Active surveillance of leprosy contacts in country with low prevalence rate.

For advanced control of leprosy in Pakistan where the World Health Organization leprosy elimination goal was achieved in 1996, we conducted surveillance of Mycobacterium leprae-seropositive patients and their contacts and drug resistant strains of M. leprae. We measured anti-PGL-I antibody level in sera from leprosy patients and their contacts for early detection of M. leprae infection. Out of 34 leprosy patients undergoing treatment, 4 lepromatous leprosy patients were antibody positive, and 6.8 to 23.7 percent of occupational or household contacts were seropositive. Furthermore, three cases (1.2%) had a high antibody titer. For surveillance of drug resistant strains of M. leprae, dapsone and rifampin were targeted. Four out of 18 polymerase chain reaction (PCR) positive samples had mutation in folP gene, and among 10 PCR positive samples, one had a mutation in the rpoB gene. These results indicate that serological analysis of patient contacts might be useful to find out high risk individuals, and there are M. leprae strains resistant to chemotherapeutic agents in Pakistan.

A second case of multidrug-resistant Mycobacterium leprae isolated from a Japanese patient with relapsed lepromatous leprosy.

Emergence of drug resistant strains of Mycobacterium leprae was reported soon after the introduction of dapsone (diamino-diphenyl sulphone, DDS) for leprosy treatment (6, 10, 11). Three cases of multidrug-resistant strains of M. leprae have been reported recently (2, 8, 9, 13). In order to prevent multiple drug resistant strains of M. leprae from developing, current leprosy control strategies are based on early detection of cases and treatment with multidrug therapy (MDT) as recommended by the World Health Organization (WHO). We report here the identification of a multidrug-resistant strain of M. leprae from a patient who received inadequate therapy for leprosy. The drug resistant profile of the isolated strain was confirmed by the mouse footpad method and the identification of mutations in genes previously shown to be associated with resistance to each drug was made.

Novel 33-kilodalton lipoprotein from Mycobacterium leprae.

A novel Mycobacterium leprae lipoprotein LpK (accession no. ML0603) was identified from the genomic database. The 1,116-bp open reading frame encodes a 371-amino-acid precursor protein with an N-terminal signal sequence and a consensus motif for lipid conjugation. Expression of the protein, LpK, in Escherichia coli revealed a 33-kDa protein, and metabolic labeling experiments and globomycin treatment proved that the protein was lipidated. Fractionation of M. leprae demonstrated that this lipoprotein was a membrane protein of M. leprae. The purified lipoprotein was found to induce production of interleukin-12 in human peripheral blood monocytes. The studies imply that M. leprae LpK is involved in protective immunity against leprosy and may be a candidate for vaccine design.

A Mycobacterium leprae isolate resistant to dapsone rifampin, ofloxacin and sparfloxacin

Mycobacterium leprae were isolated from a Japanese patient, and susceptibility to antileprosy drugs was examined by the mouse foot pad method. The isolate was susceptible to clofazimine and clarithromycin, and resistant to dapsone, rifampin, ofloxacin and sparfloxacin. Mutations were identified in the genes associated with resistance to these drugs. The risk of the emergence of leprosy with multidrug resistance is emphasized.

Mycobacterium leprae typing by genomic diversity and global distribution of genotypes

The genetic diversity and related global distribution of 51 Mycobacterium leprae isolates were studied. Isolates were obtained from leprosy patients from 12 geographically distinct regions of the world and two were obtained from nonhuman sources. Polymerase chain reaction (PCR) followed by DNA sequencing was performed targeting the rpoT gene of M. leprae. Isolates were classified into two groups based on the number of tandem repeats composed of 6 base pairs in the rpoT gene. Isolates from Japan (except Okinawa) and Korea belonged to one group, while those from Southeast Asian countries, Brazil, Haiti and Okinawa in Japan belonged to a second genotype. M. leprae obtained from two nonhuman sources (an armadillo and a mangabey monkey) revealed the latter genotype. These results demonstrate the genetic diversity of M. leprae and the related genotype-specific distribution in the world.

Dharmendra antigen but not integral M. leprae is an efficient inducer of immunostimulant cytokine production by human monocytes, and M. leprae lipids inhibit the cytokine production

Killed integral Mycobacterium leprae, Mitsuda antigen, and chloroform-treated M. leprae, Dharmendra antigen (Dh-Ag), have been used for the classification of leprosy patients based on cell-mediated immunity. Heat-killed M. leprae also were used as a component of the Convit vaccine. Human blood monocytes were stimulated with M. leprae or Dh-Ag and their cytokine-inducing ability was compared. Monocytes were cultured in the presence of fresh human serum because of the efficiency of cytokine induction and the phagocytosis of M. leprae have been shown to be optimal in the presence of fresh serum. M. leprae and Dh-Ag were equally phagocytosed by monocytes. Dh-Ag was more potent than M. leprae in the induction of immunostimulatory/proinflammatory cytokines, interleukin-1 (IL-1), IL-6 and tumor necrosis factor (TNF). In contrast, a comparable level of IL-1ra, an immunosuppressive cytokine, was induced by M. leprae and Dh-Ag. The lipids extracted from M. leprae induced none of these cytokines by monocytes. Nevertheless, when monocytes were pretreated with the lipids followed by stimulation with Dh-Ag, productions of IL-1, IL-6 and TNF were all inhibited in a dose-dependent manner. However, the lipids did not inhibit the cytokine production induced by other stimuli including BCG and lipopolysaccharide. Moreover the lipids did not affect the production of IL-1ra. These results suggest that the lipids from M. leprae are responsible for the poor cytokine-inducing ability of M. leprae, thus favoring their infection. These results also suggest that Dh-Ag rather than integral M. leprae may be useful as a vaccine candidate because Dh-Ag is able to induce a large amount of cytokines from monocytes.

Phospholipid deacylating activities in murine leprosy bacilli

1) The particulate fraction of cultivated murine leprosy bacilli (Mycobacterium lepraemurium, rough colonies of the Hawaiian-Ogawa strain) contained phospholipid deacylating activities with acidic pH optima. It hydrolyzed phosphatidylcholine and phosphatidylethanolamine at similar rates, and phosphatidylinositol oligomannosides more slowly. It also hydrolyzed 1-acyl- and 2-acyl-GPCs (sn-glycerol 3-phosphocholine) more rapidly than phosphatidylcholine. 2) Ca2+ did not stimulate either diacyl- or monoacyl-hydrolase activity. Triton X-100 and Emulgen 913 had little influence on the hydrolysis of phosphatidylcholine, but at rather high concentrations inhibited the hydrolyses of 1-acyl- and 2-acyl-GPCs. Iron ions strongly inhibited the hydrolysis of phosphatidylcholine, but caused little or no inhibition of the deacylations of 1-acyl- and 2-acyl-GPCs. 3) With 1-[stearoyl-14C]phosphatidylcholine and 2-[oleoyl-14C]phosphatidylcholine as substrates, both labeled fatty acid and lysophosphatidylcholine were produced. Labeled fatty acid appeared more rapidly from 2-[oleoyl-14C]phosphatidylcholine than labeled lysophosphatidylcholine, while labeled lysophosphatidylcholine was produced more than labeled fatty acid from 1-[stearoyl-14C]phosphatidylcholine in the early stage of incubation.