Subclinical infection by Mycobacterium leprae

International Journal of Leprosy and Other Mycobacterial Diseases, Dec 1999 by Izumi, S

Since the early 1990s, our research group has been conducting a series of epidemiological studies of leprosy in endemic pockets of Indonesia, the purposes of which were to attempt to explain why leprosy is so endemic in the area, and to collect epidemiological data that will be useful both for the global elimination of leprosy and for preventing the disease.

According to the World Health Organization (WHO), Indonesia is third highest on the list of the most leprosy-endemic countries in the world. In 1999, the number of registered cases is 29,225, with a prevalence rate of 1.41 per 10,000 population. In 1998, there were 15,337 new cases, yielding a new-case detection rate of 7.42 per 100,000 population. Leprosy is heterogeneously distributed in the country; the most endemic province is Maluku, and the majority of the patients in the province reside in North Maluku District, in the northern part of the province. We began a cohort study in the district in 1996, and have since conducted a series of epidemiologic surveys, employing serological and molecular techniques.

More than half of the healthy villagers demonstrated anti-mycobacterial antibodies, and about one-quarter of them appear to carry DNA molecules specific to Mycobacterium leprae on the surface of the nasal mucosa. We interpret these data to indicate the presence of a considerable number of M. leprae in the environment.

MATERIALS AND METHODS

Three typical agricultural villagesGamtala, Toboso, and Lolori-in North Maluku District were selected. Some demographic characteristics of the villagers are summarized in Table 1. In February 1997, the population of these three villages totaled 1417, and the mean age was 26 years. Villagers ranging in age from 5 to 60 years were selected for this study. To learn the incidence of leprosy and the predictive value of several tests, we surveyed the population of the villages twice-in February 1997 and October 1998.

Villagers were examined dermatologically by three well-trained Indonesian and Japanese specialists. Leprosy patients were classified as paucibacillary (PB) or multibacillary (MB) according to the criteria of the WHO.

One ml of venous blood was collected, and the serum, with NaN added as a preservative, was stored at -30'C until used. IgG and IgM anti-PGL-1 antibodies and IgG anti-LAM-B antibodies were measured by indirect ELISA. IgM anti-PGL-1 antibodies were also measured by a gelatinparticle agglutination test, using the Serodia-Leprae kit.

To examine the possibility that M. leprae in the environment of an endemic area play an important role in M. leprae infection of the populace, we employed a sensitive and specific technique-the nasal swab polymerase chain reaction (PCR) test. The surface of the nasal mucous membrane was swabbed with a wet, sterile cotton swab, and the material adhering to the cotton was removed by washing the swab in phosphate buffered saline (PBS) with Tween8011. The resulting suspension was centrifuged, and the sediment was treated by a lysis buffer containing proteinase-K to obtain the DNA template. An M. leprae-specific DNA fragment was then amplified by a minor modification of Plikytis' nested primer method.

RESULTS

In the course of the first survey, 936 villagers were examined and 24 new cases of leprosy-19 PB and 6 MB-were detected. Including the cases already registered, there was a total of 35 patients, yielding a prevalence of 3.7 percent. In the second survey, 861 villagers were examined, and 21 patients, 15 PB and 6 MB, were detected, yielding a prevalence of 2.4 percent (Table 2).

The incidence of leprosy in the villages was calculated by using the 644 villagers who attended both surveys as the denominator. As is also shown in Table 2, 13 healthy villagers developed leprosy during the 20 months between the two surveys, yielding an annual incidence of 1211 per 100,000 population at risk. Of the 1417 inhabitants of the villages, 342 were household contacts of patients with leprosy, suggesting that the disease affects an important proportion of the households in this area (Table 1).

Of the 885 healthy villagers studied, 304 (34.3 percent) demonstrated anti-PGL-I antibodies at the time of the first survey, and 31.4 percent were found to be seropositive at the time of the second survey. Moreover, it was found that the prevalence of antibodies did not change during the period of 20 months (Table 3). Of the populace, 54.1 percent demonstrated antibodies to PGL-I or LAM-B.

Of 890 swab samples examined, 237 were positive by PCR. This may suggest that a large number of M. leprae are floating in the air of an endemic pocket, which the people in the area breathe (Table 4).

Thirteen new cases were discovered in the course of the second survey. It was found that 11 of the 13 demonstrated at least one leprosy-related factor: six patients were household contacts; six were seropositive; three had suspicious skin lesions during the first survey; and one had an enlarged nerve at that time. However, not one of these factors appears to be useful for predicting who will develop clinical disease.