Delayed-type hypersensitivity to Mycobacterium leprae soluble antigens as a test for infection with the leprosy bacillus.

BACKGROUND: Mycobacterium leprae (M. leprae) soluble antigen (MLSA) reagents have been developed with the aim of finding a reagent, comparable to tuberculin, which could identify individuals infected with the leprosy bacillus. They have yet to be evaluated fully in human populations. METHODS: More than 15000 individuals living in a leprosy endemic area of northern Malawi were skin tested with one of five batches of MLSA prepared using two different protocols. The main difference in preparation was the introduction of a high G centrifugation step in the preparation of the last three ('second-generation') batches. RESULTS: The prevalence of skin-test positivity (delayed-type hypersensitivity (DTH)) and association with the presence of a BCG scar were greater for first (batches A6, A22) than second (batches AB53, CD5, CD19) generation reagents. The association of positivity with M. leprae infection was investigated by comparing results among known (household) contacts of leprosy cases, and among newly diagnosed leprosy patients with those in the general population. While positivity to 'first-generation' antigens appeared to be associated with M. leprae infection, positivity to later antigens was unrelated either to exposure to leprosy cases or presence of leprosy disease. There were geographical differences in the prevalence of DTH to the various batches, probably reflecting exposure to various mycobacteria in the environment. CONCLUSIONS: Our results suggest that the 'second-generation' batches have lost antigens that can detect M. leprae infections, but that they retain one or more antigens which are shared between M. leprae and environmental mycobacteria. Natural exposure to these both sensitizes individuals and provides natural protection against M. leprae infection or disease. Identification of antigens present in these groups of skin test reagents may assist in production of improved skin test reagents.

Household and dwelling contact as risk factors for leprosy in northern Malawi.

Data on household and dwelling contact with known leprosy cases were available on more than 80,000 initially disease-free individuals followed up during the 1980s in a rural district of northern Malawi. A total of 331 new cases of leprosy were diagnosed among them. Individuals recorded as living in household or dwelling contact with multibacillary patients at the start of follow-up were at approximately five- to eightfold increased risk of leprosy, respectively, compared with individuals not living in such households or dwellings. Individuals living in household or dwelling contact with paucibacillary cases were both at approximately twofold increased risk. The higher risk associated with multibacillary contact and the fact that dwelling contact entailed a greater risk than household contact if the association was with multibacillary, but not with paucibacillary, disease suggest that paucibacillary cases may not themselves be sources of transmission, but rather just markers that a household has had contact with some (outside) source of infection. When household contact was considered alone, the risks of disease were appreciably higher for younger than for older contacts and for male compared with female contacts. Despite the elevated risk of leprosy associated with household or dwelling contact, only 15% of all incidence cases arose among recognized household contacts. Given the dynamic nature of household membership and consequent misclassification of contact status, the true contribution to overall incidence of contact within household or dwelling settings is likely to be much higher than this, perhaps 30% or higher. Considering the predilection of males for infectious multibacillary forms of the disease, the transmission of Mycobacterium leprae at an early age, in particular to males, may be of particular importance for the persistence of leprosy in endemic communities. Although residential contact with a multibacillary case is the strongest known determinant of leprosy risk, the vast majority of such contacts never manifest disease, which indicates a crucial role for genetic and/or environmental factors in the transmission of M. leprae infection and/or the pathogenesis of clinical leprosy.

Comparison of bacillary indexes in slit-skin smears, skin and nerve biopsies; a study from Malawi.

Data analyzed in this paper were collected within the framework of the Lepra Evaluation Project, an epidemiological study of leprosy in Karonga District, northern Malawi. For 212 patients information on the number of skin lesions, slit-skin smear and skin biopsy results were available. Among 61 patients with a single lesion none were slit-skin-smear positive and two had bacilli detected in skin biopsies. In contrast, among 119 patients with four or more lesions 34 (28.6%) versus 59 (49.6%) had bacilli detectable in slit-skin smears or skin biopsies, respectively. In a further 47 patients skin biopsy results could be compared with split-nerve biopsy results. In 20 of 47 patients the bacterial indexes (BIs) were identical in skin and nerve biopsies, while in 26 of 47 patients the BIs were higher in nerve than in skin biopsies. This difference, which is consistent with several other studies in the literature, provides an insight into the pathogenesis of leprosy.

Does bacille Calmette-Guérin scar size have implications for protection against tuberculosis or leprosy?

SETTING: Total population study in Karonga District, northern Malawi, in which the overall vaccine efficacy of bacille Calmette-Guérin (BCG) has been found to be -7% against tuberculosis and 54% against leprosy. OBJECTIVE: To examine the relationship between BCG scar size and protection against tuberculosis and leprosy. DESIGN: Cohort study in which 85,134 individuals were screened for tuberculosis and 82,265 for leprosy between 1979 and 1984, and followed up between 1986 and 1989. RESULTS: Of the BCG scar positive individuals whose scars were measured, 31/3 2471 were later identified with tuberculosis and 81/31 879 with leprosy. In 19,114 individuals, of whom 17 developed tuberculosis, tuberculin induration was measured at first examination. Mean scar sizes increased with increasing tuberculin induration in all except the oldest individuals. Mean scar sizes were lowest in individuals aged < 10 years, highest in individuals aged 10-29 years and intermediate in older individuals. There was some evidence (P = 0.08) for an increase in tuberculosis risk with increasing scar size, which probably reflects the known correlation between scar size and tuberculin status at the time of vaccination. There was no clear association between BCG scar size and leprosy incidence. CONCLUSIONS: We find no evidence that increased BCG scar size is a correlate of vaccine-induced protective immunity against either tuberculosis or leprosy.

Geographic determinants of leprosy in Karonga District, Northern Malawi.

BACKGROUND: Geographical differences in leprosy risk are not understood, but may provide clues about the natural history of the disease. We report an analysis of the geographical distribution of leprosy in Karonga District, a rural area of Northern Malawi, between 1979 and 1989. METHODS: Cohort study of the incidence of leprosy based on two total population surveys. Area of residence was determined using aerial photographs, which allowed identification of households, as well as location of roads, rivers and the lake shore. RESULTS: Incidence rates were between two and three times higher in the north compared to the south of the district, and lowest in the semi-urban district capital. The most obvious environmental difference between these regions is the north's higher rainfall and more fertile soil. There was no overall association between leprosy incidence and population density, although highest rates were observed in the least densely populated areas. Looking at the entire district, incidence rates increased with increasing distance from a main road, but declined with increasing distance from a river or from the shore of Lake Malawi. The negative association with proximity to rivers may reflect the larger number of rivers in the north of the district. Apparent differences in incidence rates between groups speaking different languages reflected confounding by area of residence. CONCLUSIONS: There is a marked variation, not explained by socioeconomic or cultural factors, in the incidence of leprosy within Karonga District. Our results are consistent with a theme in the literature associating the environment, particularly proximity to water, with leprosy.

PIP: Researchers do not understand how geographical differences relate to the risk of contracting leprosy. The study of such differences, however, may provide clues about the natural history of the disease. The authors report findings from an analysis of the geographical distribution of leprosy in Karonga District, a rural area in Northern Malawi, between 1979 and 1989. Findings are based data from two total population surveys. The areas of residence were determined using aerial photographs, which allowed the identification of households, roads, rivers, and the lake shore. Analysis revealed that incidence rates were 2-3 times higher in the north compared to the south of the district, and lowest in the semi-urban district capital. The north has higher rainfall and more fertile soil. There was no overall association between the incidence of leprosy and population density, although the highest rates were observed in the least densely populated areas. Considering the entire district, incidence rates increased with increasing distance from a main road, but declined with increasing distance from a river or from the shore of Lake Malawi. This negative association with proximity to rivers may reflect the larger number of rivers in the north of the district. Apparent differences in incidence rates between groups speaking different languages reflected confounding by area of residence. The authors conclude that there is a marked variation, not explained by socioeconomic or cultural factors, in the incidence of leprosy within Karonga District. These results are consistent with literature which associates the environment, especially proximity to water, with leprosy.

Testing for antibody to human immunodeficiency virus type 1 in a population in which mycobacterial diseases are endemic.

During a large epidemiologic study in the Karonga District of northern Malawi, serum samples from 139 patients with incident leprosy, 124 with newly diagnosed leprosy, 277 patients with incident tuberculosis, and 2296 controls were tested for antibodies to human immunodeficiency virus. Sera were tested according to a four-test protocol using two ELISAs and two particle agglutination assays. Overall, 188 samples were considered positive, 2634 were considered negative, and 14 were indeterminate. All 18 available positive specimens from leprosy patients, a random sample of 14 positive specimens from tuberculosis patients, and 15 positive specimens from controls were tested by Western blot. There was no evidence of substantial numbers of ELISA false-positives in any patient group or among controls.

Patterns of initial and acquired antituberculosis drug resistance in Karonga District, Malawi.

There is concern that drug-resistant tuberculosis is increasing and may be concentrated among HIV-positive patients. Little information is available from developing countries, where surveillance studies are often unable to distinguish resistance in previously untreated patients (initial resistance) from resistance acquired following drug therapy, and where information on the HIV status of the patients is rare. Initial resistance patterns reflect the strains being transmitted in the community. We have studied patterns of resistance in northern Malawi, where the Lepra Evaluation Project has been collecting data on drug resistance since 1986. Initial drug sensitivity results were available for 373 new cases of tuberculosis. Initial resistance to at least one drug was found in 44 of these patients (11.8%, 95% CI 8.5-15.1): 13 were resistant to streptomycin alone, 13 to isoniazid alone, and 17 to more than one drug. Only 3 patients showed initial rifampicin resistance-1 in isolation, 1 in combination with streptomycin, and 1 with triple resistance. Drug resistance was not related to age, sex, or HIV status of the patient and there was no evidence of any increase over the period studied. There was no evidence of geographic clustering of the resistant strains, or of any increased risk of resistant strains in households with previous tuberculosis cases. Acquired resistance during follow-up was found in 5 of 329 patients with documented initially fully sensitive strains. 5 patients with initial resistance seemed to show reversion to sensitivity. The absence of an increase in drug resistance, despite an increase in tuberculosis cases over the period, is encouraging for the control programme. It emphasises the need to collect information from many areas before assuming that increases in antituberculosis drug resistance are occurring worldwide.

Epidemiological aspects of relapses in leprosy.

Life table methods in which the cumulative probability of relapse in successive periods is calculated are preferable to the presentation of overall relapse rates. Their use facilitates the comparison of relapse rates and trends from different studies independent of duration of follow-up. Results from various studies including data from Malawi indicate that, (1) unlike after dapsone monotherapy, the cumulative probability of relapse in multibacillary patients is near to zero after WHO/MDT if strict definitions of relapse are used and, (2) the cumulative probability of relapse may approach 5% in paucibacillary patients 10 years after completion of WHO/MDT. On the whole, the epidemiological relevance of relapses is insignificant and future treatment regimens should be evaluated concerning their efficacy in preventing disabilities rather than relapses.

Delayed-type hypersensitivity, mycobacterial vaccines and protective immunity.

There is a longstanding debate over the implications of natural and vaccine-induced delayed type hypertensivity for protective immunity to mycobacterial infections. The identification of correlates of vaccine-induced protective immunity should help explain the inconsistent behaviour of BCG vaccines in different populations and assist in efforts to devise improved vaccines. More than 70,000 subjects in Karonga District, northern Malawi were skin tested with soluble antigens of the tubercle and leprosy bacilli, and then followed up for five years for tuberculosis and leprosy incidence. Incidence rate ratios were calculated to compare subjects with different levels of prior skin test sensitivity, after controlling for the effects of age, sex and previous BCG vaccination. BCG vaccination protected against leprosy without persistent delayed-type hypersensitivity to tuberculin or to soluble antigens of the leprosy bacillus. In subjects who had not received BCG, hypersensitivity to tuberculin or to antigens of the leprosy bacillus was associated with strong protection against leprosy. In BCG-vaccinated and unvaccinated subjects, there was a J-shaped relation between hypersensitivity to tuberculin and subsequent rates of tuberculosis, with lowest rates associated with low grade sensitivity (induration 1-10 mm). This study shows that delayed-type hypersensitivity to mycobacterial antigens has different implications for tuberculosis and leprosy: low-level hypersensitivity (probably attributable to environmental mycobacteria) is associated with protection, but persistent vaccine-associated hypersensitivity to mycobacterial antigens is not a correlate of vaccine-derived protection against mycobacterial diseases.

Extended schooling and good housing conditions are associated with reduced risk of leprosy in rural Malawi.

Incidence rates of leprosy in Karonga District, northern Malawi, are analyzed by duration of schooling and housing conditions, controlling for age, sex, BCG scar and geographical zone of the household. There is a strong inverse relationship between the number of completed years of schooling and leprosy risk. Good housing conditions are also associated with a decreased risk of developing leprosy in this population. The effect of housing is seen most strongly in young people. It is hypothesized that schooling changes behavior and housing determines environment in ways which are relevant for the transmission of Mycobacterium leprae or for the appropriate priming of the immune system.

Incidence rates of leprosy in Karonga District, northern Malawi: patterns by age, sex, BCG status and classification.

This paper describes incidence rates by age, sex, prior BCG status and classification in Karonga District, northern Malawi. New cases (489) were identified among 83,500 individuals followed for an average of 5 years (1.12 cases per 1000 person years). Only 29 (6%) of the incident cases were multibacillary. Incidence rates generally were higher among females than males, and increased steadily with age. Although the highest incidence rates of disease were recorded among young adults without BCG scars (males 15-19; females 20-24), these peaks were less dramatic than those reported among young adults in The Philippines and Norway. In the absence of historical data and data on infection status, it is not possible to assess to what extent these peaks may reflect either greater exposure or greater susceptibility to disease among adolescents or young adults. The incidence rates of leprosy among individuals with a prior recorded BCG scar were approximately half those of individuals lacking a scar, at all ages. Since BCG had been introduced into this population only during the 1970s, this provides strong evidence for the effectiveness of BCG when given to adults. It was estimated that past vaccination of approximately 40% of the district population had reduced the overall incidence rate of leprosy by 18%, and that this impact would increase with aging of the vaccinated cohorts. A retrospective examination of the detailed records of initial examinations revealed that 62 (13%) of the incidence cases were recorded as having skin hypopigmentation or blemishes, at the site of subsequent confirmed leprosy lesions, several months or years before they were suspected of having leprosy. The nonspecificity of these lesions, some of which were probably attributable to Mycobacterium leprae infection, highlights the difficulty of diagnosing leprosy in its earliest forms.

The Karonga Prevention Trial: a leprosy and tuberculosis vaccine trial in northern Malawi. I. Methods of the vaccination phase.

In this report the methods of the Karonga Prevention Trial, a double-blind leprosy and tuberculosis vaccine trial in Karonga District, Northern Malawi, are described in detail. During a total population house-to-house survey, which lasted from November 1985 until August 1989, 121,008 people (57,892 males and 63,116 females) were vaccinated. A further 5835 people refused vaccination and 5757 were ineligible for vaccination, 2652 of them because they had a history or signs of leprosy, or because they were suspected to have early leprosy. A total of 66,145 individuals, without evidence of prior BCG vaccination, received one of the following: BCG, BCG + 5 x 10(7) killed Mycobacterium leprae, or BCG + 6 x 10(8) killed M. leprae; 54,863 individuals found with a typical or a doubtful BCG scar received either placebo or BCG, or (from mid-1987 onwards) BCG + 6 x 10(8) killed M. leprae. Side-effects were not looked for systematically, but 4 individuals self-reported with glandular abscesses, 9 with large post-vaccination ulcers (> 25 mm in diameter) and 2 with ulcers which persisted for more than 1 year. BCG vials collected from paraffin refrigerators in the field showed satisfactory concentrations of viable BCG throughout the trial. Post-vaccination skin test (RT23 and M. leprae soluble antigen) results and post-vaccination ulcer rates indicate that few mistakes were made in the field when recording the vaccine codes.

Extent, origin, and implications of observer variation in the histopathological diagnosis of suspected leprosy.

Identical slides from 100 biopsies obtained from individuals suspected of having leprosy, ascertained in a total population survey in Malawi, were examined twice, independently, by three histopathologists. Results were reported in a standard protocol, and were compared among themselves and with a standardized clinical assessment of each "suspect." The proportion of biopsies considered to show definite evidence of leprosy ranged from 29 to 55 among the six evaluations (twice by each of three histopathologists). Comparisons of variations within and between histopathologists revealed three different patterns. Two of the pathologists were very consistent as individuals, but differed markedly between themselves in that one was the least inclined and the other the most inclined to report definite evidence of leprosy. The third pathologist was less consistent, reporting appreciably more definite leprosy on the first than on the second examination of the same biopsies. Although acid-fast bacilli (AFB) were reported on at least 1 examination in 40 of the biopsies, they were observed in all six examinations of only six of the biopsies. There was greater agreement regarding classification than regarding diagnosis, except with reference to the indeterminate category which was employed more frequently by one histopathologist than by the other two. A workshop of participants at the end of the investigation highlighted several reasons for the variations observed. The fact that AFB were reported in only nine biopsies by one histopathologist but in 33 by another reveals the importance of the examination method and time in arriving at a diagnosis of leprosy. The differences in the interpretation of cellular evidence of inflammation revealed the need for further studies of nerve-related pathology in nonleprosy conditions to serve as a reference against which to judge possible evidence of leprosy per se.

Long-term active surveillance of leprosy suspects--what are the likely returns?

Data are presented from the Karonga District in Northern Malawi on the long-term follow up of 277 leprosy suspects who were not given antileprosy treatment or kept on active surveillance. Individuals who were started on antileprosy treatment within a year after leprosy was first suspected, usually on the basis of histopathology results, are excluded from this analysis, because their active surveillance would not usually cause an organizational or financial problem for leprosy control projects. After an average follow-up period of 4.5 years 35 of the 277 suspects included in the analysis (13%) were diagnosed with what we consider to be 'unequivocal' leprosy, and 3 of the 35 had developed disabilities. In 211/277 (76%) all signs of leprosy had disappeared completely. Comparing clinical certainties at first and last examinations and comparing clinical with histopathological certainties at last examinations it is estimated that up to 50% of the 35 cases of unequivocal leprosy which 'arose' in this group were attributable to misdiagnosis at the 1st or 2nd examination rather than to genuine progression of the disease. This estimate is compatible with an overall sensitivity of 90% and an overall specificity of 95% at each examination. Leprosy suspects with 1 cardinal sign of leprosy, either a typical lesion without loss of sensation, or loss of sensation in an otherwise untypical lesion, should be considered a high-risk group in that approximately 25% of such suspects (19/78) were later found with unequivocal leprosy. Policies towards such suspects should be formulated by leprosy control projects.

Efficacy of BCG vaccine against leprosy and tuberculosis in northern Malawi.

Protection afforded by BCG (bacillus Calmette-Guérin) vaccines against tuberculosis and leprosy varies widely between different populations. In the only controlled trial which assessed protective efficacy of BCG (Danish and Pasteur strains) against both diseases, there was slightly more protection against leprosy than against tuberculosis. We have studied the protective efficacy of BCG (Glaxo, freeze dried) vaccine against these two diseases in Karonga District, northern Malawi. BCG vaccination was introduced into this population in 1974. Prior information about BCG scar status was available for 83,455 individuals followed up between 1979 and 1989. 414 new cases of leprosy and 180 new cases of tuberculosis were found in this population over that period. Protection was estimated at 50% or greater against leprosy, and there was no evidence for lower protection against multibacillary (84%; 95% confidence interval 26% to 97%) than against paucibacillary (51%; 30% to 66%) disease. There was no statistically significant protection by BCG against tuberculosis in this population. These findings add to the evidence that BCG vaccines afford greater protection against leprosy than against tuberculosis.