Serology with ML Flow test in health professionals from three different states of Brazil / Sorologia com o teste ML Flow em profissionais de saude em tres diferentes estados do Brasil

BACKGROUND: In highly endemic countries, transmission and sub-clinical infection of leprosy are likely and the disease manifests itself in individuals without any known close contact with a leprosy patient. Health workers are social contacts belonging to the same network (the Health System) and some of them share the same social environment (nursing assistants) as patients with known patients and / or carriers. OBJECTIVE: To identify ML Flow seropositivity among health professionals. METHODS: We conducted a cross-sectional study using a serological survey with the ML Flow test in 450 health professionals (doctors, nurses and nursing assistants), in order to detect seropositivity in areas of high and low endemicity in municipalities from three Brazilian states (RJ, MS and RS). RESULTS: The results showed general 16% seropositivity, higher in low endemic areas, regardless of whether there was direct care for leprosy patients. Paradoxically, a statistical association was observed between the area studied and seropositivity, as the place with the lowest endemicity (CA) had the highest seropositivity rate (p = 0.033). CONCLUSION: The authors suggest these results are associated with a presence of an unspecified link to bovine serum albumin (BSA), carrier of PGL-1 in the ML Flow test, and recommend expanded seroepidemiological research utilizing tests with human and bovine albumin. .

FUNDAMENTOS: Em países altamente endêmicos a transmissão e infecção sub-clínica da hanseníase provavelmente ocorrem e a doença se manifesta em indivíduos sem qualquer contato próximo conhecido com paciente com hanseníase. Os trabalhadores de saúde são contatos sociais que pertencem à mesma rede (Sistema de Saúde) e alguns deles compartilham o mesmo ambiente social (auxiliares de enfermagem) com pacientes conhecidos e/ou portadores. OBJETIVO: Conhecer a soropositividade ao ML Flow entre os profissionais de saúde. MÉTODOS: Foi realizado um estudo transversal através de inquérito sorológico com o teste ML Flow em 450 profissionais de saúde (médicos, enfermeiros e auxiliares de enfermagem) visando conhecer a soropositividade em áreas de alta e baixa endemicidade em municípios de três estados brasileiros (RS, MS e RJ). RESULTADOS: Os resultados mostraram 16% de soropositividade em geral, mais elevada na área de baixa endemicidade, independente da assistência direta a pacientes com hanseníase. Paradoxalmente foi observada associação estatística entre a área estudada e soropositividade, apontando o lugar de mais baixa endemicidade (CA) com o maior valor (p=0,033). CONCLUSÃO: os autores sugerem a presença de ligação inespecífica a soroalbumina bovina (BSA), carreadora do antígeno PGL-1 no teste ML Flow para explicar os resultados inesperados e recomendam testagem ampliada utilizando testes com albumina humana e bovina. .

Whole-blood nested-PCR amplification of M. leprae-specific DNA for early diagnosis of leprosy.

We evaluated the sensitivity and specificity of a nested-polymerase chain reaction (PCR) method for detection of Mycobacterium leprae DNA from whole blood. Whole-blood specimens were subjected to nested-PCR amplification of M. leprae repeat DNA sequences in 49 multibacillary (MB) and 30 paucibacillary (PB) leprosy patients, 96 household contacts (HHCs), 18 tuberculosis (TB) patients, and 35 normal healthy individuals. M. leprae DNA was detected in 95.92% (47/49) of MB, 70% (21/30) of PB, and 6.25% (6/96) of HHC, but it was not detected in 18 TB or 35 normal controls. The sensitivities of the anti-bovine serum albumin (ND-O-BSA) immunoglobulin M (IgM) and antifusion protein of ML0405-ML2331 IgG for MB were 97.96% and 89.8%, and these values for PB were 70% and 53.33%. However, the ND-O-BSA enzyme-linked immunosorbent assay (ELISA) had lower specificity, with relatively high false-positive results for TB patients (16.67%) and normal healthy controls (10%). Based on these promising findings, we propose the use of nested PCR of whole-blood samples along with ELISA test for early detection of leprosy cases.

Serology with ML Flow test in health professionals from three different states of Brazil.

BACKGROUND: In highly endemic countries, transmission and sub-clinical infection of leprosy are likely and the disease manifests itself in individuals without any known close contact with a leprosy patient. Health workers are social contacts belonging to the same network (the Health System) and some of them share the same social environment (nursing assistants) as patients with known patients and / or carriers. OBJECTIVE: To identify ML Flow seropositivity among health professionals. METHODS: We conducted a cross-sectional study using a serological survey with the ML Flow test in 450 health professionals (doctors, nurses and nursing assistants), in order to detect seropositivity in areas of high and low endemicity in municipalities from three Brazilian states (RJ, MS and RS). RESULTS: The results showed general 16% seropositivity, higher in low endemic areas, regardless of whether there was direct care for leprosy patients. Paradoxically, a statistical association was observed between the area studied and seropositivity, as the place with the lowest endemicity (CA) had the highest seropositivity rate (p = 0.033). CONCLUSION: The authors suggest these results are associated with a presence of an unspecified link to bovine serum albumin (BSA), carrier of PGL-1 in the ML Flow test, and recommend expanded seroepidemiological research utilizing tests with human and bovine albumin.

Novel immunoadjuvants based on cationic lipid: Preparation, characterization and activity in vivo.

The interactions between three different protein antigens and dioctadecyldimethylammonium bromide (DODAB) dispersed in aqueous solutions from probe sonication or adsorbed as one bilayer onto particles was comparatively investigated. The three model proteins were bovine serum albumin (BSA), purified 18 kDa/14 kDa antigens from Taenia crassiceps (18/14-Tcra) and a recombinant, heat-shock protein hsp-18 kDa from Mycobacterium leprae. Protein-DODAB complexes in water solution were characterized by dynamic light scattering for sizing and zeta-potential analysis. Cationic complexes (80-100 nm of mean hydrodynamic diameter) displayed sizes similar to those of DODAB bilayer fragments (BF) in aqueous solution and good colloid stability over a range of DODAB and protein concentrations. The amount of cationic lipid required for attaining zero of zeta-potential at a given protein amount depended on protein nature being smaller for 18 kDa/14 kDa antigens than for BSA. Mean diameters for DODAB/protein complexes increased, whereas zeta-potentials decreased with NaCl or protein concentration. In mice, weak IgG production but significant cellular immune responses were induced by the complexes in comparison to antigens alone or carried by aluminum hydroxide as shown from IgG in serum determined by ELISA, delayed type hypersensitivity reaction from footpad swelling tests and cytokines analysis. The novel cationic adjuvant/protein complexes revealed good colloid stability and potential for vaccine design at a reduced DODAB concentration.

A study on a possibility of predicting early relapse in leprosy using a ND-O-BSA based ELISA.

Serological methods have been used for detecting infection with Mycobacterium leprae. We have applied a serological test to explore the possibility it could detect a bacterial relapse among patients who have been cured with chemotherapy. More specifically we used an indirect enzyme-linked immunosorbant assay (ELISA) using the natural disaccharide (ND) of the phenolic glycolipid antigen of M. leprae linked to bovine serum albumin as antigen. Antibody levels were measured in sera from normal controls, active leprosy cases, cured leprosy patients, and relapsing leprosy patients. We correlated antibody levels with the type of leprosy, the bacterial index, and with relapse among cured leprosy patients. In our hands, the ND-ELISA, when applied to screening for infection with M. leprae, had excellent sensitivity, specificity, positive and negative predictive values, and both a low false positive rate and a low false negative rate. Antibody levels gradually increased among active patients from the tuberculoid to the lepromatous end of the leprosy spectrum. There was a year-by-year fall in antibody levels in patients responding to chemotherapy. Antibody levels and the bacterial index were correlated using the Spearman's rank correlation method. Serial antibody levels were measured in 666 leprosy patients after being cured with dapsone monotherapy. Over a three year follow up, 95 multibacillary patients became antibody positive and 12 of them had bacterial relapses of their disease. In contrast, among 335 cases that remained antibody negative, only one relapse was seen. Among 44 paucibacillary cured patients who became antibody positive, there was one relapse. There were 192 such patients who remained antibody negative and one relapsed. The risk of relapse is 6.7 times higher among cured multibacillary patients compared to cured paucibacillary patients. Overall, the cumulative relapse rate among antibody positive cases was 13.7%, compared to 0.4% among antibody negative patients. We conclude that the ND-ELISA is a useful tool both for screening for early infection with M. leprae and for predicting a relapse in cured patients, particularly in cured multibacillary patients.

Identification and purification of armadillo (Dasypus novemcinctus) immunoglobulins: preparation of specific antisera to evaluate the immune response in these animals.

In this work we describe the purification and characterization of armadillo immunoglobulins. The IgM was precipitated using low-strength ionic solution and further purified by filtration through Sephadex G-200. The IgG was obtained in pure form by precipitation of serum with ammonium sulfate and DEAE-cellulose ion exchange chromatography. The purity of these immunoglobulins was evaluated by polyacrylamide gel electrophoresis. The results showed 28-kDa light chains and 55-kDa and 70-kDa heavy chains for IgG and IgM, respectively. The rabbit antibodies against these molecules were used to prepare fluorescein (FITC) and peroxidase conjugates. The FITC conjugate was used to quantify IgM-bearing lymphocytes. An average of 17% of peripheral blood lymphocytes were sIgM+ from 14 healthy animals. Additionally, in the same animals we quantified lymphocytes with the capacity to form rosettes with sheep red-blood cells; the average for this marker was 10%. Also, the production of crossreacting antibodies to BCG was evaluated in healthy and Mycobacterium leprae-inoculated animals using the peroxidase conjugates. All animals with active infection recognized BCG antigens.

Epidemiological studies in children of a low-endemic region, a high-endemic region, and dwellers of a leprosy colony: evaluation of anti-ND-BSA antibodies and lepromin response.

Children residing in a low-endemic region (LER), a high-endemic region (HER), and a leprosy colony contact population (CP) were evaluated for lepromin response as well as reactivity to the Mycobacterium leprae-specific synthetic antigen, ND-BSA. The mean reactivity to ND-BSA in the LER group (OD 0.03 +/- 0.03, N = 71) was significantly lower (p < 0.001) than that in the contact population (OD 0.14 +/- 0.09, N = 140) as well as the population residing in the HER (OD 0.09 +/- 0.08, N = 1340). ELISA-positive results were the highest (21.4%) with the CP group and lowest (0.0%) in the LER group, suggesting that it was a measure of the extent of exposure of M. leprae. In the contact population, females showed a preponderance for ELISA positivity over males (p < 0.005), a finding not observed with the HER population. The Mitsuda responses showed a Gaussian-type distribution in all of the three populations examined with the mean response being highest in the LER (6.0 mm +/- 2.9) and lowest in the HER (4.5 mm +/- 2.0) groups. The percent positivity for the Mitsuda reaction was found to be highest in the LER (93.0%) and lowest in the HER (88.3%) groups. The Mitsuda response thus appears to be independent of M. leprae exposure, and its interpretation in a given population needs consideration of several factors, such as nutritional, environmental, etc.(ABSTRACT TRUNCATED AT 250 WORDS)

Subclinical infection with Mycobacterium leprae--a problem for leprosy control strategies.

Tests for the serodiagnosis of leprosy, including those based on phenolic glycolipid-I (PGL-I), have shown poor specificity for leprosy in studies of endemic communities, despite initially promising results in studies of selected patients. During a 5 years follow-up study of a hyperendemic community in Papua New Guinea, a marked reduction in numbers of seropositive children and an increase in age of those seropositive followed introduction of multi-drug therapy. This was accompanied by a reduced case detection rate and a shift to paucibacillary disease in new cases, consistent with a reduction in transmission. Only a minority of persistently seropositive persons developed leprosy. These observations suggest that subclinical infection with Mycobacterium leprae is common in endemic communities and that PGL-I seropositivity is a marker of subclinical infection, with poor specificity for overt disease. Detection of subclinical infection may confound control strategies which use screening tests to identify asymptomatic highly infectious cases for earlier therapy.

Filter paper blood spot test for detection of anti-ND-BSA antibodies in school children.

For identifying individuals at 'high risk' for developing leprosy, a simplified technique of collection of blood samples on filter paper for detection of anti -ND-BSA antibodies, was optimised. Anti-ND-BSA antibody reactivity on the filter paper was lost on storage at room temperature, but was stable at least for a period of 8 wk at lower temperature. Among the 1495 children screened, 166 (11.1%) were lepromin negative and 122 (8.2%) positive for anti-ND-BSA antibody. In the first phase, 7 of 871 children followed up for 2 yr developed leprosy, while in the second phase 2 of 624 children followed-up for 9 yr developed leprosy. The positivity and negativity of anti-ND-BSA antibodies and lepromin in these I children indicate that the positive status for anti-ND-BSA antibodies has a better predictive value than negative lepromin reactivity. Measurement of anti-ND-BSA antibodies was also of value for monitoring the efficacy of therapy and course of the disease. Extrapolation of the two tests to the total population of children evaluated showed that an individual with negative lepromin reactivity along with presence of anti-ND-BSA antibodies is at a higher risk for developing leprosy than those who have both the tests normal. Lepromin reactivity alone was not of much value for the prediction for development of the disease. However, the presence of anti-ND-BSA antibody was a better indicator for the development of the disease. The probability of developing leprosy in a child with any one of the tests abnormal was higher as compared to a child having all the tests normal.

The diagnostic value of IgM to natural trisaccharide phenylpropionyl bovine serum albumin in leprosy patients: a preliminary report from Taiwan.

An evaluation was made of the serum anti-phenolic-glycolipid-I (PGL-I) IgM levels of leprosy patients in the Taiwan area by enzyme-linked immunosorbent assay (ELISA) with a specific synthetic PGL-I antigen and natural trisaccharide phenylpropionyl bovine serum albumin (NT-P-BSA). Fifty-five blood samples were collected from 24 tuberculoid and 31 lepromatous leprosy patients and 21 healthy age- and sex-matched subjects. Among these groups, lepromatous patients had the highest levels of IgM and anti-NT-P-BSA IgM with a good correlation between these two levels (p less than 0.01). Tuberculoid patients also had higher levels than normal subjects. Wide variation in the standard deviation and decreased levels within the cutoff value of some lepromatous patients may be due to various periods of anti-leprosy treatment. Serial anti-NT-P-BSA IgM assessments in response to anti-leprosy treatment may provide more information and serve as a guideline for therapy.

Serum IgA1 and IgM antibodies against Mycobacterium leprae-derived phenolic glycolipid-I: a comparative study in leprosy patients and their contacts.

In order to evaluate the potentials of IgA1 versus IgM as well as of native phenolic glycolipid-I (PGL-I) versus PGL-I-disaccharide coupled to bovine serum albumin (D-BSA) as antigens in the serodiagnosis of leprosy, anti-D-BSA IgA1 and anti-PGL-I IgM were investigated and compared to anti-PGL-I IgA1 in sera from patients and contacts. Anti-D-BSA and anti-PGL-I IgA1 significantly correlate in patients and contacts. The higher IgA1 positivity rates obtained with D-BSA as compared to PGL-I may suggest D-BSA as the favorable antigenic material. In patients but not in contacts anti-PGL-I IgM and IgA1 correlate, IgM predominating over IgA1. In all three antibody systems, the mean values as well as the positivity rates increased from the tuberculoid toward the lepromatous disease pole. Also, the levels of all three antibodies significantly increased with the bacterial index (BI). However, anti-D-BSA (PGL-I) IgA1 appears to be preferable to IgM with respect to sensitivity, i.e., detection of disease activity, in paucibacillary or BI-negative patients. A number of contacts were detected as seropositive with anti-D-BSA and/or anti-PGL-I IgA1 but not with anti-PGL-I IgM. This suggests that IgA1 is a better tool than IgM for the detection of leprosy in its subclinical stage.

Does the difference of the properties of trisaccharide-BSA conjugate (NT-P-BSA) of Mycobacterium leprae phenolic glycolipid influence on its seroreactivity?

The sugar content of the trisaccharide-BSA conjugate of the phenolic glycolip I of Mycobacterium leprae (NT-P-BSA) increased with the increase of the molar ratio of the trisaccharide to BSA used in the coupling reaction. The difference of the sugar content in NT-P-BSA did not give the influence on the seroreactivity and specificity in ELISA for both IgM and IgG class antibodies. During the course of the coupling reaction, about half amount of BSA was converted to dimeric form. However, there were no differences of the activity and specificity between monomeric form and dimeric form of NT-P-BSA. Based on these results, it was concluded that any lot of NT-P-BSA with variety of sugar content can be used in ELISA without any difference of the seroreactivity.

Rapid serodiagnosis for leprosy--a preliminary study on latex agglutination test.

In this study, we have developed two latex agglutination tests (LATs) with phenolic glycolipid-I (PGL-I) and natural disaccharide-octyl-bovine serum albumin (ND-O-BSA) as antigens in 110 leprosy patients (LL = 30, BL = 30, BT = 30, and TT = 20), 50 tuberculosis cases, and 30 normal controls. These two LATs were compared with corresponding ELISAs (ND-O-BSA ELISA and PGL-I ELISA) and analyzed by the chi-squared test. There were no significant differences between the two LATs (PGL-I LAT and ND-O-BSA LAT) and their corresponding ELISAs. There was an increase in the proportion of positive cases detectable which coincided with the clinical classification of leprosy, i.e., lepromatous cases were more likely to be positive than tuberculoid cases. LATs are more simple and rapid than ELISAs and have high sensitivity (77% in ND-O-BSA LAT, 80.5% in PGL-I LAT) and specificity (99% in both LATs). LATs may become useful tools for the immunodiagnosis of leprosy in the field. The stability and repeatability of LATs are discussed in detail.

Specific anti-M leprae PGL-I antibodies and Mitsuda reaction in the management of household contacts in New Caledonia.

Household contacts of leprosy patients have been tested for anti-phenolic glycolipid-I IgM antibodies (anti-PGL-I IgM) by an ELISA using the natural disaccharide (NDO) and natural trisaccharide (NTO) synthetic antigens. A group of healthy subjects without known exposure to Mycobacterium leprae served as controls. The percentages of positivity observed in multibacillary patients, paucibacillary patients, and household contacts were significantly higher than those of the negative controls. The absorbance values using NDO and NTO correlated well (range 0.59-0.91) when analysis of each subject group was performed. As reported here, NDO and NTO antigens seem to be equal in detecting leprosy cases; 100% of multibacillary and 21.43% of paucibacillary cases were detected as seropositive. For the screening of household contacts, NDO appears to be more sensitive and NTO more specific. There were more seropositive cases in the young age groups of household contacts, suggesting a higher rate of transmission of M. leprae infection in those age groups. Lepromin and anti-PGL-I IgG tests were also performed in contacts who were followed. The 2 paucibacillary subjects (1 borderline tuberculoid, 1 indeterminate) were Mitsuda negative. At diagnosis, their anti-PGL-I IgM levels were much higher than those of previous results; their anti-PGL-I IgG levels showed an increase in one and a decrease in the other. However, for the entire group anti-PGL-I IgM and anti-PGL-I IgG levels were positively correlated. The data reported here suggest that an increase in specific anti-M. leprae IgM levels in Mitsuda-negative household contacts could reveal the development of overt disease.

Use of eluates of filter paper blood spots in ELISA for the serodiagnosis of leprosy.

Blood samples were collected from 59 leprosy patients and 35 normal healthy subjects by veni-puncture and finger prick methods to obtain serum samples and blood spots on filter paper respectively. The serum samples at 1:300 dilutions and the eluates of dried blood spots at 1:40, 1:80, 1:160 and 1:320 dilutions were applied in ELISA to measure the antibody levels (IgM) against synthetic ND-O-BSA antigen. The antibody levels were found to be high in the multibacillary leprosy patients than the pauci-bacillary patients irrespective of whether serum samples or eluates were used. The OD values obtained at 1:160 dilution of the eluates were equivalent to that of values obtained at 1:300 dilution of the serum samples. The positivities differ in different dilution of the eluates, showing the highest in the 1:40 dilution and the lowest in the 1:320 dilution.

Solubilization of preformed immune complexes in sera of patients with type 1 and type 2 lepra reactions.

Serum complement activity in leprosy patients has been studied using solubilization of preformed immune complexes as an index. The solubilization capacity of sera from lepromatous patients with or without erythema nodosum leprosum (ENL) as well as from type 1 reactional patients was found markedly reduced as compared to controls. Solubilization did not improve at all in the ENL patients after remission of the reaction phase. The addition of fresh normal sera failed to bring about any significant restoration of solubilizing capacity of the deficient sera. Mycobacterium leprae sonicate significantly reduced the solubilization capacity. Our results suggest that circulating mycobacterial breakdown products possibly interfered with the capacity of the ENL patients' sera to solubilize immune complexes.