Detection of Mycobacterium leprae antigens in the sera of leprosy patients by sandwich immunoradiometric assay using monoclonal antibodies.

An immunological technique for demonstration of Mycobacterium leprae antigen in sera was developed by using specific as well as cross-reactive monoclonal antibodies. The sandwich immunoradiometric assay which we developed is a simple, robust assay that is sensitive to the nanogram level. Sera from 72 leprosy patients were screened for the presence of antigen by this assay. A total of 69% of untreated tuberculoid leprosy patients showed 35-kDa antigen positivity, and 45% of these patients showed anti-35-kDa antibody positivity. Consistently higher antigen positivity rates for the 35-, 12-, and 30- to 40-kDa components of M. leprae were observed in lepromatous leprosy patients than in tuberculoid leprosy patients. During the course of therapy the antigen positivity rate gradually declined, and the antigen could not be detected in any of the 15 patients with subsided cases of leprosy. As antigen is presumably in excess before the antibody response is evoked, our experimental approach for antigen detection is likely to be useful by itself or along with antibody detection for diagnosis of early leprosy.     

Antigens of Mycobacterium leprae identified by immunoprecipitation with sera from leprosy and tuberculosis patients.

Mycobacterial antigens which react with human B lymphocytes were investigated by immunoprecipitation of radiolabelled sonicates of Mycobacterium leprae and M. bovis (BCG) with sera from patients with leprosy and tuberculosis in the presence of Staphylococcus aureus. SDS-PAGE analysis of the immunoprecipitates demonstrated that dense bands of Mr 12,000 (12K), 15K, 27K, 32-33K, 36K and 48K were the major antigens of M. leprae recognized by antibodies in lepromatous leprosy sera. Of these, only the 15-16K band reacted significantly with sera from patients with tuberculoid leprosy and tuberculosis. Other antigens including the T cell immunogens of Mr 18K and 70K reacted with some of the BL/LL sera tested. There were differences in the pattern of antigens precipitated from BCG sonicate by leprosy sera with the 65K antigen and a high molecular weight band (greater than 94K) being readily detected. These results differ in part to these obtained by probing immunoblots of M. leprae sonicate with leprosy sera. Factors contributing to these differences are discussed.     

Cellular immune response to the cell walls of Mycobacterium leprae in leprosy patients and healthy subjects exposed to leprosy.

Cell walls of M. leprae consist of complex arrangements of carbohydrate, lipid, peptidoglycan and protein molecules. Recently, extractable proteins of a wide range of molecular weights were identified as components of the cell wall. We have examined the cellular immune responses of Nepali leprosy patients to a cell wall preparation of M. leprae enriched for these proteins. Strong lymphocyte proliferative responses to the antigens were present in half of the paucibacillary leprosy patients and in the majority of healthy control subjects with occupational exposure to leprosy. Patients with multibacillary disease responded poorly and patients with tuberculosis had intermediate responses. Proliferative responses to the cell wall protein fraction were strongly correlated to the proliferative responses to sonicates of the whole leprosy bacillus. Immunization of mice with cell wall proteins resulted in inhibition of growth of M. leprae following foot-pad inoculation with viable organisms. Therefore cell-mediated immune responses to the extractable proteins of the cell wall may play a role in protective immunity against M. leprae infection.     

Rapid and sensitive detection of Mycobacterium leprae using a nested-primer gene amplification assay.

By using a set of four nested oligonucleotide primers, a two-step polymerase chain reaction assay for the detection and identification of Mycobacterium leprae that does not require the use of radioactivity labeled hybridization probes was developed. The nested-primer procedure amplified a 347-base-pair product from M. leprae genomic DNA. No amplification products were produced from DNAs of 19 other Mycobacterium species, 19 non-Mycobacterium species, mouse cells, or human cells. Minor amplification products were observed with three additional Mycobacterium species, i.e., "M. lufu", M. simiae, and M. smegmatis. These products were easily distinguished from the M. leprae product by size and restriction enzyme cleavage patterns. The assay could amplify the 347-base-pair product from samples containing as little as 3 fg of M. leprae genomic DNA--the amount of DNA in a single bacillus. The assay also amplified target sequences in crude lysates of M. leprae bacilli isolated from tissue biopsy specimens from infected animals and humans. The entire assay, from sample preparation to data analysis, can be completed in less than 8 h.     

Mycobacterium leprae is identified in the oral mucosa from paucibacillary and multibacillary leprosy patients

In leprosy, the nasal mucosa is considered as the principal route of transmission for the bacillus Mycobacterium leprae. The objective of this study was to identify M. leprae in the oral mucosa of 50 untreated leprosy patients, including 21 paucibacillary (PB) and 29 multibacillary (MB) patients, using immunohistochemistry (IHC), with antibodies against bacillus Calmette-Guérin (BCG) and phenolic glycolipid antigen-1 (PGL-1), and polymerase chain reaction (PCR), with MntH-specific primers for M. leprae, and to compare the results. The material was represented by 163 paraffin blocks containing biopsy samples obtained from clinically normal sites (including the tongue, buccal mucosa and soft palate) and visible lesions anywhere in the oral mucosa. All patients and 158 available samples were included for IHC study. Among the 161 available samples for PCR, 110 had viable DNA. There was viable DNA in at least one area of the oral mucosa for 47 patients. M. leprae was detected in 70% and 78% of patients using IHC and PCR, respectively, and in 94% of the patients by at least one of the two diagnostic methods. There were no differences in detection of M. leprae between MB and PB patients. Similar results were obtained using anti-BCG and anti-PGL-1 antibodies, and immunoreactivity occurred predominantly on free-living bacteria on the epithelial surface, with a predilection for the tongue. Conversely, there was no area of predilection according to the PCR results. M. leprae is present in the oral mucosa at a high frequency, implicating this site as a potential means of leprosy transmission.     

Failure to induce delayed-type hypersensitivity to Mycobacterium leprae in long-term treated lepromatous leprosy patients.

Lepromatous leprosy (LL) patients whose bacillary load has decreased to almost undetectable levels by long-term chemotherapy failed to develop delayed-type hypersensitivity (DTH) to Mycobacterium leprae antigen following immunization with killed armadillo-derived M. leprae. When these LL patients were immunization with killed armadillo-derived M. leprae. When these LL patients were immunized with killed M. leprae in a mixture with live BCG, only DTH to purified protein derivative (PPD) was induced. These results are further evidence that immunological unresponsiveness to the leprosy antigen of patients with lepromatous leprosy is antigen-specific and non-reversible.     

Presence of viable Mycobacterium leprae in environmental specimens around houses of leprosy patients

Purpose: Leprosy is a chronic systemic infectious disease caused by Mycobacterium leprae, one of the first organisms to be established as the cause for disease in humans. Because of high prevalence pockets of leprosy in the endemic regions, it is necessary to identify the possible sources of M. leprae in the environment and its mode of transmission. Materials and Methods: Slit skin smears (SSSs) from lesions were collected in 70% ethanol from 50 leprosy cases staying in the leprosy resettlement village and hospital from a high endemic area. One hundred and sixty soil samples were collected from different areas around the leprosy hospital and from the resettlement village of cured leprosy patients where active cases also resided at the time of sample collection. M. leprae specific gene region (RLEP 129 bp) and 16S rRNA targets were used for polymerase chain reaction (PCR) based detection for the presence and viability of M. leprae. An rpoT region was also amplified to determine presence of numbers of 6 bp tandem repeats. Results: All the SSS samples collected from patients showed three copies of rpoT region (6 bp tandem repeat, an ancient Indian type). Fifty-two soil samples showed presence of M. leprae DNA whereas M. leprae specific 16S rRNA gene was amplified in sixteen of these samples. PCR amplification and fragment length analysis showed 91 bp, i.e., three copies of the rpoT 6 bp tandem repeats from soil samples and similar three copies observed in patient samples. Conclusion: Presence of viable M. leprae in the soil having same rpoT genotype of M. leprae noted in patients suggests that it could be the same strain of M. leprae. M. leprae found in the soil could be the one that is excreted out by the patient. Significance of its viability in the environment and its pathogenicity with respect to transmission needs to be further explored. Findings of this study might provide possible insights for further exploration into understanding transmission patterns in leprosy and also will throw light on identifying potential for existence of extra human source or reservoirs of M. leprae, if any.     

Simple colorimetric microtiter plate hybridization assay for detection of amplified Mycobacterium leprae DNA.

The detection of amplified products resulting from polymerase chain reactions (PCRs) remains a complicated process. To simplify the detection procedures, we developed a colorimetric microtiter plate hybridization assay for the specific detection of 5'-biotinylated PCR fragments of Mycobacterium leprae DNA. For this assay, an M. leprae DNA capture probe was made and immobilized on the wells of a microtiter plate. Hybridization of the biotin-labeled PCR fragments was detected through enzymatic color development. The resulting optical densities showed a logarithm-linear relationship with the amount of template DNA and corresponded to the intensity of the bands obtained through gel analysis and Southern blotting of the PCR products. The sensitivity of the assay was found to be 125 fg of genomic M. leprae DNA, or 20 lysed bacilli, revealing a detection limit similar to that of agarose gel analysis. The efficient coamplification of human DNA was used as a positive control for the presence of inhibitory substances in clinical material. For detection of human PCR products, a human DNA capture probe was also constructed for the colorimetric assay. This dual setup for hybridization, which thus detected both M. leprae and human DNA PCR products, was useful for ascertaining the presence of inhibiting substances in clinical specimens. All biopsy specimens (n = 10) from untreated patients with leprosy were positive. Apparently, this assay is more sensitive than microscopy, because biopsy specimens from half of the patients were negative upon histopathological examination. Biopsy specimens from three treated patients were negative, as were those from the three patients who did not have leprosy. We conclude that this colorimetric assay can replace agarose gel analysis and Southern hybridization, because it is as sensitive as those methods. Its advantages over conventional gel analysis and Southern hybridization are that it is less cumbersome and more rapid.     

Tau in cerebrospinal fluid: a sensitive sandwich enzyme-linked immunosorbent assay using tyramide signal amplification

Tau in cerebrospinal fluid (CSF) has been proposed as a diagnostic marker for Alzheimer's disease (AD). This paper presents a new sensitive sandwich ELISA allowing quantitation of tau from 8 microl CSF/well. A human specific monoclonal tau antibody HT7 was used as a capture antibody and a mixture of polyclonal tau antibodies, 92e and R134d was used as reporter antibodies. Tyramide signal amplification (TSA) technology was used in the last step to increase the sensitivity. With this TSA-ELISA, the lowest detection limit for tau was 14.3 pg/ml. Tau levels in CSF were found to be increased in AD patients (807+/-304 pg/ml, p<0.001) compared with controls (252+/-94 pg/ml). Thirty-five of 38 AD cases (92% sensitivity) yielded signals greater than cutoff, while only 1 of 38 control cases (97% specificity) was greater. A highly significant correlation was found between this assay and a commonly used kit, INNOTEST hTAU Antigen.     

Lymphocyte response of leprosy patients to human-derived and purified armadillo-derived Mycobacterium leprae, BCG and PPD.

The lymphocyte transformation test was applied to compare in vitro lymphocyte responses of tuberculoid (high resistant) and lepromatous (low resistant) leprosy patients to purified Mycobacterium leprae derived from experimentally infected armadillos and crude M. leprae derived from man, as well as to bacille Calmette-Guérin (BCG) and purified protein derivative (PPD). It was found that the purification procedure using enzymic digestion did not affect the immunogenicity of armadillo-derived M. leprae as compared with the crude human-derived preparation, although 2.5-5-fold higher doses of the purified organisms were required to elicitate equivalent lymphocyte responses. The result indicated the suitability of purified armadillo-derived M. leprae as the standard antigen for lymphocytes transformation tests in leprosy. The cross-reactivity studies show a close relationship between PPD and BCG, but not between M. leprae and PPD or BCG.     

Comparative assessment of the leprosy antibody absorption test, Mycobacterium leprae extract enzyme-linked immunosorbent assay, and gelatin particle agglutination test for serodiagnosis of lepromatous leprosy.

A comparative assessment of three serological methods for leprosy diagnosis (the fluorescent leprosy antibody absorption [FLA-ABS] test, the Mycobacterium leprae soluble-extract enzyme-linked immunosorbent assay [ELISA], and the M. leprae particle agglutination [MLPA] test) was carried out. The objective was to identify their performance in clinical and epidemiological diagnosis of leprosy. The study group included 45 lepromatous leprosy patients under treatment. Specificity was > 95% for all three assays, and sensitivity was 95, 58, and 74% for the FLA-ABS test, the MLPA test, and the ELISA, respectively. The only cross-reactivity for M. tuberculosis-infected patients was with the soluble-extract ELISA. Although the FLA-ABS test displayed the highest specificity and sensitivity values, it can only be used in well-developed laboratories, and the patient's clinical and epidemiological background must be considered when results are interpreted because the test remains positive after therapeutic success and could be positive for some household contacts. The MLPA test is easier to perform and interpret, and it is adequate for small laboratories and epidemiological studies intended to detect active untreated or irregularly treated leprosy cases. Therefore, the FLA-ABS and MLPA tests are complementary, and both should be used for serodiagnosis of leprosy.     

Detection of leprosy in ancient human skeletal remains by molecular identification of Mycobacterium leprae

We isolated ancient DNA from skeletal remains obtained from a South German ossuary (approximately 1400-1800 AD) and from a 10th century Hungarian cemetery partially indicating macromorphologic evidence of leprosy. In samples taken of 2 skulls from Germany and of 1 hard palate from Hungary, Mycobacterium leprae-specific fragments of RLEP1 and RLEP3 were amplified using polymerase chain reaction (PCR), thereby confirming their specificity by sequencing. In another case, PCR with primers targeting IS6110 of Mycobacterium tuberculosis gave positive results only for a mandibular specimen. No signal for any mycobacterial DNA was observed in samples from 2 Hungarian foot bones. In ancient material, osseous involvement of M leprae may be detected and distinguished from other mycobacterial infections by specific PCR. In the small bones of leprous hands and feet, not enough M leprae DNA seems to be present for detection. This supports the view that rhinomaxillary leprous alterations result from direct bacterial involvement, while osseous mutilations of hands and feet result from a nervous involvement and/or secondary infections due to small lacerations of the overlying soft tissues.     

Identification and characterization of antigenic determinants of Mycobacterium leprae that react with antibodies in sera of leprosy patients.

Antigenic determinants of Mycobacterium leprae were identified by screening a lambda gt11::M. leprae genomic library with two separate pools of sera from leprosy patients. A total of 45 recombinant clones were detected with pooled sera from 21 lepromatous (LL) leprosy patients and 5 additional clones specified polypeptides that reacted with antibodies in pooled sera from 30 borderline tuberculoid or tuberculoid leprosy patients. The recombinant clones that specified antigenic determinants that reacted with sera from LL patients were condensed into eight groups on the basis of DNA hybridization experiments among the M. leprae DNA insert fragments. In addition, 11 of the 45 recombinant clones did not hybridize to members of the eight groups nor to one another; these represent unique recombinant clones. None of the recombinant clones identified by screening with sera from tuberculoid leprosy patients hybridized to each other or to any of the 45 LL recombinant clones. The polypeptides specified by the recombinant clones were usually fusion proteins with beta-galactosidase, ranging in size from 117 to 175 kilodaltons (kDa). Members of hybridization group III specified nonfusion proteins of 45 kDa. Only members of hybridization group I reacted with any of 30 monoclonal antibodies prepared against M. leprae proteins; recombinant proteins from these clones reacted with a single monoclonal antibody directed against the M. leprae 65-kDa protein. Thus, at least 22 new antigenic determinants of M. leprae have been identified on the basis of their reactivity to antibodies in sera from LL patients or sera from tuberculoid leprosy patients or both.     

Major proteins of mycobacterial strain ICRC and Mycobacterium leprae, identified by antibodies in sera from leprosy patients and their contacts.

Sera from leprosy patients across the clinical spectrum, healthy contacts, tuberculosis patients, and healthy donors were tested for their reactivity with antigens of mycobacterial strain ICRC (a cultivable mycobacterium) and Mycobacterium leprae by immunoprecipitation technique. Using M. leprae antigens, it was not possible to distinguish between reactivities of sera from lepromatous, borderline lepromatous, borderline tuberculoid, and tuberculoid leprosy patients. All these sera identified M. antigens with molecular masses of 47, 36, 21, and 14 kDa. When the same sera were tested for their reactivities with antigens of mycobacterial strain ICRC, several differences were observed. The 21-kDa antigen of mycobacterial strain ICRC was exclusively precipitated by sera from all lepromatous leprosy patients and from those undergoing erythema nodosum leprosum reaction. Sera from all the other donors tested failed to identify the 21-kDa antigen of mycobacterial strain ICRC. The 14-kDa protein of mycobacterial strain ICRC was identified by sera from a few lepromatous leprosy patients (5 of 26) and all their contacts. Our studies indicate that antigens present on cultivable mycobacteria rather than species-specific antigens may prove to be useful in the serodiagnosis of leprosy.     

Detection of Mycobacterium leprae nasal carriers in populations for which leprosy is endemic.

In order to better understand the role of Mycobacterium leprae nasal carriage in the maintenance of infection reservoirs and transmission of leprosy, we applied a polymerase chain reaction (PCR) that detected a 531-bp fragment of the pra gene of M. leprae on nasal swab specimens collected through a total population survey from individuals living in an area in which leprosy is endemic. Among the total tested population of 1,228 people, 7.8% were found to be PCR positive. PCR positivity was shown to be randomly distributed among the population for which leprosy is endemic. No association was observed between PCR positivity, age, or sex. The observed distribution of PCR positivity among households of different sizes confirmed the expected values, with the exception of two households, each with three people with PCR-positive nasal swab specimens. Although nasal carriage does not necessarily imply infection or excretion of bacilli, the finding of nasal carriage supports the theory of a disseminated occurrence of M. leprae in populations for which leprosy is endemic.     

Comparative detection of enterovirus RNA in cerebrospinal fluid: GeneXpert system vs. real-time RT-PCR assay

Enteroviruses (EVs) constitute the most common cause of aseptic meningitis in both children and adults. Molecular techniques have now been recognized as the reference standard for the diagnosis of EV infections, and the rapidity of the molecular diagnosis of EV meningitis has been shown to be a determining factor in the management of patients. The rapid documentation of EV RNA in cerebrospinal fluid (CSF) is key to adapting patient management and the therapeutic regimen. To shorten the time needed for virological documentation, we implemented EV RNA detection in two point-of-care (POC) laboratories. Here, we present the results of the POC detection of EV RNA with the Xpert EV kit on the GeneXpert integrated system, and a comparison with the real-time RT-PCR (rtRT-PCR) assay routinely used in the core virology laboratory. From January to September 2009, a total of 310 CSF samples were tested. The rtRT-PCR gave 81 positive, 225 negative and four ‘indeterminate’ results. POC results were concordant in 81.6% (253/310). Most of the discrepancies consisted of ‘indeterminate’ results at the POC level (16%). Calculated performances (excluding the indeterminate results) of the Xpert EV kit on the GeneXpert system in POC settings were 100%, 98.9%, 97.6% and 100% for Sensibility, Specificity, positive predictive value and negative predictive value, respectively. Taken together, these results indicate that the implementation of POC detection of EV RNA can provide robust results in <4 h, and may have a significant impact on patient management, therapeutic attitude, and hospitalization costs.