A polymerase chain reaction (PCR) using heat-stable Taq polymerase is described for the specific detection of Mycobacterium leprae, the causative agent of leprosy. A set of primers was selected on the basis of the nucleotide sequence of a gene encoding the 36 kDa antigen of M. leprae. With this set of primers in the PCR, M. leprae could be detected specifically with a detection limit approximating one bacterium. This PCR appears to meet the criteria of specificity and sensitivity required for a useful tool in epidemiology and eventually for the control of leprosy.

Leprosy displays a remarkable spectrum of symptoms correlating with the T-cell-mediated immune reactivity of the host against the causative organism, Mycobacterium leprae. At one pole of this spectrum are lepromatous leprosy patients showing a M. leprae-specific T-cell unresponsiveness; at the other are tuberculoid leprosy patients displaying both acquired immunity and delayed-type hypersensitivity against M. leprae which are thought to be conferred by helper T (Th) cells. Because well-defined M. leprae antigens are crucial for the prevention and control of leprosy, we have cloned M. leprae-reactive T cells (TLC) of the helper phenotype from a tuberculoid leprosy patient. As reported here, these TLC show an unexpected diversity in the recognition of M. leprae and related mycobacteria, which is different from that exhibited by monoclonal antibodies. Half of these TLC are completely or almost M. leprae-specific, whereas the other half are cross-reactive with most or all other mycobacteria. A M. leprae protein of relative molecular mass (Mr) 36,000 (36K) defined by a M. leprae-specific monoclonal antibody stimulates 4 out of 6 TLC tested. Each of these TLC recognizes a different antigenic determinant, one of which is M. leprae-specific. The previous paper describes other M. leprae-specific T-cell clones half of which recognize an epitope on a M. leprae protein of Mr 18 K.

In order to understand better the relationship among Mycobacterium leprae, its transmission and the human host or the chain of infection which may lead to the development of leprosy, we performed a population survey in which nasal carriage of M. leprae was determined by a specific polymerase chain reaction (PCR), 2 years after an earlier survey in the same population. 1923 persons were registered, 1171 were clinically examined for signs of leprosy, and 418 were tested by PCR. The detection rate of leprosy in the study area had not changed significantly during the 2 years' observation period since the introduction of multi-drug therapy, i.e. 6/1000 compared to 7.7/1000 2 years before. Of 6 newly detected cases, 5 were diagnosed as having paucibacillary leprosy. The presence of M. leprae could be demonstrated by PCR in 2.9% (12/418) of the persons. PCR positivity was not persistent over the 2 years. All the PCR positive persons identified in the first survey were negative in the second, indicating that M. leprae nasal carriage is transient. As in the previous survey, we found evidence for widespread M. leprae nasal carriage as determined by PCR among the general population in an area in which leprosy is endemic. In addition, our data indicated that PCR positivity can occur in certain clusters in the community. This clustering seems to be time-dependent, not necessarily related to the presence of patients.

Leprosy is still a health problem in many countries. Because the causative organism, Mycobacterium leprae cannot be cultured in vitro, it is virtually impossible to assess exposure, and the onset of infection and disease. As a consequence, the chain of infection, considered as the relationships between M. leprae, transmission and human host, is poorly understood. Here, we discuss a number of organism-, host- and environmental-related factors which may be incriminated in the dynamic process of the development of leprosy disease. The use of modern molecular and immunological tools has become a valuable addition to epidemiological research. Understanding of the epidemiology of leprosy is a prerequisite for effective control of the disease.

Summary Recent advances in treatment have achieved a large drop in the prevalence of active leprosy cases, but the incidence is at best decreasing slowly. Most people within leprosy-endemic populations have been exposed to Myco bacterium leprae, but few develop disease and it seems likely that the majority of the population develops protective immunity. If the site of initial infection is in the nose, dissemination of bacilli around the body to skin and nerve implies that the initial infection is bacilliferous and it has been shown that nasal M. leprae are detectable by polymerase chain reaction (PCR) of nasal swabs. Since salivary anti-M. leprae IgA (sMLIgA) levels are correlated with protection, 5 we have surveyed groups ofleprosy patients, contacts and the general population for both their sMLIgA and nasal PCR positivity. A total of 304 subjects were enrolled in the study: PCR and mucosal challenge tests were performed in 204 of these individuals. sMLIgA was present in 66% of treated patients, 76% of leprosy workers and 72% of healthy contacts. However, only 33% of indigenous subjects were sMLIgA+, in contrast to the earlier studies showing 74% positivity. 5 PCR for M. leprae was present in both household contacts (2%) and indigenous controls (5%). In a subsequent fo llow-up study, nasal swabs were taken from 97 of those studied in the first series: three PCR+ individuals followed up after one year became negative, while of the remaining 94 PCR-individuals retested, 2

The polymerase chain reaction based on the selective amplification of a 531-bp fragment of the gene encoding the proline-rich antigen of Mycobacterium kprae was applied to nasal swab specimens from leprosy patients, occupational contacts, and endemic and nonendemic controls. To prevent false-positive amplification, we used dUTP and uracil-DNA-glycosylase in all polymerase chain reactions. False-negative reactions were detected by using a 531-bp modified template as an internal control. Amplification products were found in 55% of untreated patients, in 19%o of the occupational contacts, in 12% of endemic controls, and in none of the nonendemic controls. This study strongly suggests that not only leprosy patients but also healthy persons may carry M. leprae. We concluded that polymerase chain reaction is a reliable method to detect M. leprae in nasal specimens. The method holds promise for studying the spread and transmission of M. leprae within a population.

In order to better understand the role ofMycobacterium leprae nasal carriage in the maintenance of infection reservoirs and transmission of leprosy, we applied a polymerase chain reaction (PCR) that detected a 531-bp fragment of thepra gene ofM. leprae on nasal swab specimens collected through a total population survey from individuals living in an area in which leprosy is endemic. Among the total tested population of 1,228 people, 7.8% were found to be PCR positive. PCR positivity was shown to be randomly distributed among the population for which leprosy is endemic. No association was observed between PCR positivity, age, or sex. The observed distribution of PCR positivity among households of different sizes confirmed the expected values, with the exception of two households, each with three people with PCR-positive nasal swab specimens. Although nasal carriage does not necessarily imply infection or excretion of bacilli, the finding of nasal carriage supports the theory of a disseminated occurrence of M. leprae in populations for which leprosy is endemic.

To test for genotypic variations between different isolates of Mycobacterium leprae, the causative agent of leprosy, the 282 bp spacer region between the 165 and 235 rRNA genes was amplified using PCR, and submitted to singlestrand conformation polymorphism (SSCP) analysis. The procedure was optimized using four modified spacer fragments, containing mutations at one, three, four and six positions, respectively. Seventy-five M. leprae isolates from different sources, including isolates from leprosy patients, healthy individuals, armadillos and mouse footpads were identical in the SSCP analysis. DNA sequencing and restriction enzyme analysis performed on four and 40 samples, respectively, confirmed the results obtained with SSCP analysis.