Comparative evaluation of enzyme immunoassays based on synthetic glycoconjugates and phenolic glycolipid-I for immunodiagnosis of leprosy

Enzyme immunoassays (EIAs) based on synthetic glycoconjugates containing the terminal monosaccharide (M-BGG) or disaccharide (ND-BSA) residue of the trisaccharide component of phenolic glycolipid-I (PGL-I), for immunodiagnosis of leprosy are described. The results of the assays were compared with that of the EIA using PGL-I. All the three assays were highly specific for leprosy. The per cent positivity of active lepromatous leprosy (LL) patients with M-BGG was 78.05 in comparison to 85.36 with ND-BSA and 82.11 with PGL-I. Similarly, the positivity of tuberculoid (TT) leprosy patients in M-BGG assay was lower than that in EIAs using ND-BSA or PGL-I. However, the difference in the positivity of individual category of leprosy patients in the three EIAs was not statistically significant. The correlation between absorbance values of leprosy sera in EIAs based on M-BGG and PGL-I, as well as that in assays using ND-BSA and PGL-I was statistically significant.     

An appraisal of enzyme linked immunosorbent assay (ELISA) and serum antibody competition test (SACT) in leprosy.

Seventy-eight untreated leprosy patients, 104 treated patients and 105 healthy contacts were tested using two serological tests, SACT (serum antibody competition test based on competitive inhibition of monoclonal antibody binding to the MY2a determinant of M. leprae) and ELISA (measurement of IgM antibodies to the neoglycoproteins D-BSA and ND-BSA representing the phenolic-glycolipid antigen of M. leprae). The controls included normal healthy individuals, patients with sputum positive pulmonary tuberculosis, and active cases of rheumatoid arthritis from the department of rheumatology. The specificity of SACT was found to be very high. ELISA was found to be positive in two patients with rheumatoid arthritis, one each for D-BSA and ND-BSA ELISA. Both tests had a high sensitivity in BL and lepromatous patients. The sensitivity to both tests was considerably lower in tuberculoid and BT patients i.e., below 40%. Therefore the diagnostic value of a negative test in suspected cases of leprosy was very low employing either of the two tests. A proportion of patients with paucibacillary tuberculoid and BT leprosy were positive after six months or longer after therapy. Similarly a large number of BL and lepromatous patients were positive after considerably longer periods of treatment. The use of either tests for determining the duration of therapy is therefore limited. SACT appears to be more sensitive than ELISA with ND-BSA in detecting subclinical infection. The cumulative positivity of the two tests may be used as a measure of the infectivity of the disease in the community and for evaluating disease control methods.     

Association of mycobacterial-specific and Mycobacterium leprae specific antibody levels with clinical activity in tuberculoid leprosy: a comparative study of three serological enzyme-immunoassays

The ELISAs for polyclonal antibodies against Mycobacterium leprae (ML-ELISA) and specific antibodies against epitopes on 35 kDa protein (SACT-ELISA) and phenolic glycolipid I (PG-ELISA) of M. leprae were evaluated comparatively in a group of 88 tuberculoid leprosy patients. The overall seropositivity rate with a battery of 3 tests (68%) was not significantly higher than that obtained with ML-ELISA alone (55%) for IgG class of antibodies. Seropositivities for SACT-ELISA and PG-ELISA were, respectively, 38% and 26%. ML-ELISA for IgM class of antibodies was least sensitive, showing only 8% positivity. A significant correlation was noted between individual values of the three assays, but the positive proportions overlapped maximally in the case of ML-ELISA (IgG) and SACT-ELISA. Further, positivity for the latter two assays, particularly SACT-ELISA, showed significant associations with the extent of 'active' (largely untreated) infection. Immunoblotting revealed that the main antibody response was directed towards M. leprae antigens in the molecular weight range of 20-40 kDa and the densitometry results of this zone correlated significantly with corresponding SACT-ELISA and ML-ELISA (IgG) values.     

Use of non-conventional antigen: M. habana in detecting M. leprae antibodies from leprosy patients and contacts in FLA-ABS test

An indirect immunofluorescent (FLA-ABS) test has been developed to detect M. leprae specific antibodies in the active and subclinical cases of leprosy. An antigenically related mycobacterium, M. habana, was used as an antigen to detect M. leprae specific antibodies in the sera samples of leprosy patients. A comparison was made with M. leprae antigen using same set of sera samples. M. habana is capable of detecting anti-M. leprae antibodies in the serum samples of leprosy patients, previously absorbed with various mycobacterial antigens, cardiolipin and lecithin, almost to the same percentage as M. leprae. Possible use of M. habana antigen as an alternative to M. leprae, in the serodiagnosis of leprosy, has been discussed.     

Anti-arabinogalactan IgM/IgG ratio: a screening index for leprosy patients

The serological activities of arabinogalactan from M. smegmatis and phenolic glycolipid-1 from M. leprae were examined by Enzyme Linked Immunosorbent Assay using sera from 88 patients with leprosy (44 treated and 44 untreated) and 45 normal healthy individuals. Both IgM and IgG type of antibodies were measured against these antigens. The results confirmed the previous observation that anti phenolic-glycolipid-1 IgM antibodies are higher in lepromatous leprosy cases than in normal individuals. However, with arabinogalactan, the ratio of IgM/IgG was more than one in normal individuals and less than one in untreated LL patients. Treated patients fell in both categories. Moreover, a reverse relationship was found between anti PGL-1 IgM titers and anti arabinogalactan IgM/IgG ratio.     

Development of a mouse food pad model for detection of sub clinical leprosy

Early diagnosis of leprosy and a multi-drug therapy (MDT) regimen will block the trajectory of nerve damage, disability and deformity that are the hallmarks of this chronic disease. However, the diagnosis of leprosy is made solely by recognition of clinical signs and symptoms, requiring special expertise. These limitations also result in the under reporting of worldwide prevalence and incidence rates for leprosy. Sorely needed is an objective laboratory test for detecting early leprosy. As the antigenic burden of M. leprae can be virtually undetectable in early clinical leprosy, cell mediated immunity and antibody responses will likely be weak. So the sensitivity of new diagnostic tests is as important as specificity. Major efforts are underway employing recombinant M. leprae antigens and synthetic peptides, to develop diagnostic assays for early leprosy infection, using in vitro T cell reactivity or serological tests. We have used the initial phase of the mouse foot pad model as an 'early' model of leprosy infection to screen T cell responses against M. leprae specific antigens and synthetic peptides. Unlike human disease in animal models we can control infection progress and monitor bacillary growth relative to time course of development of T cell response to specific M. leprae antigens. The study employed splenic T cells instead of draining lymph node T cells to model the systemic response as opposed to a local one. We found that 10(5) live M. leprae is the minimum dose required for any meaningful and consistent in vitro splenic IFN-gamma response against M. leprae antigens 3 months after foot pad inoculation. Using this model we found that several M. leprae recombinant proteins, ML0840, ML2028, ML2307, ML2346, ML2478, and ML2532, induced significant levels of IFN-gamma secretion. By controlling for variables that can be confounding factors in the sensitivity of human testing, this mouse model provides an interface between M. leprae diagnostic antigen development and the screening of these antigens in humans under field conditions.     

Comparison of PCR mediated amplification of DNA and the classical methods for detection of Mycobacterium leprae in different types of clinical samples in leprosy patients and contacts

Traditional staining and microscopic examination techniques for the detection of Mycobacterium leprae, DNA amplification by polymerase chain reaction (PCR) of a 531-bp fragment of the M. leprae specific gene encoding the 36-kDa antigen, and serodiagnosis with M. leprae specific antigens (PGL-1 and D-BSA) were compared on different clinical specimens (serum samples, slit-skin smears, biopsies and swabs) from 60 leprosy patients attending the Sanatorium of Fontilles. Patients were divided into groups; (i) 20 multibacillary patients (MB) with positive bacteriological index (BI) by conventional methods and on WHO multidrug therapy (MDT); (ii) 30 MB patients with negative BI and completed minimum 2 years treatment MDT; (iii) 10 paucibacillary (PB) patients who had completed 6 months MDT at least 8 years ago. Control groups included four non-leprosy patients for PCR methods and 40 health control patients and 10 tuberculosis patients for serological methods. In the multibacillary BI positive group, there was a good correlation between all methods. All tests were negative in the paucibacillary group, although only a few patients were tested and all had been treated many years ago. One must be cautious concerning the diagnostic potential of these techniques in this type of leprosy. We also studied different combinations of leprosy diagnosis methods to determine the potential risk in a leprosy contact individuals group. The prevalence of antibodies to M. leprae antigens in serum was measured, together with the presence of M. leprae DNA in the nose and lepromin status in a group of 43 contacts of leprosy patients (12 household and 31 occupational) to evaluate the maintenance of infection reservoirs and transmission of the disease. Only two individuals were found to form a potential high risk group.     

鼻分泌物及皮肤组织中麻风菌及其PGL-1抗原的检测

为了更好地理解麻风菌鼻携带在麻风病传播、维持中的作用，以及运用鼻携带检测来评价麻风病防治效果，比较了PCR和Dot-ELISA／ECL平行检测32例活动性麻风患者、13例愈后者和143名麻风家内接触者鼻分泌物及皮肤组织中的麻风菌及其PGL-1抗原。结果显示，Dot-ELISA／ECL具有较好的敏感性、特异性，是一项适用于现场研究的简便、快速、经济的麻风流行病学工具。此外，用于免疫学试验，GVHP是一种吸附性好，适合于检测粘膜分泌物抗原的载体。     

M. leprae recombinant antigens important for T-cell reactivity.

Identification of M. leprae antigens recognized by T-cell is important for specific diagnosis, vaccine development and understanding the basic mechanisms involved in protection against and pathogenesis of leprosy. Screening of an M. leprae recombinant DNA library with antibody probes led to the identification of half a dozen M. leprae antigens recognized by B-cells. When tested for T-cell reactivity, all the antigens recognized by antibodies were shown to have T-cell reactivity. However, among these antigens 18 kDa, 65 kDa and 70 kDa heat shock proteins (hsps) were most frequently recognized by T-cell lines and clones established from healthy donors vaccinated with killed M. leprae. A 24 kDa secreted antigen of M. leprae with T-cell epitope specific for M. leprae and M. tuberculosis complex was identified by direct screening of the recombinant DNA library with T-cell clones. The recombinant T-cell antigens of M. leprae were recognized by memory T-cells of Th1 type in association with multiple HLA-DR molecules. Epitope mapping with synthetic peptides identified M. leprae-specific as well as cross-reactive T-cell epitopes on the 18 kDa, 65 kDa and 70 kDa hsp antigens. In conclusion, our studies suggest that the recombinant antigens of M. leprae could be useful as reagents for specific diagnosis as well as in subunit and recombinant vaccine design against leprosy.     

Anti-Mycobacterium leprae monoclonal antibodies cross-react with human skin: an alternative explanation for the immune responses in leprosy.

A panel of 17 mouse monoclonal antibodies (MoAb) raised against Mycobacterium leprae (M. leprae) antigens was used to detect antigenic determinants in normal human skin. An indirect immunoperoxidase technique was used. Eight of the MoAb detected epidermal antigens similar to patterns well known for human sera. Five of these MoAb detected determinants in the dermis, too. These observations may indicate a certain degree of similarity between the antigenic determinants occurring in M. leprae and in the human host. We propose that such a similarity on the one hand may facilitate the survival of M. leprae in the human host when the antigens are not recognized as "non-self," a situation which seems to occur in lepromatous leprosy, when the patients' tissues are loaded with bacteria virtually without any immune response. On the other hand, M. leprae antigens which mimic host antigens may induce an auto-immune reaction against the host's own antigens, which could explain the immune reaction in tuberculoid leprosy and during a "reversal reaction" when M. leprae is not observed in the host tissues, but extensive granuloma formation occurs.     

Demonstration of Mycobacterium leprae specific antigens in leprosy lesions using monoclonal antibodies

Cryostat sections of dermal lesions from 13 untreated patients of leprosy were studied by indirect immunoperoxidase using monoclonal antibodies (MLO4 & MLO6), defining M. leprae specific antigens. The lymphocytes and macrophages in both the tuberculoid and lepromatous granulomas showed membranous staining with the above antibodies. M. leprae organisms in the lepromatous granulomas and the cells in the section of lymph nodes of patients with tuberculosis, or sections of normal skin or psoriatic lesions did not show any staining with these antibodies. These observations suggest that M. leprae specific antigens are present and expressed on the cells infiltrating the granulomas of leprosy lesions.     

Serum samples from patients with mycobacterial infections cross-react with HIV structural proteins Gp41, p55 and p18

BACKGROUND: Infection with Mycobacterium leprae is associated with a high frequency of false positive results in a variety of serological assays. Our studies have found cross-reactivity to HIV structural proteins in serum samples from leprosy patients, irrespective of the type of disease, treatment duration, age and gender and from a few patients with active TB disease. METHODS: Western blot (WB) analysis revealed that sera from HIV negative leprosy patients across the spectrum showed high reactivity with p18, Gp41 and p55 and lower reactivity with other HIV proteins. The reactivity appeared to be specific; western blot-positive samples were negative in ELISA and in several rapid tests for HIV. Cross-reactivity was not found in sera from patients with leishmaniasis or from normal healthy individuals. RESULTS: None of the WB reactive leprosy patients seroconverted to HIV positivity within 6 months to 1 year after Western blot testing. BLAST analysis revealed that envelope antigens of HIV (Gp41, Gp120 and Gp160) contained amino acid sequences similar to M. leprae ML0470, putative integral membrane protein, Rv0740, mmpL9 (M. tuberculosis). Core (gag) antigens (p18) had similarities to ML0406, but polymerase antigens (p52) had similarities to PE_PGRS (M. tuberculosis, H37Rv). Nucleotide sequence analysis, on the other hand, did not reveal any significant homology between M. leprae or M. tuberculosis and HIV. CONCLUSIONS: The occurrence of these high false-positive rates in M. leprae-infected individuals suggests a possible complication of serodiagnosis of HIV in regions where mycobacterial infections are endemic. There is a need for caution in reporting HIV infection among leprosy patients. Our observations emphasise the value of the various rapid assay kits for HIV, where this false positivity is not observed.     

Detection of Mycobacterium leprae DNA in skin lesions of leprosy patients by PCR may be affected by amplicon size

Despite the high sensitivity and specificity of PCR for infectious disease diagnostics, it has presented low sensitivity for Mycobacterium leprae DNA detection in the tuberculoid pole (TT and BT) of leprosy. In order to demonstrate the effect of amplicon size on the efficacy of PCR detection of M. leprae DNA in skin lesions of leprosy patients, two pairs of primers targeting the M. leprae genomic DNA, RLEP3 (X17153), were used to amplify fragments of 372 and 130-bp until their PCR end-points were reached after 40 reaction cycles. Skin biopsies of leprosy lesions in 110 non-treated patients were used for bacilloscopy index (BI) analysis and PCR tests. The 130-bp fragment was detected in 73.6% of samples (81/110), and classified as TT (40%), BT (55.5%), and 100% of BB, BL and LL. The 372-bp fragment was detected in 52.7% and classified as TT (13.3%), BT (33.3%), BB (64.7%), BL (83.3%), and LL (95.2%). The BI of biopsies was positive in 39.1% of samples, classified as TT (0%), BT (2.2%), BB (64.7%), BL (91.6%), and LL (95.2%). The shorter amplicon (130-bp) has improved diagnosis by 20.9 and 34.5% in relation to the 372-bp fragment and the BI, respectively, and has shown a superior sensitivity (73.6%), specificity (100%) and accuracy (86.2%). The 130-bp amplicon could not detect % of positive BI of biopsies in BT cases. Therefore, for confirmatory diagnosis, we propose the use of PCR detection of the 130-bp genomic target, especially when the tuberculoid pole forms are considered, which has reached 51.6% of positivity in this group.     

Risk factors for developing leprosy--a population-based cohort study in Indonesia

We identified risk factors associated with increased yearly incidence rates of leprosy in five island populations. Age, sex, household size and Mycobacterium leprae-specific antibodies as well as contact factors were studied. Of 94 index patients (patients diagnosed in 2000), 43 (46%) were classified as multibacillary (MB), 17 (19%) were seropositive for PGL-1 [corrected] antibodies and 6 (7%) had M. leprae DNA in nasal swabs as determined by polymerase chain reaction (PCR) testing. All PCR positive patients were also seropositive. Forty-four of 4903 initially symptom free persons developed leprosy within 4 years, giving an incidence rate of 298 per 1000 person-years. Men had a 22 times higher risk [95% confidence interval (CI): 1.2-4.1] of developing leprosy than women. People living in households with more than 7 members had a 3.1 times higher risk (95% CI: 1.3-7.3) than households of 1-4 members. Persons who were seropositive in 2000 had a 3.8 times higher risk (95% CI: 1.1-12.6) than seronegative persons. Household contacts of MB patients had an adjusted hazard ratio (aHR) of 4.6 (95% CI: 1.6-12.9) and household contacts of PCR positive patients an aHR of 9.36 (95% CI: 2.5-34.9) compared with non-contacts. Patients with PCR positive nasal swabs, suggesting nasal excretion of M. leprae, are probably the patients with the highest transmission potential. Since all index patients who were PCR positive were also seropositive, serology seems an adequate tool to identify these patients. Preventing seropositive persons from becoming seropositive and infectious patients might break the chain of transmission.     

Comparative study of anti-PGL-1, anti-35 kDa and anti-lipoarabinomannan assays for serodiagnosis of leprosy

Three antibody assays (anti-PGL-1, anti-35 kDa and anti-LAM) were used to determine the levels of antibodies in the sera of untreated leprosy patients. All the three assays showed higher levels of antibodies in BL/LL patients as compared to I and TT/BT patients, as well as healthy controls. BL/LL patients showed positivity of 100%, 84.2% and 78.9% by anti-PGL-1, anti-35 kDa and anti-LAM assays respectively. All the three assays were negative for leprosy in healthy controls. Anti-PGL-1 assay was positive in 20% of TT/BT patients and 17.9% of I patients. Anti-35 kDa assay was negative in all the TT/BT patients and positive in 7.14% of I patients. Anti-LAM assay was positive in 13.3% of TT/BT patients and in 10.7% of I patients. Hence, while these assays are valuable in diagnosing BL/LL patients, their usefulness in diagnosing I, BT or TT leprosy is limited.     

A dot enzyme immunoassay for detection of IgM antibodies against phenolic glycolipid-I in sera from leprosy patients

A visual dipstick dot enzyme immunoassay (EIA) for diagnosis of leprosy is described. The assay is based on detection of IgM antibodies against phenolic glycolipid (PGL-I) in sera from leprosy patients. The antigen (PGL-I or synthetic disaccharide of PGL-I) was dotted on a nitrocellulose pad stuck on a plastic strip (dipstick). Sera were used at a dilution of 1:200. Peroxidase coupled mouse anti-human IgM monoclonal antibodies were used as the conjugate. A positive test gave a blue dot against a white background. The test was highly specific for leprosy, and was quite sensitive for detection of bacilliferous (BL/LL) leprosy. The antigen dotted and preblocked dipsticks stored at room temperature upto 4 months of observation period, were unable in the assay.     

Evaluation of four semi-synthetic Mycobacterium leprae antigens with sera from healthy populations in endemic and non-endemic areas.

In order to determine the frequency of occurrence of antibodies to semisynthetic antigens of Mycobacterium leprae in clinically healthy nonpatient populations and to establish a 'baseline' for comparison with antibody frequencies in both patients with a history of leprosy and their contacts, ELISAs were conducted using representative sera from two areas: a leprosy endemic area, Cebu City, Philippines and a nonendemic area for leprosy Chicago, Illinois, USA. These sera were tested, by an indirect IgM ELISA, for the presence of antibodies reacting with four semisynthetic antigens based on the phenolic glycolipid I antigen of M. leprae: ND-O-BSA (natural disaccharide with octyl linkage to bovine serum albumin), NT-O-BSA (natural trisaccharide with octyl linkage to BSA), ND-P-BSA (natural disaccharide with phenolic ring linkage to BSA) and NT-P-BSA (natural trisaccharide with phenolic ring linkage to BSA). Using an OD reading > or = 0.16 as positive, the antigen with the lowest background seroreactivity was ND-O-BSA, which reacted with 5/398 (1.3%) sera from Cebu, and 3/426 (0.7%) sera from Chicago. A total of 10 (2.5%) of 398 sera from the endemic area reacted with at least one antigen and 5 (1.3%) sera reacted with all four semisynthetic antigens. Of the 426 sera from Chicago, 12 (2.8%) were reactive with at least one antigen and 3 (0.7%) were reactive with all four semisynthetic antigens. Mean ELISA values for the 22 positive sera for each antigen ranged from 0.17 to 0.3 OD units, while the mean values for all sera in each area ranged from 0.01 to 0.04 OD units for all four antigens. Reactivity of 14 of the positive sera to some antigens, but not all four semisynthetic antigens, indicated that the carrier and linker arms might be associated with this background reactivity. Investigation of alternative linker arms and carriers is warranted. We conclude that nonspecific background reactivity to the semisynthetic antigens representing the PG-I molecule of M. leprae is 0.7-1.3%, based on a > or = 0.16 OD cutoff value. From these data it was concluded that reactivity in individuals free of leprosy was low enough to warrant use of these antigens in a diagnostic setting, such as screening household contacts and highly endemic populations. When incidence and prevalence of leprosy are low, testing with these antigens would not be cost effective, unless applied to high risk individuals.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mycobacterium leprae reactive T cell clones from lepromatous leprosy patients after prolonged dapsone chemotherapy

The proliferative responses of peripheral blood mononuclear cells (PBMC) to Mycobacterium leprae and BCG were studied in two groups of leprosy patients: a group of 8 lepromatous patients who had been on treatment for more than 20 years (TLL) and a group of 8 untreated lepromatous leprosy patients (ULL). The mean response to M. leprae of the TLL group was 6195 cpm with 5 of the 8 patients responding positively. The mean response to M. leprae of the ULL group was 617 cpm, with only 1 patient showing a positive response. The corresponding proliferative responses to BCG were 19,908 cpm in the TLL group and 7908 in the ULL group. Thirteen M. leprae reactive clones were established from 2 TLL patients and 5 M. leprae reactive clones were established from 2 tuberculoid leprosy patients. Seven of these clones, 4 from the TLL patients and 3 from the tuberculoid (TT) patients could be studied further. Three of the TLL clones responded specifically to M. leprae, while one of the clones exhibited a broad cross-reactivity to other mycobacteria. All of these clones were of the CD4+CD8- phenotype. Our findings suggest that responsiveness to M. leprae can be detected in vitro in a proportion of LL patients who have undergone prolonged chemotherapy, and that this response involves M. leprae reactive CD8+CD8- T cells, of which some appear to be specific to M. leprae.     

麻风皮损石蜡切片中酚糖脂抗原表达

用免疫组化染色技术检测了６７例各型麻风患者皮肤活检石蜡切片中酚糖脂（ＰＧＬ－Ｉ）抗原的表达，除２例结核样型外有６５例标本ＰＧＬ－Ｉ抗原呈阳性表达。麻风杆菌和有关的可溶性抗原染色在形态学上呈５种染色模式。在瘤型、偏瘤型和中间界线类麻风中，ＰＧＬ－Ｉ阳性与抗酸染色阳性基本一致。而在结核样型和偏结核样型麻风中，ＰＧＬ－Ｉ阳性高于抗酸染色。本研究结果表明免疫组化技术有较高的特异性和敏感性，为麻风病理学诊断和病因学研究提供了新方法。     

Presence of M. leprae in tissues in slit skin smear negative multibacillary (MB) patients after WHO-MBR

This study looked for M. leprae in the lymph node, nerve and skin of multibacillary (MB) leprosy patients who become slit skin smear negative after the completion of WHO-MBR. Twenty-five WHO-MBR-treated multibacillary leprosy patients were studied; borderline lepromatous (BL) leprosy (n = 11) and lepromatous (LL) leprosy (n = 14)). Fifteen patients had reaction (erythema nodosum leprosum 11, upgrading reaction 4) either at presentation or during therapy. All patients attained slit skin smear negativity after WHO-MBR (range 24-39 months. Sixteen (64%) patients with multibacillary leprosy showed fragmented bacilli in skin and nerve biopsy or lymph node aspirates after WHO-MBR. Lymph node aspirates alone revealed M. leprae in seven patients, followed by nerve in two and skin in one patient. Four cases showed M. leprae at all sites followed by nerve and skin or lymph node in one case each. A pretreatment bacteriological index (BI) of 4+ or more was significantly associated with the presence of M. leprae at the end of treatment. Also, significantly more lymph node aspirates contained M. leprae in comparison with nerve or skin biopsies. All seven cases in whom treatment was extended beyond 24 months showed M. leprae in tissues even after attaining slit smear negativity. In conclusion, M. leprae persist in tissues after 2 years of WHO-MBR and patients with an initial BI of 4+ or more need to be closely followed up after stopping MDT.