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.

IL-18 induction of IgE: dependence on CD4+ T cells, IL-4 and STAT6.

Overproduction of immunoglobulin E (IgE) and T helper cell type 2 (TH2) cytokines, including interleukin 4 (IL-4), IL-5 and IL-13, can result in allergic disorders. Although it is known that IL-4 is critical to the polarization of naïve CD4+ T cells to a TH2 phenotype, both in vitro and in many in vivo systems, other factors that regulate in vivo IL-4 production and TH2 commitment are poorly understood. IL-18, an IL-1-like cytokine that requires cleavage with caspase-1 to become active, was found to increase IgE production in a CD4+ T cells-, IL-4- and STAT6-dependent fashion. IL-18 and T cell receptor-mediated stimulation could induce naïve CD4+ T cells to develop into IL-4-producing cells in vitro. Thus, caspase-1 and IL-18 may be critical in regulation of IgE production in vivo, providing a potential therapeutic target for allergic disorders.

An epidemiological study on Mycobacterium leprae infection and prevalence of leprosy in endemic villages by molecular biological technique.

One of the most important unsolved questions in epidemiology of leprosy is the highly uneven geographic distribution of the disease. There are many hyperendemic "pockets" in endemic countries. Little is known about the reasons why leprosy is hyperendemic in these areas. We conducted, therefore, a series of epidemiological studies on Mycobacterium leprae infection and prevalence of leprosy in North Maluku district, Maluku Province, Indonesia where leprosy is highly endemic. It was found that considerable number of general inhabitants are seropositive to various mycobacterial antigens and 27% of the villagers were carrying leprosy bacilli on their surface of nasal cavity. These results suggested the importance of M. leprae in the residential environment in infection of the leprosy bacillus and the resulting transmission of the disease. Based on these observations, we conclude that new preventive measures are essential for global elimination of leprosy in addition to early diagnosis and multidrug therapy (MDT).

Mycobacterium leprae DNA in daily using water as a possible source of leprosy infection.

Some environmental factors were suspected to be sources of leprosy infection according to the results of total survey in the highly endemic villages in Indonesia. M. leprae DNA were detected by PCR from 21 out of 44 water sources used daily by villagers. Prevalence of leprosy among the people using PCR-positive water for bathing and washing was significantly higher than that among the people who used PCR-negative water. No significant difference in prevalence was, however, recognized in case of usage of negative or positive water for drinking. Water was regarded as a reservoir and infectious source of M. leprae. Transmission of leprosy through the contaminated water was strongly suggested by epidemiological analysis.

Serodiagnosis of Hansen's disease/leprosy by enzyme-linked immunosorbent assay using cord factor (trehalose 6,6'-dimycolate) as an antigen.

IgG and/or IgM antibodies against mycobacterial cord factor (trehalose 6,6'-dimycolate, TDM) in sera of 65 patients of Hansen's disease (21 cases with smear-positive and 44 cases with smear-negative) and 60 healthy individuals were tested by enzyme-linked immunosorbent assay (ELISA) with TDM purified from Mycobacterium tuberculosis H37Rv as an antigen. Of 65 patients with Hansen's disease, 58 cases (89.2%) had positive results (21 samples from 21 patients, 100% with acid-fast bacilli positive in the lesion, and 37 samples from 44 patients, 84.0% with acid-fast bacilli negative Hansen's disease diagnosed clinically). The sensitivity and specificity of anti-cord factor ELISA were higher than those of anti-phenolic glycolipid-I (PGL-I) agglutination test. Among the total, 34 patients were classified clinically into three types of the disease, lepromatous leprosy (LL), borderline lepromatous (BL) and borderline tuberculoid (BT). The antibody titer showed LL > BL > BT, indicating that the elevation of anti-cord factor antibody titers appeared to be parallel with the degree of humoral immune response against M. leprae. By using semisynthetic cord factor consisting of a single subclass of mycolic acid from M. tuberculosis, it was revealed that sera from patients with Hansen's disease were highly reactive against alpha-mycoloyl cord factor (alpha-TDM) and less reactive against methoxy mycoloyl TDM (methoxy TDM), differed from sera of tuberculosis patients, which were highly reactive against both methoxy and alpha-mycoloyl cord factor (alpha-TDM). Most of sera from patients with Hansen's disease were more reactive against TMM than TDM, differed from sera of tuberculosis patients which were highly reactive against TDM. ELISA using TDM as an antigen is simple, reproducible and useful for the rapid serodiagnosis of Hansen's disease, especially for smear-negative cases.

Two-dimensional electrophoretic analysis of humoral responses to culture filtrate of Mycobacterium bovis BCG in patients with leprosy and tuberculosis.

Sera from 3 lepromatous (LL), 3 borderline lepromatous (BL), 3 mid-borderline (BB), 3 borderline tuberculoid (BT), 2 tuberculoid (TT) and 4 tuberculosis (TB) patients and 3 healthy individuals were examined for their reactivities against the proteins in the culture filtrate of BCG separated by two-dimensional electrophoresis (2-DE). The sera were obtained from patients who were untreated. Sera from LL and BL patients reacted strongly with the antigen 85 (Ag85) complex and MPB51. Sera from LL and BL patients also weakly reacted with the newly identified 29-, 24- and 23-kDa spots. Sera from the other patients reacted similarly, but the levels of reaction were remarkably lower than those form LL and BL patients. Mycobacterium leprae antigens that are analogous to BCG AG85 and MPB51 are suggested as the main targets for the humoral immunity of untreated patients. The reactivities of sera with newly identified antigens may provide the potential for predicting the severity and prognosis of diseases.

Alpha-1-antichymotrypsin is not associated with the increased frequency of apolipoprotein-E-epsilon-4 allele in elderly non-demented leprosy patients.

In our previous study, elderly leprosy patients showed a low prevalence of senile dementia of the Alzheimer type, but the frequency of apolipoprotein E (APO-E) epsilon 4 was elevated in non-demented elderly leprosy patients. Recent study has shown that Alzheimer's disease risk associated with APO-E epsilon 4 is significantly increased by the alpha 1-antichymotrypsin (ACT) genotype AA. Therefore we examined an association between ACT polymorphism and the APO-E epsilon 4 allele in 350 leprosy patients. None of our data showed an association of ACT genotype and APO-E epsilon 4 allele in leprosy patients. The allelic frequencies of the ACT gene did not differ even between demented patients with leprosy and age-matched controls. Our present data suggest that ACT polymorphism is not associated with the increased frequency of APO-E epsilon 4 in leprosy patients.

[Present situation of leprosy in highly endemic area of tropical Asia--a seroepidemiological study of Mycobacterium leprae infection in general inhabitants].

One of the most important unsolved problems in epidemiology of leprosy is the heterogeneous geographic distribution of the disease. There are highly endemic area called "Pocket" in the endemic countries. Little is known why leprosy is so endemic in the area. We conducted, therefore, an epidemiological study on M. leprae infection and distribution of leprosy bacilli in the environment by using serological and molecular biological techniques. It was found that considerable number of general inhabitants in the pocket are infected with leprosy bacilli and more than 20% of the villagers are carrying M. leprae on the surface of the nasal cavity; suggesting that leprosy bacilli in the residential environment play an important role in high prevalence of leprosy in the endemic area. New preventive measures such as chemoprophylaxis, in addition to MDT, will be needed for global elimination of the disease.

Transepidermal elimination of lepromatous granuloma: a mechanism for mass transport of viable bacilli.

A 35-year-old male with lepromatous leprosy showed significant progression of the disease on initial examination. Along with typical lepromatous skin lesions, many scar-forming lesions were present, mainly on his extremities. Some lesions showed erosive surfaces. From clinicopathological findings, these lesions were suspected to be due to the partial excretion of intradermal lepromatous granulomata by 'transepidermal elimination'. Increased local volume, which might be due mainly to rapidly growing lepromatous infiltration before chemotherapy, is suspected of triggering this phenomenon. There is no doubt that many fresh Mycobacterium leprae were included in these excretions. After the initiation of chemotherapy, no new scar-forming lesions were observed.

A serologic Mycobacteria leprae gelatin particle agglutination (MLPA) in the diagnosis of leprosy: comparison with conventional enzyme-linked immunoassay and bacterial index.

A gelatin particle agglutination test (MLPA) for the detection of anti-phenolic glycolipid-1 (PGL-1) antibodies was compared with the slit skin smear method in the diagnosis of leprosy. MLPA and BI tests showed a good agreement rate of 88.1% and MLPA and ELISA tests showed an excellent agreement rate 96.2%. This MLPA test is simple and reliable, it will be very convenient for the medical practitioners, it would be of great benefit for leprosy patient as well because this test would look like a routine blood examination compared with slit skin smear method which is widely known diagnostic tool for leprosy.

Significantly increased frequency of the apolipoprotein E epsilon 4 allele in elderly non-demented leprosy patients.

Apolipoprotein E (apo E) genotypes in 350 leprosy patients were examined and compared with those of 870 age-matched controls. The allelic frequencies of the apo E gene did not differ between demented patients with leprosy and controls. However, the frequency of apo E epsilon 4 allele was significantly higher in non-demented leprosy patients than in controls (P < 0.001). Of special interest is that the prevalence of E3/4 genotype in non-demented leprosy patients increased significantly with age, being 14.1%, 24.4%, and 28.3% in the 60s, 70s, and 80s, respectively (P < 0.05). These data suggest that apo E epsilon 4 is not a risk factor for senile dementia in elderly leprosy patients, and there exist factors to overcome the risk of apo E4 in leprosy patients.

Evaluation of the Mycobacterium leprae particle agglutination (MLPA) test as a tool in the epidemiology of leprosy in high prevalence village in South Sulawesi, Indonesia.

This study reports on the usefulness of an IgM phenolic glycolipid-1 (PGL-1) Mycobacterium leprae particle agglutination (MLPA) test for serodiagnosis of leprosy in a prospective longitudinal community survey in a high prevalence village in South Sulawesi, Indonesia, IgM PGL-1 MLPA has simple and limited value as a screening method for detection of transmission of leprosy in the community. Many normal persons in the community in a high prevalence area had increased IgM anti PGL-1 antibodies, presumably as a consequence of early subclinical infection. In conclusion, this study demonstrated that the MLPA test can only reliably detect anti-PGL-1 antibodies in the community and may be useful for follow-up study of subclinical infection of leprosy among individuals living the an endemic area.

[Uveitis in leprosy patients].

We examined 24 dermatologically cured leprosy patients with ongoing uveitis (UV+) and 22 age and type matched controls (UV-) to study the late phase leprous UV. All patients have been skin smear negative for more than 10 years. The history of chemotherapy, 5 years before and after a accomplishing bacterial negativity, was evaluated and represented by "SCORE". It was found that anti-PGL-I and anti-LAM-B antibodies were significantly higher in UV+ group compared to the controls. The mean SCORE of chemotherapy in UV+ group was significantly lower than in the controls. Iris pearls were seen in 10 cases or 42% out of 24 UV+ patients. No iris pearls were seen in control group. These results suggest that insufficient chemotherapy and consequent incomplete elimination of bacilli are the risk factors for leprous UV in the quiescent stage of the disease.

Decreased beta-amyloid and increased abnormal Tau deposition in the brain of aged patients with leprosy.

We examined the brains of 37 leprosy patients (mean age, 76.3 +/- 7.8 years), 5 patients with Alzheimer-type dementia (mean age, 79.0 +/- 9.5 years), and 23 age-matched non-dementia controls (mean age, 77.6 +/- 5.4 years). The frequency of beta-amyloid (A beta)-positive cases was lower (27.0%) in leprosy patients (n = 37) than in controls (47.8%; P = 0.05, Z = 1.49). When senile plaque subtypes were examined, type III (classical) plaques were significantly fewer (P < 0.05) in leprosy subjects compared with controls. Interestingly, neurofibrillary tangles in the temporal cortex were much more frequent in leprosy patients than in controls (P < 0.05). However, hippocampal CA3 pyramidal neurons in leprosy patients were well preserved. These data indicate that 1) leprosy patients have a low risk of A beta deposition but a high risk of abnormal tau deposition, 2) abnormal tau deposition is unrelated to A beta deposition in leprosy, and 3) neuronal loss is unrelated to abnormal tau deposition. It is not clear at present whether the result is related to the disease process itself, antileprosy treatment, environmental factors, or the genetic background in leprosy patients.

An epidemiological study of leprosy infection by serology and polymerase chain reaction.

A population-based study has been carried out in two adjacent villages in a highly leprosy-endemic area of South Sulawesi, Indonesia. The prevalence of clinical leprosy was 10.0 per 1000 inhabitants. A total of 1015 serum samples and 1228 nasal swab specimens were collected. IgM antibodies in blood to phenolic glycolipid-I (PGL-I) of Mycobacterium leprae were demonstrated by the gelatin particle agglutination test (MLPA) and by indirect ELISA (IgM-PGL). IgG antibodies to PGL-I (IgG-PGL) and lipoarabinomannan-B (IgG-LAM) were measured by indirect ELISA. The presence of M. leprae in nasal swab specimens was established by a polymerase chain reaction (PCR). The seropositivity rates in the population were 32% for MLPA, 30.8% for IgM-PGL, 6.7% for IgG-PGL, and 11.6% for IgG-LAM. Seropositivity rates for MLPA and IgM-PGL were highest in the younger age groups. There was no difference in seropositivity in any of the tests between household contacts of leprosy patients and noncontacts. The seropositivity rates in the MLPA and IgM-PGL were not randomly distributed among all households. The presence of M. leprae by PCR was demonstrated in 7.8% of the nasal swab specimens. No correlation was found between the results of the PCR and serology. This study indicates that M. leprae is widespread in the population, and that in endemic areas many individuals carry M. leprae in their nasal cavities without having obvious symptoms of leprosy.

Immunopathological stain of lipoarabinomannan-B (LAM-B) for diagnosis of leprosy.

We developed an immunopathological staining of LAM-B antigen in formalin-fixed paraffin-embedded tissues, and compared it with, PGL-I immunostaining, Fite Faraco's stain and periodic acid carbol pararosaniline (PACPR) stain. Out of the total 28 leprosy cases, 27 were positive to LAM-B immunostaining while 23 were positive to PGL-I stain. Fite's stain was positive in 21 cases while PACPR stain was positive in 24 cases. In scrofuloderma, LAM-B antigen was observed only in the granuloma while no other positive findings were noted with other stains. Normal skin did not give any positive findings with any of the stains. Other dermatoses showed no positive findings to any of the stains tested. LAM-B staining was observed in the nerve even in the absence of bacilli in leprosy tissues. Presence of LAM-B in the cutaneous nerves is helpful in discriminating leprosy from other mycobacterioses. Considering the high sensitivity of LAM-B and the predilection of M. leprae for the nerves, we concluded that LAM-B staining can be a useful new tool in the prompt diagnosis of leprosy, especially in suspected or early cases.

[Modification of Harada's method for rapid staining of mycobacteria].

Harada employed periodic acid-carbol pararosaniline and periodic acid-methenamine silver stain for demonstrating chromophobic bacilli which do not get stained with conventional carbol fuchsin or counter stain. This staining method takes considerable time for complete oxidation with periodic acid. We have succeeded in reducing the oxidation time by using hydrogen peroxide treatment prior to periodic acid and with the use of acidified sodium hydrogen sulfite treatment before carbol pararosaniline stain. We also found that in methenamine silver stain, combined use of semicarbazide and microwave treatment can shorten the whole staining time up to four hours without losing ito sensitivity.

Demonstration of PGL-I & LAM-B antigens in paraffin sections of leprosy skin lesions.

An investigation on the demonstration of PGL-I and LAM-B antigens in thirty-four paraffin embedded skin biopsies taken from leprosy patients who covered the whole spectrum of the disease and in four control specimens was carried out. Neither the PGL-I antigen nor the LAM-B antigen was demonstrated in the normal skin specimens that were used as negative control; and only the LAM-B antigen appeared in the tuberculosis specimens in which the PGL-I antigen was negative. The PGL-I antigen was demonstrated on thirty-three leprosy samples except one TT sample and the LAM-B antigen, on all samples by immunochemical staining technique. The antigens were identified as intracytoplasmic bacillary staining, in solitary, granular as well as debris patterns; and as soluble antigenic staining, in vacuolar or amorphous pattern. In LL and BL cases, the antigens were detected predominantly from macrophages and peripheral nerves in all five staining patterns; in BB cases, from macrophages mostly in the granular as well as debris patterns, from the nerves in the vacuolar pattern; while in TT and the majority of BT cases, they were mainly from nerve remnants inside the granuloma in the vacuolar or amorphous staining pattern. In addition, it is interesting to note that the immunochemical staining was able to differentiate the foamy change from the hydropic degeneration. We also found that the antigens distributed in arrector pili muscles and the walls of muscular vessels were obviously related to the unmyelinated nerve fibers innervating the smooth muscle cells.(ABSTRACT TRUNCATED AT 250 WORDS)