supernates is 5 g per litre. The phenolized, diluted suspension of organisms is then dispensed into suitable containers, and autoclaved at l200C for 15 minutes. The usual concentration of AFB in integral lepromin is between 4.0 x 10 7 and 1.6 x 108 AFB per ml. Preparations with counts in this range have given a high degree of repro ducibility of skin reactivity. The final product is tested as follows: (1) each filling lot is tested for sterility according to the requirements given in Part A, Section 5 of the General Requirements for the Sterility of Biological Substances (217); (2) each filling lot is tested for innocuity by intraperitoneal injection of 0.2 ml into each of five mice and of 0.5 ml into each of two guinea pigs. The animals must remain healthy during an observation period of seven days; (3) each filling lot is tested for skin-reactivity by intradermal injection of 0.1 ml into the skin of each of two guinea pigs. The filling lot is satisfac tory if there is no necrosis at the site of injection; WHO/CDS/LEP/86.4 page 138 (4) the total protein concentration of each filling lot is determined. It should not be higher than that found in batches of lepromin shown to be safe for human use; (5) the phenol content of each filling lot is determined using an approved chemical method of analysis, and should not exceed 5.5 g per litre; (6) the number of killed M. leprae contained in an accurately measured portion is determined microscopically by standard procedures. Lepromin should be stored at refrigerator temperature (4 0C). WHO/CDS/LEP/86.4 page 139 APPENDIX 4 - PREPARATION OF REAGENTS Acid Alcohol To prepare acid alcohol, employed in the standard acid-fast stain described by Shepard, dilute 665 ml 95% ethanol with 285 ml distilled water, and then add slowly and carefully 9.5 ml concentrated HCl. Ammonium Sulfamate To prepare a 10% solution of ammonium sulphamate, for analysis of dapsone, 10 g of the compound are dissolved in distilled water, and distilled water is added to a final volume of 100 ml. The solution is stable at 40C for three months. Bouin's Fluid Bouin's fluid, employed in the procedure for pyridine extraction, has the following composition (31): Saturated aqueous picric acid 50 ml Formalin 10 ml Glacial acetic acid 5 ml Distilled water 35 ml Carbolfuchsin First, one prepares a 5% solution of phenol, by adding, in order, 1400 ml distilled water, 14.8 ml 95% ethanol, and 85.2 ml liquefied phenol. Also, one prepares a stock solution of basic fuchsin, by dissolving 20 g basic fuchsin in 190 ml 95% ethanol. This stock solution should be allowed to "age" for one month before it is used. The "working" solution of carbolfuschsin stain is then prepared by mixing 180 ml 5% phenol with 20 ml of the stock solution of basic fuchsin; the working solution should be allowed to age for two weeks before use. For the best mycobacterial staining, the basic fuchsin should have a light absorption maximum no lower than 552 nm, or new fuchsin (C.I. 42520) should be used (73) . Cardiolipin-Lecithin Solution Four hundred mg each of cardiolipin and lecithin are dissolved in 100 ml 100% etha- nol. Celestine Blue To prepare celestine blue, 2.5 g ferric ammonium sulphate are dissolved in 50 ml distilled water, after which 0.25 g celestine blue B is added; the mixture is boiled for 3 minutes, filtered and cooled. The solution keeps for 2-3 weeks in a dark jar. Citrate Buffer, pH 5 One first prepares a 0.1 M solution of disodium citrate by dissolving 21 g citric acid monohydrate in 200 ml 1 N NaOH, and making the solution up to 1 1 by the addition of distilled water. Then, one adds 96.3 ml of this solution to 3.7 ml 0.1 N NaOH. Creatinine For use as a standard in measurement of the dapsone/creatinine ratio, 100 mg creatin oC. ine are dissolved in 100 ml 0.1 N HCl. This solution is stable indefinitely at 4 WHOjCDSjLEPj86.4 page 140 Dapsone For determination of dapsone, a standard solution is prepared by dissolving 100 mg in 100 m1 ethanol. This solution is stable at 40C for periods as long as three months. Diluent A (for Abe's FLA-ABS test) One volume of cardiolipin - lecithin solution is mixed rapidly with 19 volumes of PBS. Diluent B (for Abe's FLA-ABS test) Nine volumes of diluent A are mixed with 1 volume of 1% BSA (wjv) in PBS. Dimethy1aminobenza1dehyde-Oxa1ate Reagent (for spot-test for dapsone in urine) To prepare this reagent, 0.2 g p-dimethy1aminobenza1dehyde, 1.0 g oxalic acid and 0.1 g naccono1 (dodecy1benzenesu1fonic acid, sodium salt) are dissolved in 100 m1 50% eth anol. Stored in the dark, the solution is stable for for one month. Ehr1ich's Haematoxylin To prepare Ehr1ich's haematoxylin, 2 g haematoxylin are dissolved in 100 m1 absolute ethanol; to this solution are added 100 m1 glycerol, 100 m1 distilled water, 10 m1 glac ial acetic acid, and finally 10 g aluminium potassium sulphate. The stain should be allowed to "ripen" during a period of several weeks by permitting it to stand, in a loosely stoppered large flask, in a warm place exposed to sunlight; during this time, the flask should be shaken frequently. The stain should be filtered before use. Eosin A stock solution of eosin is prepared by dissolving 5 g eosin Y in 100 m1 tap water. This is diluted 1:5 with tap water to provide a working solution. Ethanol, 70% Because it is very expensive, one does not dilute 100% ethanol, to produce 70% or 95% ethanol. Rather, one begins with 95% ethanol, which is the constant boiling mixture. The simplest way in which to dilute 95% ethanol to prepare 70% ethanol, for example, is to dilute 70 ml of 95% ethanol to 95 ml. Formalin, Buffered The composition of buffered formalin is: Formaldehyde (40%) 10 m1 NaH2P04.2H20 (Sodium diacid phosphate) 0.35 g Na2HP04.12H20 (Disodium acid phosphate) 0.65 g Distilled water 90 m1 Formalin, Neutral Neutral formalin for fixation of histopathological specimens is prepared as follows: WHO/CDS/LEP/86.4 page 141 Formaldehyde 100 ml NaH2P04 4.0 g Na2HP04 6.5 g Distilled water 900 ml Formol Calcium Formol calcium, employed in the procedure for pyridine extraction, is pre pared as follows: Formalin 50 ml Calcium chloride or calcium acetate 2 g Distilled water to 100 ml Formol Milk Formol-milk is prepared by centrifuging cow's milk at 3000 rpm for 15 minutes. The subnatant, cream-free layer is carefully removed with a Pasteur pipette, 1.5 ml formalin is added to a 10 ml portion of skim milk, and the volume is made up to 100 ml with dis tilled water. This reagent may be stored indefinitely at 4oC. Gelatin-Phenol Gelatin-phenol consists of 0.5% gelatin and 0.5% phenol in distilled water. It is prepared by first dissolving 0.5 g gelatin in 100 ml distilled water, with heating. Then, 0.56 ml liquefied phenol is added. Gelatin-phenol is stored at room temperature; before every use, complete solution of the gelatin should be ensured by inspection and warming if necessary. Hanks' Balanced Salt Solution (HBSS) Hanks' balanced salt solution has the following composition: CaC12 200 mg KCl 400 mg MgS04 100 mg NaCl 6800 mg NaHC03 2200 mg NaH2P04·H20 140 mg Glucose 1000 mg Phenol red 17 mg Distilled water to 1000 ml HEPES Buffer To prepare 0.15 M HEPES buffer, 35.7 g HEPES are dissolved in 1 1 distilled water and adjusted to pH 7.2 with NaOH. Hydrochloric Acid Concentrated HCl contains 40% HCl (w/v) , and is 13.1 N. To prepare 1 N HC1, there fore, one dilutes 7.63 ml concentrated HCl with distilled water to a final volume of 100 ml. In the case of concentrated HC1, as for all concentrated mineral acids and strong bases, one should add the acid to water, and never water to the acid, which is very dan gerous, because of the great heat of solution. Moreover, the acid should be added slowly, while cooling the resulting solution with running water or ice. Once having diluted the concentrated acid, further dilution requires much less care. Thus, one prepares 0.1 N HCl from 1 N HCl simply by diluting the 1 N solution ten-fold with distilled water. WHO/CDSjLEP/86.4 page 142 Lowy's Fixative Lowy's fixative is prepared as follows (118): Formaldehyde solution (40%) 10 ml Mercuric chloride 2 g Glacial acetic acid 3 ml Distilled water 87 ml Heat may be required to dissolve the mercuric chloride; one first dissolves the mercuric chloride in the water, and allows the resulting solution to stand one day before adding the remaining components. The final solution may produce a sediment on standing; this is not important, and it can be left. The fixative should be stable for three months. Methylene Blue Methylene blue stain is prepared by first dissolving 0.6 g methylene blue in 60 ml 95% ethanol, and, at least two weeks later, adding 140 ml distilled water. N-l-Naphthy1ethylenediamine Dihydrochloride For determination of dapsone, 2 g N-l-naphthylethylenediamine dihydrochloride are dissolved in 100 m1 of a solution prepared from equal volumes of acetone and distilled water. This solution is stable at 40C for three months. Phosphate Buffer, pH 7.4 To 80.4 ml M/15 Na2HP04 (11.88 g Na2HP04.2H20 per 1 distilled water) one adds 19.6 ml M/15 KH2P04 (9.08 g KH2P04 per 1). Phosphate-Buffered Saline Phosphate-buffered saline (PBS) is prepared as follows: NaH2P04 0.45 g/l Na2HP04·l2H20 3.28 g/l NaCl 8.0 g/l. Adjust the pH to 7.2 by dropwise addition of HCl or HaOH. Picric Acid A saturated aqueous solution of picric acid is prepared by gently heating 20 g crys talline picric acid in 1 1 distilled water until all of the solid material has dissolved, and permitting the resulting solution to cool. Polyethylene Glycol Palmitate To prepare polyethylene glycol (PEG) monopalmitate, dissolve 100 g PEG in 600 ml toluene, and distil off about 100 ml toluene from the solution to remove traces of mois ture. Add 2 g triethylamine previously distilled over phthalic anhydride. Add 5 g palmi toyl chloride in 50 ml toluene dropwise with continuous stirring. Reflux mixture gently for 15 minutes, then filter. Cool to 30C and collect precipitated derivative by vacuum filtration. Recrystallize twice from absolute ethanol. The derivative contains one palmitoyl group per PEG molecule. Saline, Physiological Physiological saline is 0.9% NaCl (w/v) in distilled water. One simply dissolves 9 g NaCl in 1 1 distilled water. WHOjCDSjLEPj86.4 page 143 Sodium Hydroxide NaOH has a molecular weight of 40. Therefore, to prepare a 1 N solution of NaOH, one dissolves 40 g NaOH (in pellet form) in 1 1 distilled water. Because this process gener ates considerable heat, it should be carried out slowly, with cooling of the resulting solution. Further dilution may be made with less care. Solutions of NaOH should be protected from contact with air, which results in precipitation of Na2C03. Also, because NaOH reacts with glass, bottles containing solutions of NaOH should not be closed with glass stoppers, but with rubber stoppers or corks. Sodium Nitrite For determination of dapsone, a 1% (wjv) aqueous solution of sodium nitrite is prepared by dissolving 100 mg NaN02 in 10 ml distilled water. This solution should be prepared immediately before each use. Tribromoethanol A stock solution of tribromoethanol is prepared by dissolving the material in amylene hydrate (2-methylbutan-2-ol, tertiary amyl alcohol) in a ratio of 1 g tribromoethanol to 0.5 g (0.625 ml) amylene hydrate; to prepare 5 ml of stock solution, enough to anaesthe tize 1000 mice, one dissolves 8 g tribromoethanol in 5 ml amylene hydrate. This stock solution is stable for many months when stored in the dark at room temperature (up to 250C). However, it slowly becomes toxic with aging, and should therefore be made up freshly every six months. The working anaesthetic solution is freshly prepared on the day of surgery as follows: Saline - 50 parts Ethanol - 4 parts Tribromoethanol stock solution - 1 part (added drop by drop with shaking to a warm solution of saline and ethanol). TRIS-Buffered Saline, pH 8.0 This buffer is prepared by adding 250 ml 0.2 M TRIS base (24.2 g per 1) to 13.4 ml 0.2 N HC1, and diluting to 1 1 with distilled water. 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WHO/CDS/LEP/80.4 page 154 INDEX 2,3-dimethy1rhamnose, 116 Antibody titres, 6 2-methy1butano1, 73 Antigen, 2, 50, 116, 119, 120, 121, 3 ,4-dihydroxydipheny1a1anine , 110 122, 123, 124, 125, 126, 146 3,6-di-O-methy1g1ucose, 116, 124 Antigenicity, 116, 146 3,6-di-O-methy1g1ucose, 116, 124 Antigens, 2, 4, 106, 115, 116, 117, 3-methy1rhamnose, 116 118, 120, 121, 122 4,4'-diacetamidodipheny1su1phone Anti1eprosy drugs, 78 5-aminosa1icy1ic acid, 128 Antilymphocyte globulin, 50, 145 Ab initio, 109 Antimicrobial, 3, 26, 30, 67, 69, Aberration, 15 77, 78, 79, 109, 147 Aberrations, 15 Antiseptic,S, 11, 12, 13, 14, 30 Acanthosis, 41 Antitubercu10sis drug, 67 Accident, 11 Aperture, 15 Accidents, 6 Apochromatic, 15, 27, 29, 63 Acedapsone, 69, 74, 75, 76, 147, Aqueous ammonium su1famate, 72, 150, 151 128 Acetic acid, 73, 138, 139, 141 Aqueous methylene blue, 65 Acetone, 12, 72, 124, 128, 141 Aqueous picric acid, 128, 138 Acety1isoniazid, 130, 144 Aqueous sodium nitrite, 72, 128 Achromatic, 15 Arabinoga1actan, 114 Acid-fast stain, 2, 21, 24, 37, 63, Arabinomannan, 116, 148 67, Ill, 138 Armadillo, 3, 4, 48, 57, 66, 109, Activity, 3, 11, 12, 22, 38, 41, 110, 111, 115, 116, 117, 121, 122, 42, 43, 46, 59, 67, 77, 79, 110, 124, 131, 132, 147, 148 123, 144, 149, 150, 151 Armadillos, 3, 57, 110, 115, 120, Adenine, 112 131, 136, 144, 146, 148 Adrenaline, 30 Aromatic amino groups, 127 Adult-thymectomized, 49, 50, 52 Arthropod, 3, 7, 11, 148 Adult thymectomy, 50, 55 Arthropods, 57, 58, 148 Aerosol, 6, 8 Artificial media, 3 Aerosols,S, 6, 8 Aspiration, 55, 130 AFB, 5, 7, 12, 21, 24, 25, 27, 28, Assay, 73, 113, 116, 118, 121, 126, 37, 43, 46, 48, 51, 63, 64, 65, 66, 128, 129, 148, 149 67, 68, 73, 74, 75, 78, 79, 80, 82, Athymic, 50, 51, 53, 61, 144, 146 107, 108, 109, 120, 121, 122, 131, Atlanta, 3, 27 136 Auto-oxidation, 110 Air, 6, 7, 8, 13, 14, 23, 24, 34, Autoclave, 12, 13, 14, 118 35, 58, 61, 62, 65, 111, 119, 123, Autoc1aved, 5, 13, 22, 58, 60, 62, 129, 142 120, 121, 136 Air pressure, 61 Autoclaving, 5, 8, 12, 13, 14, 58, Air transport, 34 62 Alcohol, 8, 12, 31, 33, 34, 36, 73, Automatic micropipette, 63 106, 135, 138, 142 Automatic pipettors, 6 Alkaline, 12, 127, 129 Axon, 40 Amyl alcohol, 73, 142 Bacilli, 2, 7, 29, 38, 39, 43, 44, Anaesthetic, 30, 31, 33, 54, 142 122, 136, 145, 147, 148, 149, 150, Anaesthetized, 31, 33, 55, 56 152 Analysing samples, 72 Bacillus,S, 13, 24, 106, 143, 148, Animal inoculation, 30, 34, 67 149 Annular, 22, 30 Back-extracted, 72, 73 Antibodies, 115, 116, 117, 118, Bacterial degeneration, 107 122, 124, 125, 126, 145, 146, 152 Bacterial morphology, 26, 28 Antibody, 6, 39, 116, 118, 122, Bacterial population, 2, 21, 29, 123, 124, 125, 126, 128, 143, 146 73, 74, 82, 146 WHO/CDS/LEP/86.4 page 155 Bacterial protein, 110 Calculations, 48, 83 Bacterial suspensions, 24, 77 Calculator, 91 Bactericidal, 73, 79, 81, 82, 144, Cambridge rocker microtome, 37 148, 151 Campo-Aasen, 111, 143 Bacteriological index, 25 Capillary micropipettes, 63 Bacteriopausa1 effect, 80 Carbolfuchsin, 24, 37, 138, 146, Bacteriopause, 79 149 Bacteriostatic, 79, 80 Care, 4, 11, 19, 23, 29, 30, 31, Baker, 111 53, 57, 60, 71, 128, 140, 142, 144, BALB/c, 49, 53, 54, 61, 109 148 Baohong, 1, 146 Carry-over, 106 Barksda1e, 111, 145, 146 Case-finding, 3 Barrier, 62 Caseation, 44 Base, 66, 112, 114, 133, 142, 146, CBA, 49, 50, 54, 61, 109 149, 150 Cebu, the Philippines, 69 Bases, 112, 113, 140 Cedar wood oil, 36 Bedding, 13, 58, 60, 61, 62 Celestine blue, 37, 138 Bench tops, 11, 13 Cell culture, 5 Benzene, 73, 124 Cell-free culture media, 5 Bercovier, 1 Cell-mediated immune response, 50 Br, 2, 3, 21, 22, 25, 26, 29, 30, Cell wall, 107, 114, 116, 151 66, 73, 74, 76 Cell walls, 106, 107, 114, 144 Bibulous paper, 37 Cellular infiltrate, 39, 40, 41, Binford, 49, 143, 148, 152 44 Biology, 3, 117, 145, 146, 147, Centers for Disease Control, 150, 151 Atlanta, GA, USA, 3 Biopsy, 4, 7, 8, 26, 27, 30, 31, Centrifugation, 6, 8, 21, 72, 73, 33, 34, 35, 37, 42, 47, 48, 63, 64, 115, 122, 123, 130, 134 66, 67, 68, 69, 73, 77, 107, 111, Centrifuge, 6, 8, 72, 73, 133, 134 136, 150 Centrifuging, 134, 140 BL, 26, 44, 46, 121 Ceramic circle, 17 Blender, 71, 72, 122 Cerocebus sp, 57 Blood, 22, 38, 39, 40, 41, 56, 73, Cesium chloride gradient, 112 107, 108, 130 CFW, 49, 109 Body fluid, 106 Chemicals, 4, 14, 133 Boeck, 21, 144 Chemoprophylactic treatment, 11 Bone marrow, 49, 50, 54, 56, 57, Chemotherapeutic regimen, 28 143 Chemotherapeutic regimens, 4, 83 Bone-marrow reconstituted, 49 Chemotherapy, 1, 2, 3, 4, 7, 21, Bouin's fluid, 111, 138 25, 27, 28, 30, 67, 75, 78, 82, 83, Bovine serum albumin, 63 107, 124, 129, 143, 144, 145, 147, Bratton, 73, 127, 128 148, 150, 152 Bratton-Marsha11, 73 Chimpanzee, 57, 146 Breakage, 6 Chlorination, 58, 62 Breeding, 53, 57, 58, 60, 61 Chloroform, 130 Breeding nucleus, 60, 61 Chromatic aberration, 15 Brenner, 114, 143 Chromatography, 112, 113, 124, 125, Brightness, 15 129 Brush, 19 CidexR, 14 BSA, 63, 64, 65, 66, 125, 139 Circle, 17, 18, 63, 64, 65, 123 Buchanan, 1, 118, 143, 145, 148, Classification, 4, 21, 26, 30, 36, 150, 152 38, 44, 45, 46, 112, 114, 118, 120, Buffer, 36, 110, 111, 112, 126, 136, 150 133, 134, 138, 140, 141, 142 Classifying, 2, 3, 120, 127 Buoyant density, 112, 134 Cleaning, 5, 12, 13, 58, 60 Cage, 58, 60 Clinical trials, 27, 28, 69, 77, Cages, 12, 13, 53, 58, 60, 62 127, 150 Calculation, 26, 84, 86, 91, 99, Clinically active, 21 102, 121, 126 WHO/CDS/LEP/86.4 page 156 Clofazimine, 28, 69, 70, 72, 73, Cultured, 108, 131 78, 129, 130, 143, 145, 147, 148 Customs regulations, 34 Clothing, 8, 11, 60, 61 Cut, 22, 30, 31, 34, 36, 37, 43, Clumps, 38, 64, 108 55, 56, 66, 136 Coalescing papules, 21 Cystoscopes, 14 Collagen bundles, 39, 42 Cytological detail, 36 Colorimetric , 127, 128, 144 Cytoplasm, 37, 38, 39, 40, 44, 45, Colour, 15, 22, 37, 111, 124, 126, 107 128, 129, 130 Cytosine, 112 Colston, 3, 79, 144, 145, 147 D-DOPA, 110 Community, 6, 71 DADDS, 69, 150, 151 Compliance, 3, 4, 127, 128, 129, Danielsen, 21, 144 130, 145, 146 Dapsone, 3, 7, 21, 26, 27, 28, 67, Compound, 15, 124, 125, 138 68, 69, 70, 71, 72, 73, 74, 75, 78, Confidence limits, 99, 100, 108 80, 81, 82, 106, 109, 110, 116, Congenital, 51 127, 128, 129, 130, 138, 139, 141, Congenitally Athymic, 50, 53, 61, 142, 145, 146, 147, 148, 149, 151, 144 152 Congentially, 51 Dapsone-resistance, 3, 69, 109, Conjugate, 128 130 Connective tissue involvement, 42 Dartos muscle biopsy, 33 Contagion, 6 Davey, 25, 144, 147, 150 Contagiosity, 6 De Man, 99, 148 Container, 8, 11, 12, 13, 23, 34, De Man's tables, 99 58, 60, 129 Dead, 2, 7, 21, 24, 25, 39, 53, 63, Contaminants, 34, 59 74, 75, 111, 149 Contaminated wastes, 8 Decalcification solution, 64 Contamination, 7, 8, 11, 12, 13, Decalcified, 64, 67 58, 59, 60, 62, 66, 71, 72, 131 Decolourized, 37, 65 Contingency table, 84 Decontamination, 11 Continuous technique, 77, 78, 79 Defects, 15 Control, 1, 3, 4, 7, 23, 69, 70, Deformity, 2 71, 77, 79, 80, 83, 123, 125, 129, Dehydrated, 37 130, 132, 145, 150, 152 Delayed hypersensitivity reactions, Controls, 69, 83, 110, 111, 126, 46 129 Deoxyribonucleic acid, 106, 112, Convit, 111, 121, 143, 144, 148 143, 145, 146 Coplin jar, 23, 24, 37 Dermatitis, 41, 43 Cork, 55 Dermis, 22, 37, 39, 40, 41, 42, 43, Corynebacteria, 114 46 Count, 29, 107, 108 Desiccator, 19, 111 Counterstained, 37, 65 Desquamating, 22 Counterstaining, 24 Destaining, 24 Counting chamber, 56, 107, 108 Developing countries, 1, 2 Counting error, 107, 108 Deviation, 102, 108 Creatinine, 127, 128, 138 Diagnosis, 7, 21, 30, 37, 42, 43, Criteria, 13, 28, 44, 106, 117, 46, 122, 143, 148 121, 124 Diagnostic Specimens, 34 Cross-infection, 54 Diameter, 6, 15, 17, 18, 19, 20, Cryostat, 111 22, 31, 33, 39, 63, 64, 65, 107, Cuff of lymphocytes, 43 110, 119, 120, 129, 130 Cultivation, 3, 48, 63, 106, 107, Diaphragm, 8, 15, 19 109, 117, 147 Diazotisablle Culture,S, 63, 67, 69, 86, 106, Diet, 58, 59, 62, 69, 70, 71, 72, 107, 109, 116, 117, 145 73, 78, 80, 109, 116 Culture media,S, 63, 69, 106, 109, Dihydroxyphenylalanine, 106, 110 116 Diluted, 26, 27, 64, 65, 67, 69, 72, 77, 79, 86, 111, 120, 122, 123, 125, 126, 128, 129, 136, 139, 140, 142 WHO/CDS/LEP/86.4 page 157 Dilution, 7, 11, 65, 66, 67, 71, Enzyme, 110, 113, 125, 128, 133, 72, 77, 79, 86, 102, 123, 124, 127, 148, 149 140, 142, 146 Eosin, 37, 40, 42, 139 Dimethy1g1ucose, 116 Eosinophi1s, 39, 47 Disability, 2 Epidemiological, 3, 7, 121, 122 Disease-free mice, 60 Epidermis, 22, 33, 37, 39, 40, 41, Dissecting board, 64 43, 44, 136 Distribution, 85, 86, 107, 108 Epinephrine, 110 Diuresis, 127 Epithelioid cells, 38, 39, 44, 45, Divergence, 113, 114 46 DNA, 106, 112, 113, 114, 117, 132, Epitopes, 116, 117 143, 146, 149 Equation, 84, 86, 91, 102 DOPA, 106, 110, 111, 149 Equipment, 4, 5, 12, 13, 14, 22, DOPAchrome, 110 28, 62, 63, 118 Dosage, 54, 69, 70, 71, 74, 77, 78, Erinaceus europaeus, 57 81, 83, 130, 152 Erythema, 39, 42, 119 Double-stranded, 112, 113 Erythema nodosum 1eprosum, 39 Doubling time, 27, 48, 109, 116, Erythrocytes, 25 147 Ethanol, 24, 37, 55, 64, 66, 71, Doubling time, 27, 48, 109, 116, 72, Ill, 135, 138, 139, 141, 142 147 Ether, 54, 124 Dowex 50, III Ethionamide, 69, 70, 72, 78, 80, Down-grading, 44 81, 82, 129, 130, 145, 150 Draper, 1, 3, 144, 145 Ethyl acetate, 72 Draper's procedure, 3 Ethyl alcohol, 36 Droplet, 3, 6, 7 Ethylene Oxide Sterilization, 14 Drug, 2, 3, 26, 28, 30, 48, 58, 67, Europe, 2 69, 70, 71, 72, 73, 75, 77, 78, 79, Expansion, 38, 39, 42, 55, 119 80, 82, 83, 84, 116,127, 129, 130, Experiment, 12, 78, 83, 123 145, 150 Experimental Chemotherapy, 67, 82, Drug 83, 144 Intake, 127 Experimental group, 83 Resistance, 30, 82 Extinction, 72 Drug-administration, 72, 79, 80, Extracts, 72 83 Extremities, 2 Drug-containing mouse diets, 48, Fat, 30, 31, 39, 42, 58, 59, 63, 73 64, 65, 66, 136 Drug-screening, 77, 82 Female, 54, 60 Drug-susceptibility, 67, 69, 70 Fenner, 48, 145 Dubos-Midd1ebrook, 109 Fera11y infected armadillos, 110 Dup1exes, 113, 114 Fibrob1ast nuclei, 42 Ear lobes, 21 Fie1dstee1, 50, 143, 144, 145 Early Leprosy, 43 Filter, 13, 53, 62, 111, 112, 113, Ears, 49, 51, 83 123, 124, 129, 130, 134, 135, 141 Ehrlich's haematoxylin, 37, 139 Filter-paper, 13, Ill, 112, 129, Electron-microscopic, 25, 107, 146 130 ELISA, 118, 124, 128, 152 Filters, 14, 61, 113 Ellard, 1, 145 Fisher's, 84, 86, 91, 121 Embedding medium, 36 Fisher's exact probability Endemic, 1, 2, 3, 4, 5, 6, 118, calculation, 84, 86, 121 123, 124 Fisher test, 92 Endoneuria1 zone, 40 Fixation, 23, 27, 36, 42, 64, 106, Endoneurium, 40, 41 139 ENL, 39, 41, 46 Fixative, 35, 36, 37, 141 Enteric, 58 FLA-ABS test, 6, 118, 122, 139, Enterobacteriaceae, 114 143, 146 Envelope, 35, 116 Environment, 7, 149 WHO/CDS/LEP/86.4 page 158 Flaming wire loops, 6 HBSS, , 56, 57, 63, 64, 65, 79 Fluorescence, 116, 124 HC1, 12, 24, 37, 58, 67, 72, 124, Focus, 15, 17 128, 129, 130, 138, 140, 141, 142 Foetal calf serum, 77, 125 Heat-fixation, 23 Fold-multiplication, 108, 109 Heat-fixing slides, 6 Foot-pad, 28, 48, 57, 63, 64, 67, Heat-sealing, 35 69, 74, 75, 108, 118, 145, 146, Heating,S, 23, 24, 62, 63, 106, 147, 150, 152 112, 114, 119, 140, 141 Forceps, 23, 30, 31, 55, 56, 118, Hedgehog, 57 119, 129 Heiffor pattern, 37 Foreign tissue, 77 Heterologous, 113, 114 Formaldehyde, 23, 63, 65, 139, 140, High speed blenders, 6 141 Hilobatus lar, 57 Formalin, 14, 23, 24, 27, 29, 36, Histiocytes, 38 42, 64, 138, 139, 140 Histopathological, 4, 30, 31, 34, Formol-mi1k, 63, 140 36, 37, 38; 41, 42, 43, 44, 46, 57, Formula, 64, 112 64, 67, 68, 139, 144 Fractional areas, 104 Homogenate, 63, 64, 136 Freezing,S, 144, 151 Homogenates, 66, 115, 134 Friable, 22 Homology, 113, 114, 117, 145, 146 Fungal contamination, 66 Homozygous, 61 Fungal infection, 41 Hosts, 38, 62 Fungus, 19 Hot-Air Sterilization, 13, 14 Gamma rays, 14 Housing, 57, 58, 62 Gaugas, 50, 145 HPLC, 129 Gauze squares, 12, 22, 64 Huikeshoven, 128, 146, 152 Gelatin-phenol, 23, 140 Human, 3, 26, 57, 63, 64, 66, 77, Generation time, 26, 68, 71, 147 107, 111, 115, 116, 121, 122, 123, Genetic drift, 61 125, 137, 145, 148, 150, 152 Genome, 112, 114, 143, 146 Humidity, 58 Genomes, 112, 113 Husbandry, 48, 53, 57, 61, 62 Gerbil, 49 Hyaluronidase, 31 Germ-free, 50, 61, 62, 63 Hybrid, 113, 114 Gibbon, 57, 152 Hybridization, 113, 114, 117, 143, Glassware, 12 145 G1obi, 107 Hydrogen peroxide, 126, 128 Gloves, 11, 14, 22 Hydroxyapatite column, 113, 114 Glutaraldehyde, 14 Hyperchromicity, 112 Glycine, 106, 115 Hyperplasia, 41, 44 Glycolipid, 115, 118, 124, 133, Ice, 34, 66, 122, 127, 135, 140 143, 146, 152 Ice bath, 66 Granuloma, 38, 39, 40, 41, 42, 43, ID 50 44, 45, 46 ID50, 79, 86, 102, 103 Guanine, 112 Identification, 35, 75, 106, 112, Guld, 1 116, 143, 149, 150 Gunders, 57, 146 Identify, 40, 48, 106, 115, 126, Habitat, 40 128 Haematoxylin, 37, 40, 42, 139 Image, 15, 19 Hair-follicles, 40, 41 IMMLEP, 3, 4, 131, 152 Half-life, 127 Immune, 2, 3, 39, 41, 44, 47, 48, Halvorson, 86, 146 49, 50, 51, 52, 53, 54, 57, 61, 62, Hamster, 49, 152 66, 67, 75, 76, 77, 82, 124, 136 Hansen, 3, 21, 25, 146 Immune-competent, 48, 51, 57, 61, Harem-mating, 60 62, 76, 77, 82 Harvest, 27, 48, 53, 67, 68, 69, Immune-Deficient, 3, 48, 49, 50, 78, 83, 84, 147 51, 52, 53, 54, 61, 62, 66, 67, 75, Harvests, 49, 69, 70, 77, 79, 81, 76, 77, 82 83, 84, 85 Immune deficit, 2 WHO/CDS/LEP/86.4 page 159 Immunological, 2, 3, 38, 41, 42, Isog1utaminy1-meso-diaminopime1y1 118, 150 D-a1anine, 115 Immuno1ogica11y intact mice, 48, Isolated, 3, 27, 31, 32, 57, 69, 50, 51, 52, 66, 69, 75, 77, 82 106, 110, 116, 124, 146, 149 Immunosuppressed, 5, 49, 50, 75, Isolates, 106, 150, 151 143 Isoniazid, 130 Immunosuppressive drugs, 41 IsoprodianR, 130 In vitro, 48,117 Ito, 3, 147 In vivo, 3 Johnson, 114, 146 Incineration, 11 Jop1ing, 3, 30, 44, 118, 147, 150 Incision, 30, 31, 32, 55 Keloid, 42 Incubation, 2, 67, 110, Ill, 113, Killed, 2, 5, 7, 13, 28, 64, 67, 125, 126, 129, 147 68, 73, 75, 79, 81, 109, 110, 122, Indo1e-5,6-quinone, 110 123, 137 Inert solid, 72 Killing, 5, 28, 68, 79, 130 Infection, 2, 3, 5, 6, 7, 8, 38, Kinetic technique, 77, 79 39, 40, 41, 42, 49, 51, 52, 53, 54, Kirchheimer, 3, 143, 145, 146, 147, 57, 61, 62, 73, 109, 116, 122, 124, 148, 149 131, 143, 144, 145, 146, 147, 148, Kit, 128 149, 150, 151, 152 Koch's postulates, 106 Infections, 6, 40, 52, 53, 54, 57, Koh1er, 15, 27, 29, 63 60, 61, 69, 149, 151 L-a1anine, 115 Infectious, 2, 3, 5, 6, 7, 8, 79, L-a1any1-D- , 115 102, 109, 151, 152 L-DOPA, 110, III Infectivity, 5, 6, 26, 28, 68, 69, Label, 35, 60 107, 146, 148, 151 Labels Infiltrate, 39, 40, 41, 43, 44, 46, Laboratory, 1, 3, 4, 5, 6, 7, 8, 67, 109 11, 12, 13, 14, 15, 21, 23, 27, 34, Infiltrated skin, 21 35, 36, 37, 48, 57, 58, 59, 61, 67, Infiltration, 21, 39, 40, 41, 42, 69, 117, 127, 131, 144, 146, 148, 43, 44, 45 149, 152 Inflammatory cells, 39, 40 Laboratory-associated infections, Ingestion, 127, 129, 130, 145 6, 149 Injection, 54, 69, 72, 74, 118, Laboratory techniques, 1, 3 119, 120, 121, 136, 150 Lactoperoxidase, 116 Inocula, 5, 26, 28, 52, 60, 62, 66, Laminar-f1ow racks, 62 75, 76, 77, 145, 147 Langerhans, 44 Inoculated, 8, 11, 26, 28, 48, 51, Lens, 15, 18, 19, 29 52, 53, 58, 64, 65, 66, 67, 68, 69, Lenses, 15, 18, 19, 29, 63 70, 74, 75, 77, 80, 81, 82, 83, 86, Lepromatous, 2, 7, 21, 25, 26, 27, 91, 102, 106, 107, 108, 109, 117, 28, 38, 39, 40, 41, 42, 44, 45, 46, 129, 131, 147 48, 67, 68, 73, 74, 75, 82, 83, Inoculation, 3, 5, 7, 8, 11, 27, 106, 107, 109, 110, 116, 120, 122, 28, 30, 33, 34, 48, 49, 52, 53, 54, 123, 124, 126, 131, 136, 143, 145, 57, 60, 63, 64, 65, 66, 67, 68, 69, 147, 148, 149, 150, 151, 152 73, 74, 75, 76, 77, 79, 80, 83, Lepromin, 2, 3, 4, 106, 110, 118, 109, 110, 117, 131, 143, 146, 147, 120, 121, 136, 137, 143, 150 150, 152 Leprosy, 1, 2, 3, 4, 5, 6, 7, 8, Inoculum, 5, 27, 28, 48, 50, 51, 12, 21, 22, 23, 25, 26, 27, 28, 30, 62, 64, 66, 67, 69, 73, 75, 76, 77, 31, 38, 39, 40, 41, 42, 43, 44, 46, 79, 80, 81, 102, 106 48, 53, 61, 63, 67, 68, 73, 74, 76, Insusceptibility, 57 78, 82, 106, 109, 110, Ill, 115, Interferon, 77 116, 117, 118, 120, 121, 122, 123, Interpupillary distance, 19 124, 125, 126, 127, 129, 130, 131, Intracellular parasite, 38 136, 143, 144, 145, 146, 147, 148, Iodogen, 116 149, 150, 151, 152 Irradiation, 5, 11, 14, 50, 54, 55, Leprosy Unit, 1, 4, 120 56, 62, 143 Lesions, 2, 7, 21, 22, 38, 39, 41, 42, 43, 44, 46, 48, 49, 52, 69, 75, 107, Ill, 116, 148 WHO/CDS/LEP/86.4 page 160 Lethally irradiated, 49 Marble chips, 36 Levelling table, 63 Marsha11, 73, 127, 128 Levy, 1, 145, 147, 148, 151, 152 Mast cells, 39 Liberia, 57 Mathematical tables, 86 Lidocaine, 30 McCormick, Ill, 148 Lifespan , 62 McRae, 26, 27, 148, 150, 151 Light,S, 11, 15, 19, 22, 33, 36, MDT, 129, 130 107, 112, 123. 129, 138, 149 MED, 69, 77, 78 Lignocaine, 30, 31 Median infectious dose, 79, 102 Linkage, 116 Mercuric chloride, 37, 141 Linked, 114, 116, 128, 148 Heriones unguiculatus, 49 Lipid, 39, 114, 115, 124, 125, 143 Heso-diaminopime1ic acid Liquid nitrogen,S, 144 Hesocricetus auratus, 49 LL, 26, 45, 46, 117, 121 Metabolic products, 127 Localization of granu1oma, 41 Metabolite, 129 Logarithmic phase, 79 Metabo1ites, 127, 128, 129, 130, Loop, 8, 23, 65 152 Lowe, 1, 25, 148 Methylene blue, 24, 37, 65, 141 Lowenstein-Jensen, 63, 109, 116 MI, 2, 21, 26, 27, 28, 29, 30, 73, Lowenstein-Jensen medium, 63, 116 74, 76 Lowy's fixative, 36, 141 MIC, 78, 109, 110 Lungs, 55 Mice,S, 7, 8, 11, 12, 24, 27, 28, Lyrnphocyte transformation, 2, 118 30, 48, 49, 50, 51, 52, 53, 54, 55, Lyrnphocytes, 39, 40, 41, 43, 44, 56, 57, 58, 60, 61, 62, 63, 64, 65, 45, 46, 47, 50, 51, 54, 109 66, 67, 68, 69, 70, 71, 72, 73, 74, H. avium, 107, 115 75, 76, 77, 78, 79, 80, 81, 82, 83, H. kansasii, 110, 115, 149 84, 86, 91, 102, 106, 107, 109, H. leprae, 2, 3, 4, 5, 6, 7, 8, 116, 117, 131, 136, 142, 143, 144, 11, 12, 15, 17, 18, 21, 23, 24, 25, 145, 146, 147, 148, 149, 150, 151, 26, 27, 28, 29, 30, 33, 34, 38, 40, 152 42, 43, 48, 49, 50, 51, 52, 53, 54, Mick1e tissue disintegrator, 63 57, 58, 60, 61, 62, 63, 64, 65, 66, Microbe 67, 68, 69, 70, 71, 73, 74, 75, 76, Microbes 77, 78, 79, 80, 81, 82, 83, 84, 86, Microbial persistence, 73, 82 91, 102, 103, 106, 107, 108, 109, Microbiological safety cabinet, 8, 110, Ill, 112, 114, 115, 116, 117, 10, 11, 63, 66 118, 120, 121, 122, 123, 124, 130, Micrometry, 17, 83 131, 132, 133, 134, 136, 137, 143, Microscope, 4, 12, 15, 16, 17, 18, 144, 145, 146, 148, 149, 150, 151 19, 20, 21, 22, 23, 24, 29, 36, 63, H. lepraemurium, 107, Ill, 115, 64, 65, 67, 122, 123, 144, 148, 143, 145 149 H. lufu, 110, 150 Microscopy, 15, 65, 107, 112, 143, H. marinum, 48, 115, 144 146, 149, 151 H. tuberculosis, 6, 24, 114, 115 Microtitre, 125, 128 H. ulcerans, 48, 115, 149 Middle Ages, 2 Macrophages, 25, 38, 39, 42, 43, Minimal effective dosage, 77 44, 45, 46, 48, 107, 109, 150 Minimal inhibitory concentration, Macule, 22, 30 78 Magnification, 15, 18, 19, 25, 27, Mitsuda reaction, 110, 117, 120 123 Model, 82, 83, 121, 143, 147, 150 Malaysia, 27, 57 Molecular biology, 3, 117 Male, 54, 60 Monitoring, 4, 21, 30, 121, 127, Man, 62, 73, 99, 109, 122, 127, 128, 129, 130 131, 148 Monkey, 57, 148 Monoc1ona1 antibodies, 115, 116, 117, 145, 146, 152 Monocytes, 38 Mononuclear cells, 38, 40 WHO/CDS/LEP/86.4 page 161 Monotherapy, 3, 7, 26, 73, 74, 75, Needle-lumen, 8 76 Neonatal1y Thymectomized, 50, 52, Morphologic appearance 53, 61, 62, 77, 83, 145 Morphological features" 106 Nerve biopsy, 31, 33 Morphological index, 2, 25, 27 Nerves, 2, 31, 40, 41, 43, 44, 45 Most probable number, 79 Neutrophilic polymorphonuclear Motile, 38, 39 leucocytes, 39 Mouse, 1, 2, 3, 11, 13, 26, 28, 33, Neutrophils, 41, 43, 46, 47 48, 49, 50, 51, 52, 53, 55, 56, 57, NIMR, 50 58, 59, 60, 61, 62, 63, 64, 66, 67, Nitric acid, 12 68, 69, 70, 71, 72, 73, 74, 76, 77, Non-cultivability, 109 78, 80, 82, 83, 86, 102, 106, 108, Non-enzymatic oxidation, 110 109, 110, 116, 117, 118, 144, 145, Non-random, 108 146, 147, 150 Non-solid, 26, 27, 29, 51 MPN, 79, 86, 99, 100, 102 Non-specific immunity, 57 Mucous, 2, 8, 21 Norepinephrine, 110 Muir, 25, 148 Normal curve, 102, 104 Mu1tibaci11ary, 2, 21, 24, 25, 74, Normal mice,S, 7, 67, 74, 75, 109, 76, 130 116 Mu1tidrug, 129 Nose blows, 7, 21, 23 Multiple harvests, 83 NQ bacillus, 24 Multiplication, 3, 5, 24, 26, 27, Nuclear detail, 37 38, 39, 48, 49, 50, 51, 52, 57, 60, Nude mouse, 3, 50, 51, 53, 109, 61, 66, 68, 69, 70, 74, 75, 77, 78, 117, 144, 145 79, 80, 81, 82, 83, 84, 86, 91, Null hypothesis, 85, 86 102, 107, 108, 109, 116, 117, 147, Nutrition, 57, 58, 146 150, 152 O-dipheno1oxidase, 110 Murine viruses, 66 Objective lens, 15, 19 Mutation, 51, 53 Ocular lens, 15, 19 Mutations, 61, 82 Oculars, 15, 19, 27, 29, 63 Mycobacteria, 110, Ill, 114, 115, Oedema, 40, 41, 42, 43, 44, 46, 116, 122, 131, 143, 144, 145, 146, 119 148, 149, 151 Oil, 12, 15, 19, 25, 27, 29, 36, Mycobacterium, 2, 5, 106, 115, 37, 59, 63, 65 143, 144, 145, 146, 147, 148, 149, 01igosaccharides, 116 150, 151, 152 One-tailed test, 91 Mycobacterium leprae, 2, 5, 106, Optical density, 128 143, 144, 145, 146, 147, 148, 149, Organism, 3, 5, 27, 29, 38, 40, 42, 150, 151, 152 48, 60, 68, 75, 86, 106, 107, 112, Mycolic acids, 114, 115, 117, 144 113, 116, 129 Mystromys, 49 Organisms, 2, 3, 5, 7, 8, 11, 12, Mystromys mystromys, 49 13, 21, 23, 24, 25, 26, 27, 28, 29, N-1-naphthy1ethylenediamine 33, 38, 39, 40, 42, 48, 49, 50, 51, dihydrochloride, 72, 128, 141 52, 53, 57, 58, 60, 61, 65, 66, 67, N-acetylg1ucosamine, 114 68, 69, 70, 73, 74, 75, 76, 77, 79, N-acy1muramic, 114 80, 81, 82, 83, 84, 86, 106, 107, N sodium hydroxide, 128 108, 109, 110, 111, 113, 114, 115, NA, 15 116, 117, 122, 123, 124, 129, 131, Nakagawa, 73, 151 136, 147 NaOH, 66, 67, 124, 133, 138, 140, Organs, 2 142 Overlapping of values, 84 Nasal, 2, 5, 7, 21, 23, 48, 144, Overt, 5 146, 149, 150 Oxidation, 13, 24, 106, 110, 111, National Institute for Medical 147, 149 Research, 50 Oxytetracycline, 54, 62 Necrosis, 43, 44, 47, 119, 136 p., 1, 5, 17, 59, 62, 64, 76, 84, Necrotizing vasculitis, 46 85, 86, 91, 112, 139, 143, 144, Needle, 8, 11, 55, 57, 64, 118, 145, 146, 147, 148, 149, 150, 151, 119, 120 152 WHO/CDS/LEP/86.4 page 162 Pain, 2 Pouring, 6 Paper, 13, 14, 19, 34, 37, 58, 62, Prabhakaran, 110, 149 65, 86, 111, 112, 129, 130 Precautions,S, 6, 7, 8, 54, 60 Papillae, 41 Preservation, 36, 43, 127 Paraffin, 36, 37, 67, 111 Prevalence, 2, 6, 147 Paramedical workers, 127 Previously-untreated patients, 21, Parasites, 58, 60 27, 69, 109 Parent colony, 61 Primary resistance, 69, 109 Parfoca1, 17 Primates, 57 Passaged, 73, 83 Probability values, 84 Pasteurization, 5 problem patient, 69 Pathogen, 3, 8, 50 Procaine, 30 Pathogenesis, 38, 150 Processing, 34, 36, 134, 136, 148 Pathogenic organism, 60 PropipetteR, 8 Pathological specimens, 35 Proportional bactericide, 79, 80, Patient 81, 82, 83, 86 education, 127 Proportions, 26, 37, 76, 77, 83, compliance, 3, 127, 130 107, 122 Paucibaci11ary, 2, 22 Protionamide, 69, 70, 72, 78, 129, Peanut oil, 37 130 Pentobarbital, 54 Pseudoepithe1iomatous hyperplasia, Peracetic acid, 62 44 Perineurium, 40, 41, 43, 44, 45 Pulmonary, 6, 7, 127 Periodate oxidation, 24, 147 Punch method, 31 Periodic acid, 24 Puncture, 7, 11 Peroxidase, 125, 128 Pyridine extractability, 24, 148 Perry, 1 Quarantine, 34 Persisters, 75 Quarantined, 57 Persisting, 2, 40, 66, 67, 73, 74, Quarters, 58, 60, 61 75, 77, 82 Quiescent, 42 PG, 116, 124, 125, 126 Rabbit serum, 77 Phagocytic macrophages, 38 Rabbits, 128 Phago1ysosomes, 39 Radiation, 14, 50 Phenolic, 110, 111, lIS, 116, 118, Radioactive disintegrations, 108 124, 133, 143, 146, 152 Radioactivity, 111, 113 Phenolic substrates, 110, 111 Radioisotopic Procedure, 110 Phosphate buffer, 36, 110, 141 Rat, 49, 50, 52, 53, 83, 144, 145, Phospholipid, 111 146 Pigmentation, 129, 130 Reaction, 3, 22, 38, 40, 41, 42, Pipette, 6, 8, 9, 11, 23, 55, 57, 47, 72, 106, 110, 111, 113, 114, 63, 130, 140 117, 119, 120, 121, 123, 124, 126, Pipette-AidR, 8 128, 145, 146, 148 PIano lens ' Reagents, 4, 122, 138 Plaques, 21 Reassociation, 113, 114, 117, 146 Plasma, 39, 40, 44, 45, 57, 78, Red blood cells, 107, 108 107, 109, 127 Rees, 1, 3, 21, 25, 49, 63, 64, 70, Plastic, 13, 23, 31, 34, 35, 58, 75, 121, 143, 144, 145, 146, 147, 62, 125, 134 148, 149, 152 Platinum wire, 63 Regimens, 4, 27, 83, 85, 127, 130 Pleomorphic, 107 Regression, 38, 39, 42, 46 Poisson distribution. , 107, 108 Relapsed, 7, 28, 109 Polar, 2, 45, 106, 110, 120 Reproducible, 27, 113 Poly I:C, 81 Research, 1, 3, 4, 12, 16, 23, 37, Po1yethy1ene films, 14 38, 48, 50, 63, 67, 69, 118, 131, Po1yinosinic:po1ycytidy1ic acid, 144, 145, 146 81, 147 Resistance, 3, 5, 30, 48, 69, 71, 82, 109, 130 Resistant, 3, 11, 21, 58, 60, 69, 70, 71, 82, 109, 116, 129, 130, 146, 147, 148, 149 WHO/CDS/LEP/86.4 page 163 Resolution, 15, 40, 42, 43 Shipment, 34, 127 Respiratory, 3, 6, 7, 61, 144, 145, Significant lesion, 67 148 Single-stranded, 112, 113, 114 Rete pegs, 41 Sites, 21, 22, 25, 30, 31, 43, 121, Reversal, 44, 46 147 Rhamnose, 116, 125 Skin, 2, 3, 4, 6, 7, 21, 22, 23, Rid1ey, 1, 3, 25, 30, 44, 118, 147, 26, 27, 30, 31, 33, 36, 37, 38, 40, 149, 150 41, 43, 44, 45, 48, 55, 61, 64, 67, Rid1ey's logarithmic scale, 25 68, 73, 75, 106, 107, 111, 118, Rifampicin, 7, 28, 69, 70, 72, 73, 119, 120, 121, 122, 129, 130, 131, 74, 75, 76, 78, 127, 129, 130, 145, 136, 146, 148, 150, 152 146, 148, 149 - Skin lesions, 2, 7, 21, 22, 43, 75 Rodents, 3, 5, 48, 49, 50, 57, 58, Skin scrapings, 21, 22, 23, 107 61, 66, 77 Skin-test, 2, 4, 106, 118, 119, Rotary, 37, 64 120, 121, 136 Russe11, 75, 150 Skinsnes, 111, 151 Sl nuclease digestion, 113 Slide micrometer, 19, 65 Sacrificed, 56, 67, 68, 69, 83 Slides, 6, 12, 22, 24, 37, 63, 65, Safety, 4, 5, 6, 8, 9, 10, 11, 12, 123 54, 63, 66, 123, 136, 143 Slosarek, 111, 151 Saline, 5, 23, 64, 81, 122, 123, Smear, 4, 6, 21, 22, 23, 24, 25, 136, 141, 142 29, 30, 65, 108, 111, 122, 123 Salmonella sp., 58, 59 Smear-positive, 6 San Francisco, 69 Smears, 2, 7, 8, 21, 22, 23, 24, Sanchez, 111, 148 25, 47, 63, 64, 65, 66, 75, 111, Sanitary surveillance, 86 123, 149 Sansarricq, 1, 143, 149 Sodium citrate, 130 Satellite foci, 39 Solid bacilli, 29 Scales, 17, 18 Solid ratio, 2, 21, 25, 26, 27, 28, Scalpels, 22 29, 47, 68 Scarring, 42 Solidly-stained organisms, 26 Schwann cell, 40, 43 Solids, 27, 28, 29, 68, 71, 72 Scientific Working Group on Solubility, 72 Chemotherapy, 1, 3, 131 Solvents, 5, 11, 12 Scintillation fluid, 111 Sonication, 6 Scrapings, 21, 22, 23, 107 Source, 1, 3, 5, 6, 7, 11, 13, 14, Screening, 3, 67, 77, 82, 83, 110, 30, 38, 43, 55, 58, 60, 61, 131 124, 148 Species, 48, 49, 57, 106, 107, 109, Secondary dapsone resistance, 109 110, 112, 114, 115, 118, 131, 143, Secretions, 2, 3, 5, 7, 21, 23 145, 146 Sectioning, 36, 37, 146 Specific-pathogen-free, 50 Sections, 7, 30, 36, 37, 40, 42, Specimen, 30, 31, 33, 34, 35, 36, 43, 47, 53, 63, 64, 67, 107, 109, 37, 47, 48, 63, 64, 66, 67, 69, 73, 111, 147, 148 111, 127, 128, 136 Self Specimens, 7, 8, 12, 21, 23, 26, medication , 127 27, 30, 33, 34, 35, 36, 37, 48, 63, administered, 127, 129, 130 64, 66, 67, 68, 75, 77, 107, 111, Sensation, 2 123, 127, 136, 139 Serial dilutions, 86, 91 Spectrophotometer, 72, 73, 114, Serially-diluted inocula, 26 126, 128 Serological, 4, 57, 124, 143, 152 Spectrophotometrically, 73 Serum, 63, 77, 122, 123, 124, 125, SPF, 50, 53, 61, 62 126, 145, 152 Spherical aberration, 15 Sex, 58 Spirit lamp, 22, 118 Shadow-casted, 107 Spore-StripsR, 13 Shepard, 1, 3, 17, 18, 21, 26, 27, Spot, 17, 110, 111, 128, 129, 139, 28, 48, 63, 67, 70, 73, 75, 79, 86, 149 102, 108, 138, 143, 148, 150, 151 WHO/CDS/LEP/86.4 page 164 Spurs, 39 Suture, 31, 55 Sputum, 2, 7, 67 Sweat-glands, 40, 41 Stage, 17, 18, 19, 20, 29, 38, 39, Swing-out lens, IS, 19, 29 40, 41, 47, 63, 64, 65, 134 Syngeneic, 50, 54, 56 Staining, 2, 23, 24, 27, 36, 37, T-1ymphocyte-dep1eted, 53 42, 67, 73, 75, 106, 116, 138, 144, T-1ymphocytes, 50, 51, 54 146, 148, 149, 151 T200X5R, 50, 51, 54, 55, 61, 62 Standard deviation, 102, 108 T900R mice, 50, 51, 54, 62, 75, 77 Staphylococcus aureus, 129 Table, 25, 26, 27, 28, 36, 44, 45, Statistical tables, 86, 149 58, 59, 63, 66, 67, 69, 70, 71, 73, Statistical technique, 84 74, 75, 76, 78, 80, 82, 84, 85, 86, Statistical techniques, 48, 83 87, 88, 89, 90, 91, 92, 99, lOO, Sterile, 8, 14, 22, 23, 30, 31, 34, 101, 102, 103, 104, lOS, 114, 115, 54, 55, 62, 63, 64, 66, 129, 133, 119, 131, 132 136 Tables, 86, 91, 99, 144, 149 Sterility, 62, 66, 136, 152 Talking, 7 Sterilization,S, 12, 13, 14, 62, Taxonomic criteria, 106 63 Taxonomy, 114, 115 Stigma, 6 TOR, I, 152 Storage,S, 8, 34, 58, 60, 62, 127, Temp-TubesR, 13 144 Temperature, 6, 13, 24, 27, 37, 48, Storrs, 3, 143, 147 57, 58, 62, 67, Ill, 112, 113, 114, Streptomycin, 129 123, 125, 126, 127, 137, 140, 142, Sub-lethal dose, 54 ISO, 151 Subclinical infection, 6, 143, 146 ten Dam, 1 Subcultures, 106 THELEP, I, 4, 152 Subepidermal zone, 39, 41, 43 Thermal denaturation, 112 Subpapi11ary blood vessels, 39 Thioamides, 127 Substage Thiomersa1, 127 Condenser, 15 Thymectomized, 3, 49, 50, 51, 52, Illuminator, 15 53, 54, 55, 61, 62, 77, 83, 145, Substrate, 126, 128 149 Su1furic acid, 65, 125 Thymectomized-Irradiated, 3, 50, Sulphonamides, 129 51, 53 Sunahara, 73, 151 Thymectomy, 50, 52, 54, 55, 56, Sundaresan, 1 145 Sungei Bu10h Leprosarium, 27 Thymine, 112 Sunlight, 6, 139, 152 Thymol, 127 Superimposed, 29 Thymus-directed 1ymphocytes, 50 Supernate, 63, Ill, 136 Thymus gland, 51, 53, 55 Surgical mortality, 55 Time-TapeR, 13 Surgical procedure, 7 Tissue, 21, 22, 30, 31, 33, 34, 35, Surveillance, 86, 127 36, 37, 40, 42, 47, 63, 64, 66, 77, Susceptibility, 3, 11, 53, 57, 67, 106, 110, Ill, 115, 119, 120, 122, 69, 70, 71, 72, 109, 110, 145, 147, 123, 124, 131, 132, 133, 134, 136, 149 145, 147, 148, 149, 151 Susceptible, 51, 53, 54, 57, 69, Tissue 70, 73, 74, 75, 109, 110, 129, 149 Fluid, 22 Suspending fluid, 66, 67 Grinders, 64 Suspension,S, 8, 11, 26, 63, 64, Specimens, 21, 36, 37 65, 66, 69, 73, 75, 77, 79, 83, 86, Titration, 28 102, 107, 108, 109, 110, Ill, 122, Touching, 11, 29, 65, 129 123, 125, 134, 136, 148 Transmission, 3, 143, 149, 150 Suspensions,S, 8, 24, 27, 75, 77, Transverse-band structures, 107 86, 122, 123, 134 Treatment, 3, 4, 6, 7, 11, 21, 24, 25, 26, 28, 36, 39, 46, 54, 62, 66, 67, 68, 69, 73, 74, 75, 76, 79, 81, 82, 84, 85, 123, 127, 128, 130, 134, 144, 145, 147, 148, 149, 150, 151, 152 WHO/CDS/LEP/86.4 page 165 Treatment Work surfaces, 11 failure, 127, 147 Xylol, 19, 36, 37 regimens, 127 Young, 1, 38, 39, 42, 46, 55, 152 Tribromoethanol, 54, 142 Zaire, 110 Trichloracetic acid, 111, 128 Ziegler, 86, 146 Trypan blue solution, 57, 142 Ziehl-Neelsen technique, 24 Tryptose agar, 63 Tuberculoid, 2, 22, 25, 38, 39, 41, 43, 44, 46, 106, 110, 120, 122, 124 Tuberculosis, 6, 7, 24, 114, 115, 121, 127, 145, 148 Tumour cells, 46 Ulcerate, 46 Ultraviolet, 5, 11, 152 UNDP/World BankjWaO Special Programme (TDR), 1, 3, 4, 131 United States, 3 Untreated, 2, 7, 21, 25, 27, 28, 41, 46, 48, 67, 68, 69, 70, 77, 79, 80, 82, 83, 109, 116, 122 Upgrade, 46 Urea, 129 Urine, 127, 128, 129, 130, 139, 144, 145, 146, 148, 152 Vaccine, 4, 8, 75, 83, 122, 131 Vacuolated cells, 39 Vacuoles, 39, 42, 44, 45, 46, 47 Vacuum pump, 55 Variation among mice, 60 Vasculitis, 41, 46 Vein, 31, 57 Ventilation, 7, 61 Vermin, 58, 62 Vernier, 17, 18, 63 Vesicles, 39, 119 Viable, 2, 3, 5, 6, 7, 8, 11, 21, 27, 28, 29, 33, 42, 48, 53, 57, 63, 66, 67, 69, 73, 74, 75, 76, 77, 81, 82, 83, 86, 91, 102, 103, 109, 152 Virus Viruses, 14, 58, 60, 62, 66 Vitamins, 58, 59, 62 Vortex mixer, 72, 73 Wang, 5, 146, 152 Water, 11, 12, 13, 14, 23, 24, 37, 54, 55, 58, 60, 61, 62, 64, 65, 66, 72, 73, 86, 111, 118, 122, 123, 125, 126, 128, 129, 133, 134, 138, 139, 140, 141, 142 Waters, 25, 57, 73, 74, 75, 145, 149, 152 Wax, 31, 32, 33 WHO, 1, 3, 4, 5, 7, 8, 21, 24, 68, 69, 73, 99, 107, 109, 111, 117, 121, 124, 126, 128, 129, 130, 131, 136, 143, 152 WHO Study Group on Chemotherapy of Leprosy, 129, 130, 152 11111"111"111"11'"111"111""'"'""111'""*000'-10717*