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Lepr Rev (2011) 82, 389– 401

Comparison of three immunological tests for leprosy
diagnosis and detection of subclinical infection
JANAINA LOBATO*, MARIANA PENA COSTA*,
E´ RICA DE MELO REIS*,
MARIA APARECIDA GONC¸ ALVES*,
JOHN S. SPENCER**, PATRICK J. BRENNAN**,
LUIZ RICARDO GOULART*,***
& ISABELA MARIA BERNARDES GOULART*
*Universidade Federal de Uberlaˆndia – Centro de Refereˆncia
Nacional em Hansenı´ase Av. Aspirante Mega, 77 – Bairro Jaragua´,
38413-018 – Uberlaˆndia, MG, Brazil
**Mycobacteria Research Laboratories, Department of
Microbiology, Immunology and Pathology, Colorado State
University, 1682 Campus Delivery, Fort Collins, CO 80523, USA
***Dept. Medical Microbiology & Immunology, Genome &
Biomedical Sciences Facility, GBSF Suite 5503, room 5521,
University of California – Davis, Davis, CA, 95616, USA
Accepted for publication 22 September 2011
Summary
Objective: Our aim was to compare the performance of three serological assays
in leprosy patients and their household contacts utilising two quantitative ELISA
tests using native PGL-I (PGL-1 ELISA), synthetic ND-O-HSA (ND-O-HSA
ELISA), and the semi-quantitative lateral flow test (ML Flow).
Methods: Comparisons among three immunological assays, PGL-I ELISA, ND-OHSA ELISA, and ML Flow were performed in 154 leprosy patients, 191 household
contacts and 52 health subjects.
Results: The sensitivity results of the PGL-1, ND-O-HSA, and ML Flow were
68·83%, 63·84%, and 60·65%, respectively, with specificity of 98% for both ELISA
assays. The native and synthetic PGL-I ELISA assays detected antibodies in 22·73%
and 31·82% of the paucibacillary (PB) patients, respectively and the ML Flow test did
not detect antibodies in this group. The ML Flow test was able to discriminate
patients into PB or multibacillary (MB) forms, while the native PGL-I and ND-OHSA was correlated with the bacillary load and the Ridley-Jopling clinical forms.
L.R.G and I.M.B.G are Senior Co-authors.
Correspondence to: Luiz Ricardo Goulart, PhD – Professor, Dept. Medical Microbiology & Immunology,
Genome & Biomedical Sciences Faculty, GBSF Suite 5503, Room 5521, University of California – Davis, Davis,
CA 95616, U.S.A. (Tel: þ 1 530 752 8113; Fax: þ1 530 754 7240; e-mail: lrgoulart@ucdavis.edu)
0305-7518/11/064053+13 $1.00

q Lepra

389

2 Leprosy is a disease with a wide spectrum of clinical forms for which there is still no gold standard diagnostic test. the world prevalence of 211. which evaluates the specific cellular immune response against Hansen’s bacilli. skin.15 – 18 Although PGL-I antibody levels in tuberculoid patients are low or negative.1 If not properly diagnosed or treated. the native PGL-I. respectively. In household contacts.12 – 14 These soluble forms of PGL-1 can also be more readily incorporated into lateral flow devices for the rapid detection of antibodies in multibacillary patient sera.6 – 9 A significant inverse correlation has been shown between the antibody titer and the Mitsuda test.or trisaccharides coupled to bovine or human serum albumin and a variety of neoglycoconjugates with up to 40 to 50 residues per molecule. and lepromatous (LL). 17%. with a sensitivity higher than that of the native PGL-I antigen. The presence of circulating IgM antibodies has been correlated with the clinical spectrum and bacterial index in leprosy patients. The PGL-I is an antigen found within the cell wall of the bacillus. considering immunpathological parameters and bacterial load. and ML Flow assays detected seropositivity of 25%. the reliability of this classification criterion has been questioned.3 Currently. and other organs. It is a disease associated with deformity and disability.903 cases registered at the beginning of 2010 still makes the disease a serious public health problem.4 However. borderlineborderline (BB). generating social stigmas and discrimination against patients and their respective families. sensitive and specific diagnostic laboratory tests would be of great use in the detection of leprosy patients in early stages of the disease. aiding in prescribing the correct treatment regimen to prevent subsequent nerve damage and disability. Various clinical manifestations can present in leprosy. and the multibacillary (MB) group with six or more lesions. leprosy patients are classified as tuberculoid (TT). with the paucibacillary (PB) group presenting five lesions or less. which is able to induce the production of antibodies in the host.5 In endemic areas of leprosy.390 J.10. ND-O-HSA. borderline tuberculoid (BT). leprosy can result in permanent physical impairment. Conclusions: The use of ELISA and ML Flow tests are thus recommended as additional tools in the diagnosis and classification of the clinical forms. Lobato et al. Despite the success of multidrug therapy (MDT) introduced in the mid-1980’s. Assays that detect PGL-I antibodies can utilise the native glycolipid. synthetic PGL-I di. and 10%. borderline-lepromatous (BL). since patients with less than five lesions may be classified as MB based on an assessment of the Bacterial Index (BI) results. According to the spectrum. leprae-specific antigens to be isolated and characterised was the phenolic glycolipid (PGL-1). and besides.11 The main antigenic determinant of PGL-I recognised is the trisaccharide portion of the molecule. the use of the quantitative PGL-I assay to assess the titer plays an important role in detecting . Introduction Leprosy is a chronic infectious disease caused by Mycobacterium leprae. an obligate intracellular parasite that affects peripheral nerves. the PGL-I ELISA may be used to detect subclinical infection in leprosy. and its detection is usually based on clinical signs and symptoms. patients are classified based on the number of skin lesions.6 One of the first M.

and the PGL-1 ELISA was performed at the beginning and end of the MDT. . The number of skin lesions and the bacterial index of the skin smear were used to determine the operational classification (OC). and limit further lines of transmission. borderline-lepromatous (BL).20 Consequently.6 Household contacts of leprosy patients that are positive for PGL-I display about a 7·2-fold higher risk of developing leprosy relative to PGL-I antibody-negative household contacts. and lepromatous leprosy (LL). as per Ridley-Jopling’s spectrum. The patients were diagnosed according to dermato-neurological clinical exams and laboratory tests. 1988). ETHICAL ASPECTS The present study was approved by the Research Ethics Committee of the Federal University of Uberlaˆndia (UFU) under protocol # 099/2003. considering as paucibacillary (PB) patients who had up to five skin lesions and a negative bacilloscopy. and those who progress to disease predominantly develop the more severe MB form. As negative control of the ELISA tests we used 52 sera samples from healthy individuals who have no contact with leprosy patients. the identification of household contacts of leprosy patients who are positive for the PGL-I antibody could lead to an earlier detection of infection. and multibacillary (MB). borderlineturberculoid (BT). those with more than five lesions and/or positive bacilloscopy (WHO. Material and Methods SUBJECT OF STUDY AND SAMPLES The study population was comprised of 191 serum samples of household contacts and 154 serum samples of newly diagnosed leprosy patients seen at the National Reference Center of Leprosy and Sanitary Dermatology of the Teaching Hospital – Federal University of Uberlaˆndia (CREDESH/HC/UFU). Minas Gerais. The rest of the BT (48·33% – 29/60) patients and 100% of the BB (28). more prompt diagnosis and treatment. and LL (31) patients were classified as MB. determining the proper duration and regimen of MDT.Immunological tests for leprosy 391 subclinical infection. borderline-borderline (BB).3 into the five clinical forms: tuberculoid (TT). The other two serology tests (ML Flow and ND-O-HSA ELISA) were not performed at the end of treatment due to the scarcity of these tests. Household contacts were those individuals who reside or resided with the patient in the last 5 years. Of the 154 patients diagnosed with leprosy. we have compared the performance of three serological assays in leprosy patients and their household contacts using two quantitative ELISA tests using native PGL-I. BL (21). Brazil. Patients and household contacts who voluntarily agreed to participate in the study were enrolled after giving their Informed Consent.19. synthetic ND-O-HSA. To this end. and the semi-quantitative lateral flow test (ML Flow). 100% (14/14) of the TT patients and 51·67% (31/60) of the BT patients were considered PB. and in the prevention of relapses.19 This study was undertaken in order to develop a standardised protocol for a more sensitive and specific serological ELISA assay to detect the PGL-I antibody in patients and household contacts screened at Reference Centres. 84 were followed during treatment. Based on these criteria.

Serum samples were added in duplicate using a dilution of 1:100 (native PGL-I) and 1:300 (ND-O-HSA) in PBS/BSA 1%. For the ML Flow (a lateral immunochromatographic flow test for anti-PGL-1 detection to investigate the Mycobacterium leprae immune response). NUNC) were coated with native PGL-I diluted in absolute ethyl alcohol. sera from three individuals mentioned above were selected and included in all test plates. We used two positive controls on each plate.21 EI values above 1·1 were considered positive. which is a natural trisaccharide antigen linked to bovine serum albumin. The antibody titers were expressed as the ELISA index (EI) according to the following formula: EI ¼ ODsample/ODcut-off. The absence of a line in the test zone indicated a negative result. The reaction was stopped by the addition of H2SO4 4N. For the lateral flow assay. and after 5 minutes the result was read by three independent readers. knees. ANTIGENS Two antigens were used for ELISA antibody detection. Ltd. of low EI (E. and from one active lesion. microtiter plates (Maxisorpw. 5 ml of whole blood was collected from the tip of the left index finger.15 BACTERIAL INDEX The bacterial load was calculated from the average of skin smears obtained from seven sites: the right and left earlobes. The Netherlands). ANTIBODY TITER Briefly. MO) was added to the plates in the dilution of 1:10.22 based on the number of bacilli detected by field microscopy.000 (ND-O-HSA). Lobato et al.0). in which the cut-off point was determined by the average OD of the negative controls plus three standard deviations (ND-O-HSA ELISA) or four standard deviations (native PGL-1 ELISA).I ¼ 2. as described previously. for the PGL-I antibody detection ELISA assays. obtained from Colorado State University through the NIH/NIAID Leprosy Contract N01 AI 25469. respectively.I ¼ 7. Sigma) enzyme substrate was added to the plates and incubated at room temperature for 10 minutes in the dark. Rayto Life and Analytical Sciences C. For the negative control. while a positive result was graded in intensity from 1þ to 4þ .392 J. using 100 X oil immersion lens. Two positive and three negative controls were included in each plate. Amsterdam. TP-READER. Germany) at 492 nm. the ND-O-HSA ELISA used the synthetic natural disaccharide octyl linked to human serum albumin (ND-O-HSA).0).000 (PGL-I ELISA) and 1:2. This is the antigen used in the ML Flow kit (KIT Biomedical Research.. one low positive. leprae-infected armadillo tissues from which the bacteria had been purified and utilised in PGL-1 ELISA. followed by washing. The optical density (OD) was obtained in a microplate reader (THERMO PLATE. The substrate o-phenylenediamine dihydrochloride (OPD. the native PGL-I isolated by organic extraction of M. and a strong positive of high EI (E. The bacterial index (BI) was calculated according to Ridley’s Logarithmic Scale. or synthetic ND-OHSA in Phosphate Buffered Saline (PBS). where: BI ¼ 0 (no bacilli in . at concentrations of 10 mg/ml and 0·2 mg/ml. St. The blood was placed in the test cartridge together with the buffer solution. antigen NT-P-BSA was used. elbows. The anti-human IgM-peroxidase conjugate (Sigma Chemical Co. incubated for 1 hour at 378C. The ELISA specificity was calculated based on tests of 52 serum samples from healthy individuals. Louis.

fair agreement.0. definitely positive – 8 to 10. ANALYSIS OF MITSUDA TEST AND THE BACTERIAL INDEX A classification of the clinical forms using Ridley & Jopling criteria3 is shown in Table 1.000 bacilli in 1 field). 0·05 were considered statistically significant. 0·40 – 0·59. and BI ¼ 6 (. represented in the following frequencies: 9·09% (14/154) were TT. Results Patients were classified into five clinical forms. 1. STATISTICAL METHODS Pearson’s coefficient was used to verify the degree of correlation among the tests and was applied to determine the degree of significance of the differences among the average of the serological and BI results. and strongly positive – readings . from the TT pole to the LL pole. no agreement. SP). when appropriate. patients with the LL form. and 20·13% (31/154) were LL. BI ¼ 1 (average of 1 to 10 bacilli in 100 fields). An intradermal injection of 0·1 ml was applied at the upper third of the anterior surface of the right forearm of patients and household contacts. 38·96% (60/154) were BT. Traversing the spectrum of the disease. 0·80 –1·00. poor agreement. almost perfect agreement. while at the other pole of the disease. BI ¼ 2 (average of 1 to 10 bacilli in 10 fields). Kappa values and their interpretations varied as follows: . A negative correlation was noted between the Mitsuda test results and the BI of the skin smear covering the spectrum of clinical forms. presented a negative Mitsuda and a average value for skin smear BI of 5·16. The diameter of the granulomatous lesion was measured in millimeters for quantitative and qualitative analyses. Bauru.10 mm or a granulomatous lesion of any diameter with ulceration. BI ¼ 4 (10 to 100 bacilli in 1 field). the average Mitsuda intradermal test result was 11·15 mm and the BI was equal to zero (BI ¼ 0). from .Immunological tests for leprosy 393 100 immersion fields). doubtful – 0 to 3 mm. Student’s t test was used to analyse the differences between the average of the serological tests at the beginning and end of the MDT. 0 –0·19. 0·20 – 0·39. In patients with the TT form. SP. BI ¼ 5 (100 to 1. MITSUDA TEST The Mitsuda antigen is a heat-killed suspension of 6 £ 107 bacilli/ml produced and donated by Dr. Maria Esther Nogueira Sales from the Lauro de Souza Lima Institute. 18·18% (28/154) were BB. The Kappa test was applied to evaluate the concordance results. BI ¼ 3 (1 to 10 bacilli in 1 field). Values of P. 13·64% (21/154) were BL. substantial agreement. in patients in the borderline group. moderate agreement.24 Differences between the groups were assessed by chi-square. Brazil (ILSL-Bauru.23 Patients and contacts were categorised into classes according to the World Health Organization (WHO) using the following criteria: negative – no reaction. 0·60 – 0·79. The concordance between the laboratory tests was calculated by dividing the number of congruent cases by the total number of patients. and the presence or absence of a granulomatous response on the 28th day following injection was determined.000 bacilli in 1 field). weakly positive – 4 to 7 mm.

As to the dynamic range of the results of the three serologic tests. Clinical and laboratory traits of patients according to clinical forms of leprosy Clinical Forms Tests TT BT BB BL LL Mitsuda positivity (%) Mitsuda averages (mm) BI positivity (%) BI averages ML Flow positivity (%) ML Flow averages ML Flow magnitude PGL-1 ELISA positivity (%) PGL-1 ELISA averages (EI) PGL-1 ELISA magnitude (EI) ND-O-HSA ELISA positivity (%) ND-O-HSA ELISA averages (EI) ND-O-HSA ELISA magnitude (EI) 100 11·15 0 0 0 0 0 0 0 0·1 –0·8 35·71 1·11 0·3–2·8 86·66 5·49 9·43 0·05 32·65 0·47 0–3 38·33 1·59 0·2–11·4 38·33 1·33 0·3 –6·4 7·69 0·92 100 1·90 94·73 2·5 0 –4 92·86 4·25 0·2 –12·2 57·14 1. According to the operational classification (PB and MB). and between EI average and clinical forms (r ¼ 0·97 . In the classification of clinical forms. 76·36% (84/110) for ND-O-HSA ELISA. varying from 0·1 to 31·0 for the native PGL-1 and from 0·3 to 27·8 for the ND-O-HSA. and 76 and 71% in accuracy.394 J. ANALYSIS OF SEROLOGICAL TESTS IN PATIENTS WITH NO PRIOR TREATMENT The sensitivity. 63·64% (98/154) for ND-O-HSA ELISA. ND-OHSA ELISA in 31·82% (14/44). with a gradual increase of EI accompanying the increase in BI towards the LL pole. The detection of IgM antibodies against PGL-1 in patient serum in the three tests analysed were 68·83% (106/154) for native PGL-1 ELISA. In the group of MB patients. BI: Bacterial Index. 0·05). the reactivity of the three tests was 86·36% (95/110) for PGL-1 ELISA. 98 and 98% for specificity. Lobato et al. The results of the ML Flow test varied from zero to four (0 to 4þ ) and did not differentiate between patients with clinical forms BT and BB. The PGL-1 ELISA displayed a significant positive correlation between positivity and clinical forms (r ¼ 0·92. respectively. along with an increase in BI (Table 1). The ELISA test showed to the dynamic range of the ELISA Index (EI) wider. and the ML Flow test showed no reactivity in any of the PB patient sera samples (0/44). BT – borderline tuberculoid. PGL-1 ELISA was reactive in 22·73% (10/44). and accuracy of the two ELISA tests were calculated. and 60·65% (74/122) for the ML Flow test. differentiating the five clinical forms. among the 44 PB patients. BB – borderline-borderline.89 0·2–8·4 0 0 100 4·22 100 3·81 3–4 100 9·96 1·4–28·0 95·24 5·87 1·1–23·6 0 0 100 5·16 100 3·96 3–4 100 11·21 2·68–31·0 100 7·62 1·8–27·8 Clinical forms: TT – tuberculoid. in which native PGL-1 ELISA and ND-O-HSA displayed. P . 68·83 and 63·84% for sensitivity. and between BL and LL (Table 1).3 it was demonstrated that the seroreactivity for the three serum tests showed a gradual increase in positivity according to the spectrum of the disease in the direction of the LL pole (Table 1). a gradual decrease in the cellular immune response was observed. and 90% (99/110) for ML Flow. Table 1. and LL – lepromatous. the two quantitative ELISA tests showed greater range that the semi-quantitative ML Flow. the BT to the BL form. specificity. BL – borderlinelepromatous.

001).05). There was a significant correlation for ND-O-HSA ELISA (r ¼ 0. a positive and significant correlation was demonstrated for PGL-1 ELISA (r ¼ 0·98.88.48 and k ¼ 0.0001).53. for ND-O-HSA ELISA. Average values for the native PGL-I ELISA. 0·05) and (r ¼ 0·93. the concordance was substantial (k ¼ 0. 0·05) and (r ¼ 0·96. . 0·05). and between the ND-O-HSA ELISA and the ML Flow test.94. respectively. 0·05). with k ¼ 0. Between PGL-1 ELISA and the ML Flow. which displayed reduced PGL-1 levels before treatment. P . all clinical forms showed a significant decline in PGL-1 upon MDT treatment (Figure 2).75. Average of serological tests 20 PGL-I N-D-HSA 15 ML-Flow y = 1·8992x – 1·3969 R 2 = 0·94 10 y = 1·0265x – 0·5427 R 2 = 0·77 y = 0·6665x – 0·1737 R 2 = 0·90 5 0 0 0–0·99 1·0–1·99 2·0–2·99 3·0–3·99 4·0–4·99 5·0–5·99 Bacterial index (BI) Figure 1. ANALYSIS OF THE PGL-1 ELISA SERUM TEST IN PATIENTS AFTER MULTIDRUG THERAPY With the exception of the TT clinical form. Accordingly.05) (Figure 1) as well. 0. the correlation was also significant (r ¼ 0·95. and for ML Flow (r ¼ 0. 0. respectively (P . signifying that the greater the number of bacilli. concordances were moderate and significant. P . the greater the quantity of IgM anti-PGL-1 antibodies. P . 0·05).Immunological tests for leprosy 395 P . the quantification of IgM antibodies in the three tests. it was observed that 26% (49/191) were contacts of PB and 74% (142/191) were contacts of MB patients. As to the quantitative results of the serological tests and the BI of the skin smear of the patients. 0. respectively. using the EI for the ELISA tests and the positivity intensity for the ML Flow test. P . The ML Flow test positivity and average also showed a correlation per clinical form: (r ¼ 0·91. P . P ¼ 0. Between the PGL-1 ELISA and the ND-O-HSA ELISA.001) (Table 2). ANALYSIS OF THE CELLULAR AND HUMORAL IMMUNE RESPONSE IN HOUSEHOLD CONTACTS Each of the household contacts was categorised according to their relationship with the index case. ND-O-HSA ELISA and ML Flow assays. The concordance among the three serological tests was calculated using Kappa’s coefficient (Table 2). 0. P . according to the bacterial index of leprosy patients. P .

i. Discussion This present study proposed a comparison among three serological tests for the detection of specific anti-PGL-1 antibodies of M. Average ELISA Index values for the native PGL-I assays. Positivity of the three tests was not different in household contacts of MB patients when compared to contacts of PB patients.396 J. 0·0001. and 10% (19/191) for the ML Flow. according to clinical forms of the leprosy before and after MDT treatment. The cellular immune response was evaluated by the Mitsuda test.4 mm among household contacts. Table 2. greater reactivity for PGL-I ELISA with 25% (49/191) of the contacts followed by 17% (32/191) for the ND-O-HSA ELISA. seeking to assess their use in diagnosis and/or in subclinical leprosy Average PGL-I ELISA index 15 Before treatment After treatment 10 5 0 TT BT BB Clinical form BL LL Figure 2. although with no statistical differences between the tests (Table 3). .e.. Analysis of the Kappa agreement index among three serological assays in leprosy patients ML Flow Positive (N) PGL-1 ELISA Positive (N) Negative (N) Total PGL-1 ELISA Positive (N) Negative (N) Total ND-O-HSA ELISA Positive (N) Negative (N) Total * Negative (N) 69 10 4 38 73 48 ND-O-HSA ELISA Positive (N) Negative (N) 62 17 12 30 74 47 ML FLOW Positive (N) Negative (N) 60 14 13 34 73 48 Total Kappa 79 42 121 0·75* Total 79 42 121 Kappa 0·48* Total 74 47 121 Kappa 0·53* P . leprae in treatment-naı¨ve leprosy patients and their household contacts. Lobato et al. which showed an average of 7. following the same positivity pattern found in patients.

(D-BSA) and trisaccharide (T-BSA) residues. The two ELISA tests showed a positive correlation with the bacterial load. and the ML Flow test. the ND-O-HSA ELISA test. leprae. 6-dio-methyl B D-glycopyranoside. which possess the non-reducing sugar 3. since the antigenic determinant of PGL-1 is represented by the trisaccharide terminal.Immunological tests for leprosy 397 Table 3.15. and disease. and among the synthetic antigens those that result in best reactivity with patient sera are those with di. several markers for epidemiology and control of leprosy have been evaluated. infection. infections. The native PGL-1 ELISA showed greater sensitivity and accuracy. it has a small scale of variation ranging from 0 (negative) to 4þ (highly positive). although the difference between the tests showed no statistical differences. the cellular immune response and the bacillary load were inversely proportional. This differentiation of patients in the spectrum and the correct classification are essential for a work of this nature since it guarantees reliability of the serology results presented. showed variation in the scale of amplitude of results. The lowest sensitivity observed with ND-O-HSA ELISA may be due to the use of the synthetic disaccharide antigen. especially for the PGL-1 ELISA test.. with similar specificity.3 i. i. as well as their amplitudes. analysis of the BI of the skin smear and Mitsuda test demonstrated that the clinical forms of the patients are consistent with the classification of Ridley & Jopling.25 In order to identify these points involved in transmission. making this test an important tool for the determination of the Ridley and Jopling forms. according to the clinical forms of Ridley and Jopling.30 – 32 The two ELISA tests.e. Positivity of the three serological assays for household contacts according to the operational classification of their index case (leprosy patients) OC Index Case MB PB Total Contacts (N) Positivity in PGL-1 ELISA N (%) Positivity in ND-O-HSA ELISA N (%) Positivity in ML Flow N (%) 142 49 191 39 (27·46) 10 (20·41) 49 (25·65) 25 (17) 7 (14) 32 (17) 14 (10) 5 (10) 19 (10) MB: Multibacilary. This is the first study comparing seropositivity of patients with Hansen’s disease with the PGL-1 ELISA test. . since the ML Flow test is semiquantitative. as well as for the control of therapeutic.6 Nevertheless. as is demonstrated in the present paper. as well as the early onset of the disease.e. with a growing EI accompanying the spectrum of the disease and it also showed amplitude of EI that was able to better differentiate between the five clinical forms of leprosy.. increased in proportion to the BI. PB: Paucibacilary. with a gradual increase of EI according to clinical forms of the disease. including the use of molecular and immunological methods in the detection of M.26 – 29 In order to eliminate the bias of an incorrect clinical classification. since there is considerable difficulty in leprosy in identifying the three reference points that are involved in the transmission of the disease. as well as to monitor patient treatment. which makes it difficult to precisely differentiate the levels of antibodies among the clinical forms and to monitor treatment (unpublished results). and the average of the EIs. in an effort to differentiate individuals with subclinical infections. the onset points of exposure.3 The native PGL-1 ELISA test showed greater seropositivity when compared to the other tests.

the assumption of persistent bacilli present in patients already treated with MDT cannot be excluded. this may be due to the difference in protocols and antigens. containing only one fraction of the glycolipid.49 However.18. studies performed with leprosy patients in Brazil. as is shown in literature15. which could favour sub-treatment and relapses. BB. in this study.18.37. probably because the two antigens used in the tests are trisaccharides.17. showing that the quantitative test had good sensitivity to the decrease in antibodies that correlate with the drop in bacterial load promoted by the MDT. and may represent a continual stimulus in antibody production.398 J.36 which gives it the status of an important tool to be utilised by healthcare teams not specialised in leprosy in the care of field patients. The persistence of these antibodies at high levels of MDT in MB patients may be due to the inefficiency of macrophages in cleaning out dead M.17.38 As to the two ELISA tests displaying moderate concordance. the origin or trigger of this immunological persistency must be determined. The results of the ML Flow test showed an excellent correlation with the operational classification and the BI. seeking to classify the patient after diagnosis. conjugated with a human seroalbumin.42 – 44 which have demonstrated a gradual reduction in IgM-anti PGL-1 antibodies in patients under treatment. and Nigeria with the ML Flow test.36 The analysis of concordance among the three tests showed substantial agreement between PGL-I ELISA and the ML Flow. the present study evaluated the serum response of household contacts of patients with leprosy to the three anti-PGL-1 tests. leprae as a slow growth bacillus.46 This fact may occur since the clearance of bacilli from the body is a slow process.35 Recently. with a structure different from the native PGL-1 and from the synthetic trisaccharide antigen.33 – 35 The high positivity observed in MB patients to the ML Flow test may be due to the association of the anti-PGL1 antibodies in the more bacillary forms of the disease with the synthetic trisaccharide antigen PGL-1 contained in the said test. As the ELISA PGL-I test showed greatest sensitivity and accuracy.33 – 35 The ML Flow is a limited test for the detection of anti-PGL-1 antibodies in sera of PB patients. Nepal.47 Moreover. reducing the risk of allocating patients to inadequate treatment schemes. as had already been reported in literature. The ND-O-HSA is a terminal disaccharide epitope of the synthetic PGL-1. noted that the use of the test helped in the operational classification of the disease. such as is reported in literature. the native ELISA anti-PGL-1 displayed the greatest seropositivity.36 Nonetheless. LL. leprae and their antigenic fragments from lesions. as well as a greater correlation .17. considering that this is the group considered as having the greatest risk of becoming ill when compared to the general population of non-contacts.25.37 New glycoconjugated antigens are correlated with the native PGL-1 in the detection of antibodies due to the presence of carbohydrate epitopes of PGL-1 that are critical in the antigen-antibody bond. and BL) and of the clinical form.32.33. Lobato et al. These serum monitoring in patients’ data corroborate other studies6. the semi-quantitative ML Flow test demonstrated an excellent performance for discriminating patients as to the operational classification (PB and MB)15.12.49 – 52 Among the three serology tests analysed in sera of household contacts. In order to approach the problems with the point of first exposure and/or with the point of onset of infection.45.39 – 41 The decline of the EI in the PGL-1 ELISA test after MDT treatment was observed in patients of the borderline group (BT.7 corroborating the determination of the epidemiology of M. This present study demonstrated that there was persistence of IgM anti-PGL-1 antibodies at the end of treatment in multibacillary clinical forms. demonstrating a positive correlation between the levels of antibodies and the bacterial load. considering that it is a simple and quick test to perform.

Samira Bu¨hrer Sekula and KIT Biomedical Research. Similarly. The Netherlands. while the ML Flow could be made available to the basic non-specialised healthcare services.3 determining the power of the PGL-1 ELISA test in discriminating titers of antibodies correlated to the bacterial index of patients.53. it could be implanted in the routine of leprosy reference centres of endemic countries. and the Ministry of Health. Brazil. in order to accrue experience of their potential benefit for patients when used on a large scale in routines of leprosy control programmes. 85: 337– 348. Smith WC.61 Briefly. Jopling WH. the serological PGL-1 ELISA test could be offered by reference centres to patients referred by healthcare services. Int J Leprosy. negative Mitsuda. Weekly Epidemiological Record. demonstrating the novel pattern of anti-PGL-1 antibody detection in leprosy patients according to the clinical forms of Ridley-Jopling. CAPES. as well as to facilitate the identification and assessment of individuals with a higher risk of becoming ill for intervention with chemoprophylaxis. its indication for monitoring household contacts seems obvious. Int J Lepr. 1966. Sensitivity and specificity of methods of classification of leprosy without use of skin-smear examination. Classification of leprosy according to immunity: a five group system. Geneva. 847. the authors would also like to thank Dr. and no BCG scar have a 24·26-fold higher risk of developing leprosy. favouring the monitoring of treatment. Amsterdam. Croft RP. Global Strategy for Further Reducing the Leprosy Burden and Sustaining Leprosy Control Activities (Plan period: 2006–2010) 2005. 2010. World Health Organization.Immunological tests for leprosy 399 with BI and clinical forms of the Ridley and Jopling spectrum. as has been demonstrated by prior study of our group.20 Another study also demonstrated that contacts with positive ML Flow. 1998. FAPEMIG. Switzerland.54 – 57 and prognosis. FINEP. Seropositive contacts have demonstrated a risk 7·2 times higher of becoming ill when compared to seronegative contacts for ELISA anti-PGL-1. favouring allocation of patients to treatment regimens for PB and MB. Acknowledgements Grants were provided by the following agencies: CNPq. Nicholls P. for the ML Flow assays that were provided as a donation. The authors are also grateful for the kind donation of antigens by Dr. . References 1 2 3 4 5 World Health Organization. 53: 1–17. Since the ELISA anti-PGL-1 test demands laboratorial structure and specialised human resources. as well as its performance for detection of subclinical infection in contacts. since these services do not have bacilloscopy test available for all patients. Ridley DS. within a network of facilities between Basic Healthcare Units and Reference Centres. besides the fact that the reliability of these tests have been questioned in some control programmes.58 – 60 The present work corroborates and advances knowledge as to prior study.53 There are already enough studies in literature that recommend the use of serological tests in the control of leprosy. 66: 445– 450. 34: 255–273. Richardus JH. Technical Report Series 1994. Study Group Chemotherapy of leprosy. World Health Organization. John Spencer (generated through the NIH/NIAID N01 AI 25469 Leprosy Research Contract). and even as an aid in diagnosis7.

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