A sensitive and specific ES-31 antigen detection based fluorometric assay for confirmation of Mycobacterium tuberculosis in cell culture.

Confirmation of presence of M. tuberculosis bacilli on microscopic examination is very important in diagnosis of tuberculosis. The present study was undertaken to find the usefulness of mycobacterial ES-31 serine protease as a marker to detect tuberculosis bacilli using fluorescein isothiocyanate conjugated anti-ES-31 serine protease antibody. This immunofluorescence method was compared with Ziehl-Neelsen and auramine-O staining methods for detection of tuberculosis bacilli. Slides were prepared for each serially diluted tuberculosis H37Ra bacilli (1 x 10(7) bacilli/ml to 5 bacilli/ml). Slides for each dilution group were stained by ZN method, auramine-O and immunostaining methods using fluorescein isothiocyanate conjugated anti-ES-31 serine protease antibody. ZN staining method showed efficacy for detection of M. tuberculosis H37Ra upto 1 x 10(4) bacilli/ml while auramine-O method showed upto 1 x 10(2) bacilli/ml. The presence of bacilli was indicated by green fluorescence on immunostaining using anti-ES-31 antibody conjugate and this method was effective upto 10 bacilli/ml. The slides which were negative for ZN (1 x 10(3) cells/ml) and auramine-O (100 cells/ml) method showed positivity on restaining with immunofluorescent staining method. The results of this preliminary study showed that immunofluorescent staining method using specific anti-ES-31 antibody conjugate was more sensitive for detection of tuberculosis bacilli than ZN and auramine-O methods in samples of laboratory strain. The utility of this method will be studied further in clinical specimens.

Potential of mycobacterial excretory secretory protein antigens (sevatb ES-31, ES-43, EST-6 and ES-20) as biomarkers to detect Mycobacterium tuberculosis bacilli.

There is a need for a simple and reliable method to identify Mycobacterium tuberculosis from nontuberculous mycobacteria (NTM). The utility of mycobacterial ES-31, ES-43, EST-6 or ES-20 antigen as a biomarker for differentiation of Mycobacterium tuberculosis bacilli from nontuberculous mycobacteria was explored using Fluorescein isothiocyanate conjugated antibodies against these antigens. Detection of these antigens was done from M.tb H(37)Ra and H(37)Rv DSS antigen. The presence of antigen in bacilli using FITC labelled antibody was indicated by green fluorescence on the cell surface while, its absence by no fluorescence under microscope. In M.tb H(37)Ra and H(37)Rv bacilli, fluorescence was observed on addition of FITC labelled anti ES-31 and anti ES-43 antibody; whereas no fluorescence was observed in case of EST-6 and ES-20 antibody conjugates. However all the antigens were detected in detergent soluble sonicate antigen of tubercle bacilli on addition of FITC conjugates. Fluorescence was not observed for ES-31, ES-43, EST-6 and ES-20 antigen in any of the tested NTM as well as in Escherichia coli. SEVA TB ES-31 and ES-43 may be used as biomarkers to distinguish M.tuberculosis bacilli from NTM.

Inhibition of Mycobacterium tuberculosis secretory serine protease blocks bacterial multiplication both in axenic culture and in human macrophages.

To study the possible importance of mycobacterial ES-31 serine protease for bacterial cell growth, the effect of serine and metalloprotease inhibitors, anti-tubercular drugs such as isoniazid and anti-ES-31 antibody, was evaluated on mycobacterial ES-31 serine protease in vitro and on bacilli in axenic and macrophage cultures. Serine protease inhibitors such as pefabloc, 3,4 dichloroisocoumarin, phenyl methyl sulfonyl fluoride (PMSF) and metalloprotease inhibitors such as ethylene diamine tetracetic acid (EDTA) and 1,10 phenanthroline inhibited 65-92% serine protease activity in vitro. Isoniazid showed 95% inhibition on mycobacterial ES-31 serine protease. These inhibitors also showed decreased bacterial growth in axenic culture and inhibition was further confirmed by a decreased amount of ES-31 serine protease in culture filtrate. In human macrophage culture, highly inhibitory pefabloc, 1,10 phenanthroline and isoniazid inhibited infectivity of virulent as well as avirulent M. tuberculosis bacilli to macrophages. It was observed that addition of mycobacterial ES-31 serine protease to macrophage culture enhanced the entry of bacilli and their multiplication in human macrophages. However, the addition of anti-ES-31 serine protease antibody strongly inhibited the mycobacterial growth as observed by decreased CFU count, showing the importance of mycobacterial ES-31 serine protease for entry of bacilli and their multiplication.

Study of M. tuberculosis ES-31 and ES-20 antigen levels in different pathogenic grades of lymph node tuberculosis.

OBJECTIVE: To study tuberculous excretory-secretory (ES) 31 and ES-20 antigens in different pathogenic grades of lymph node tuberculosis (TB). DESIGN: The study group included lymph node TB patients showing granuloma with mature epithelioid cells based on cytology findings (strong immune response group, SI) and patients showing no granuloma formation and acellular necrosis (weak immune response group, WI). Sandwich ELISA was performed using affinity purified antibodies against Mycobacterium tuberculosis ES-31 and ES-20 antigens to assay free and immune complexed antigen levels in the serum of these patients. RESULTS: Higher levels of immune complexed ES-31 (geometric mean titre [GMT] 848) and ES-20 (GMT 1818) antigens than free ES-31 (GMT 462) and ES-20 (GMT 647) were observed in WI patients. There were higher levels of immune complexed ES-20 antigen levels (GMT 1818) in WI patients than in SI lymph node TB patients; the difference was significant (P < 0.05). CONCLUSION: Elevated levels of immune complexed ES-20 antigen in patient's serum may be a useful immunological marker for weak immune response patients in lymph node TB.

Isolation of Mycobacterium tuberculosis protein antigens ES-3 1, ES-43 and EST-6 of diagnostic interest from tubercle bacilli by affinity chromatography.

Immunodiagnostically useful M. tuberculosis H37Ra protein antigens ES-31, ES-43 and EST-6 were isolated from detergent soluble sonicate (DSS) antigen using monospecific antibodies by affinity chromatography and compared with similar antigens isolated from M. tuberculosis culture filtrate for seroreactivity in tuberculosis sera by Indirect Enzyme Linked Immunosorbent Assay. Recovery of affinity purified ES-31, ES-43 and EST-6 antigen from DSS antigen was approximately 3, 3.5 and 4% respectively, compared to 10, 9 and 6.3% from culture filtrate. Affinity purified ES-31, ES-43 and EST-6 antigens from both culture filtrate as well as DSS antigen showed similar seroreactivity with overall sensitivity 85, 80 and 75% respectively and specificity of 85% at optimum concentration of 50 pg protein of each antigen. The results suggest that DSS antigen may be a promising antigen source for isolating antigens of diagnostic interest obviating the need for cumbersome, time-consuming culture techniques of mycobacteria.

Detection of enzymes dehydrogenases and proteases inBrugia malayi filarial parasites.

Lymphatic filariasis caused mainly by infection fromW. bancrofti andB. malayi remains a major cause of clinical morbidity in tropical and subtropical countries. Analysis ofB. malayi mf, infective larval and adult worm lysates for the activity of enzymes led to the demonstration of activities of three key enzymes of carbohydrate metabolism viz., Malate dehydrogenase (MDH), Malic enzyme (ME) and Glucose-6-phosphate dehydrogenase (G6PDH) in all the three stages of the parasite. The specific activity of all the three dehydrogenases was significantly high in mf lysate compared to their activity in lysates of the other two stages (P<0.001). Analysis by native polyacrylamide gel to their activity inlysates of the other two stages (P<0.001). Analysis by native polyacrylamide gel electrophoresis (PAGE) using 7.5% non-gradient gel showed the presence of two isoforms of each of the three enzymes (MDH, ME & G6PDH) in mf lysate, while only one form of each enzyme was present in L(3) larval and adult worm lysates. Further proteolytic enzyme activity was demonstrated both in microfilarial and infective larval lysates ofB. malayi. While both mf and L(3) larval lysates showed optimal protease activity at alkaline pH of 9.0, the mf lysate showed increased activity also at pH 3.0. The infective larval lysate was markedly inhibited by Tosylamide-L-Phenylalanine chloromethyl ketone (TPCK), a thiol protease inhibitor, while the protease activity in mf lysate was significantly inhibited by both TPCK and a serine protease inhibitor Phenyl Methyl Sulphonyl Flouride (PMSF). In sodium do-decyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), using gelatin copolymerized gel, the microfilarial lysate showed 3 protease molecules of 40 kDa, 180 kDa and 200 kDa and the L(3) larval lysate had 6 protease molecules of 18, 25, 37, 49, 70 and 200 kDa size.

Detection of free and immune-complexed serine protease and its antibody in TB patients with and without HIV co-infection.

OBJECTIVE: To understand the usefulness of detecting tuberculous IgG antibodies against mycobacterial excretory-secretory 31 kDa serine protease antigen (SEVA TB ES-31) and circulating free and circulating immune-complexed (CIC) serine protease in TB patients with and without HIV infection. DESIGN: Serum was collected from 144 individuals: patients with TB, with TB-HIV co-infection and HIV infection only, and ill and healthy controls. SEVA TB ES-31 antigen, a serine protease isolated from Mycobacterium tuberculosis H37Ra culture fluid, was used in indirect penicillinase ELISA to detect tuberculous antibodies. Similarly, affinity purified anti-ES-31 antibody was used in sandwich ELISA to detect circulating free and CIC serine protease. RESULTS: There was less sensitivity for tuberculous antibody in HIV-infected TB patients (46%) than in those with TB alone (87%) using mycobacterial serine protease. However, the sensitivity of detection of TB in the presence of HIV increased to 87% by concomitant detection of circulating free and CIC serine protease antigen. CONCLUSION: Detection of free and CIC tuberculous serine protease antigen along with antibody is more useful for detecting TB in the presence of HIV co-infection.

Effectivity of crude versus purified mycobacterial secretory proteins as immunogen for optimum antibody production.

Monospecific antibodies have been successfully utilized in antigen detection, which is better indicator of active infection. Mycobacterium tuberculosis excretory secretory (M tb ES) antigens such as ES 31, ES 41 and ES 43 (31 kDa, 41 kDa and 43 kDa protein, respectively) have been shown to be present in Mycobacterium tuberculosis H37Ra culture filtrate and are of diagnostic interest. To study the immunogenic potential of crude versus purified antigen, goat was immunized with M tb detergent soluble sonicate (DSS) antigen as well as purified antigen fraction (ESAS 7) containing ES 31 antigen. Both anti-DSS IgG antibody and anti ESAS 7 IgG antibody were found to be reactive with ES 31 antigen upto 1 ng concentration of antibody by ELISA. Crude DSS antigen was found to be quite effective in producing high titre antibodies and showed further high reactivity with other ES antigens (ES 41 and ES 43) of diagnostic interest.

Immunodiagnosis of bancroftian filariasis: comparative efficiency of the indirect hemagglutination test, indirect fluorescent antibody test, and enzyme-linked immunosorbent assay done with Wuchereria bancrofti microfilarial antigens.

The indirect hemagglutination test (IHAT), indirect fluorescent antibody test (IFAT) and enzyme linked immunosorbent assay (ELISA) have been applied to the serodiagnosis of bancroftian filariasis employing Wuchereria bancrofti mf antigens and the efficiency of these tests in the detection of antibody has been compared. Each test was found to be marginally superior to the other two tests with a particular group of endemic sera for the detection of filarial antibody. In other words, the IHAT, IFAT and ELISA showed a greater number of positive reactions with endemic normal, microfilaremia and clinical filarial sera, respectively. ELISA is simple, sensitive and can be used in seroepidemiological studies for bancroftian filariasis employing soluble antigens.

Parasite antigens in sera and urine of patients with bancroftian and brugian filariasis detected by sandwich ELISA with monoclonal antibodies.

We developed a sandwich enzyme-linked immunosorbent assay to detect circulating parasite antigens in human lymphatic filariasis. The assay utilizes a polyclonal rabbit antifilarial antiserum to capture, and a monoclonal antibody to identify, circulating parasite antigen. Using this assay, we found that greater than 95% of sera from microfilaremic donors with bancroftian or brugian filariasis, approximately 60% of sera from microfilaremic patients with hydroceles, chyluria, or elephantiasis, and 15%-20% of sera from asymptomatic residents of filariasis-endemic areas evidently contain filarial antigens. Antigen was also detected in the urine of some microfilaremic patients. Serum levels of antigen detected with one monoclonal antibody, ES34, correlated well with microfilarial density in night blood. In contrast, less than 5% of sera from residents of areas where lymphatic filariasis is not endemic reacted in the assay, even though approximately one-third of the donors whose sera were tested were known to be infected with intestinal nematodes. The assay was designed to be flexible enough to allow the parallel use of multiple monoclonal antibodies with different specificities and simple enough to be applicable in most areas where lymphatic filariasis is endemic.

Isolation and characterisation of in vivo released 41 kDa mycobacterial antigen in pulmonary and bone and joint tuberculosis and its identification with H37Ra in vitro released antigen.

OBJECTIVE: To isolate and characterise in vivo released 41 kDa mycobacterial antigen in pulmonary and bone and joint tuberculosis (TB) and its identification with in vitro released ES-41 kDa antigen. DESIGN: Circulating antigen was isolated from confirmed pulmonary tuberculosis serum (PTS) and bone and joint tuberculosis serum (BJS) by trichloroacetic acid precipitation and further fractionation by fast-protein liquid chromatography (FPLC). RESULTS: Fractionation of PTS and BJS by gel filtration column gave six protein fractions each. PTS-G3 and BJS-G3 showed maximum antigenic activity with ELISA. Further fractionation of PTS-G3 and BJS-G3 on cation exchange FPLC gave four different fractions each, of which BJS-G3B was seroreactive similarly to in vitro released 41 kDa antigen (ES-41) isolated from culture medium, whereas PTS-G3C was slightly less seroreactive. BJS-G3B could inhibit binding of in vitro released ES-41 to affinity purified antibodies in inhibition ELISA at lower concentrations than PTS-G3C (2 vs. 20 ng/ml), showing the identical nature of the antigens. Biochemical characterisation showed that circulating antigen PTS-G3C, BJS-G3B and in vitro released ES-41 antigen were lipoproteins in nature. CONCLUSION: This study helped to demonstrate the presence of 41 kDa antigen in the serum of pulmonary and bone and joint TB patients and its identification with H37Ra in vitro released 41 kDa antigen.

Fractionation, characterization and diagnostic potential of filarial antigens isolated from hydrocoele fluid in bancroftian filariasis.

Filarial antigen was isolated from hydrocoele fluid and fractionated on Ultrogel AcA 44. Six protein fractions (HFA C1 to HFA C6) were chromatographically separated from the column. Of the 4 antigenic fractions (HFA C1, HFA C2, HFA C3 and HFA C5), HFA C3 was a major reactive fraction with filarial serum immunoglobulin G. Analysis of HFA C3 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) yielded 18 bands of Mr 18,000-160,000. Two fractions, HFA C3-7 and HFA C3-9 of Mr 48,000-55,000 and 32,000-39,000 obtained by gel elution, were antigenic in an enzyme-linked immunosorbent assay (ELISA). SDS-PAGE immunoblotting confirmed the ELISA by identifying 3 immunoreactive bands of Mr 51,000, 38,000 and 35,000. The diagnostic potential of HFA C3-7 and HFA C3-9 was compared in serodiagnosis of active infections and diseased states. HFA C3-9 showed greater sensitivity in detection of filarial specific IgM antibody in cases with microfilaraemia. Physicochemical characterization indicated the protein nature of HFA C3-9.

Detection of antimicrofilarial ES antigen-antibody in immune complexes in Bancroftian filariasis by enzyme immunoassay.

An enzyme immunoassay using penicillinase conjugated to Wuchereria bancrofti microfilarial ES antigen has been developed to detect specific antibody in circulating immune complexes in Bancroftian filariasis. Immune complexes were prepared by 3% polyethylene glycol (PEG) precipitation. 44 sera belonging to different groups were tested. 16 of 19 clinical filarial and two of 16 endemic normal sera but none of the non-endemic normal sera showed the presence of antimicrofilarial ES antigen-antibody in immune complexes.

Diagnostic potential of fractionated Brugia malayi microfilarial excretory/secretory antigen for bancroftian filariasis.

Due to the non-availability of sufficient parasite material from Wuchereria bancrofti, a heterologous filarial antigen from Brugia malayi has been investigated for the diagnosis of bancroftian filariasis. B. malayi microfilarial excretory/secretory antigen (BmmfES) effectively inhibited the binding of circulating filarial antigen fractions (CFA2-1, 9, 11 and 12) from microfilaraemic cases, and of W. bancrofti microfilarial excretory/secretory antigen, to the immunoglobulin G (IgG) fraction of anti-filarial serum immunoglobulins. BmmfES was separated by ion-exchange chromatography on DEAE cellulose into 2 fractions, BmE DE1 and BmE DE2. BmE DE1 was marginally superior to whole BmmfES and BmE DE2 in detecting filarial IgG antibodies in human sera. 230 human sera from different groups of patients were screened against BmE DE1, which detected specific IgG in 83% of sera from microfilaraemic donors, 83% of sera from patients with clinical filariasis, 17% of sera from normal residents of an endemic area, and in none of the sera from persons living in a non-endemic area. The assay system using BmE DE1, with a sensitivity of 83% and specificity of 83%, should be very useful in detecting microfilaraemia, particularly in active clinical infections where the parasite is usually not seen.

Humoral immune response to infective larval antigen in Brugia malayi infected Mastomys natalensis.

The sequential changes in the humoral immune response against infective larval antigens during the course of Brugia malayi infection in Mastomys natalensis have been studied using enzyme linked immunosorbent assay. IgM antibody against B. malayi infective larval excretory secretory (ES) antigen was detected in the peripheral circulation within a week of infection, whereas IgM antibody against B. malayi infective larval somatic antigen and IgG antibody against both somatic and ES antigens were detected on day 20 post-inoculation. Thereafter, the antibody levels showed a steady increase until day 150. A gradual decrease of IgM antibody level was observed upto day 360, whereas IgG antibody level was decreased upto day 250 and then maintained almost the same level upto day 360. Wuchereria bancrofti cross reactive antigen as well as B. malayi infective larval ES antigen were detected in blood circulation on day 20, the level increased upto day 150 and then remained almost the same upto day 360 with slight variations. Studies of antigen and antibody levels in microfilaraemic and amicrofilaraemic animals show that there is no significant difference in antibody level whereas elevated antigen titre was observed in active infection with microfilaraemia.

Microfilaraemia, filarial antibody, antigen and immune complex levels in human filariasis before, during and after DEC therapy. A two-year follow-up.

A study on the effect of DEC therapy on microfilaraemia and the immune status in 27 patients with W. bancrofti infection was carried out for two years. Persistence of microfilaraemia was observed in 4 out of 27 cases after one course of DEC therapy and were treated again for one week. On further follow-up, none was microfilaraemic upto Day 60. The mean filarial antibody titres of IgM and IgG showed a gradual decrease as assessed by enzyme linked immunosorbent assay (ELISA). The mean titres of circulating microfilarial excretory-secretory (ES) antigens and immune complexes (ICs) showed an initial increase during therapy, followed by a gradual fall upto Day 60. Filarial antigen was detected in urine of all the carriers during therapy. Excretion pattern of antigen in urine showed correlation with DEC dose. Reappearance of microfilariae (mf) in circulation in 12 patients after a year showed that DEC had temporary attenuating effect on adult worms or no effect on developing larvae, suggesting further treatment and follow-up of patients. Parasitological and immunoscreening at the end of 2 years showed that the presence of mf ES antigen in blood correlated with the appearance of microfilariae in blood.

A 43-kDa circulating filarial antigen fraction of Wuchereria bancrofti in immunoprophylaxis against Brugia malayi in jirds.

A 43 kiloDaltan (kDa) antigen fraction (CFA2-6) isolated from microfilaraemic plasma of bancroftian filarial patients showed selective reactivity with sera samples collected from endemic normals. Antibodies raised against this antigen showed a strong reactivity with the surface of Brugia malayi infective larvae as well as microfilariae. Similar antigenic determinants were detected in the parasite extracts, but not in the excretory-secretory products. Further analysis was done on the immunoprophylactic potential of CFA2-6 in inducing immunity against Brugia malayi in Meriones unguiculatus (jird) in vivo. A strong protective response of approximately 84% was observed against the development of the filarial parasite in the jirds immunized with CFA2-6. The immunized jirds also showed a significant clearance (87%) of microfilariae inoculated intravenously. Approximately 65% of infective larvae failed to survive in jirds transferred with anti-CFA2-6 serum compared to the jirds transferred with sera from the control jirds. Passive transfer of anti-CFA2-6 antibody to the jirds followed by intravenous inoculation of microfilariae resulted in the reduction of 77% of circulating microfilariae. This study suggests that the 43-kDa CFA2-6 could stimulate a strong protective immune response against infective larvae and microfilariae in experimental animals.

Detection of S-100 protein and anticeramide antibodies in leprosy patients by ELISA.

The status of assay for S-100 antigen protein and anticeramide antibodies in serum in understanding nerve damage in different forms of leprosy were evaluated by the enzyme immunoassay. Based on the clinical and smear examination, patients were classified as indeterminate (Ind), tuberculoid (TT), borderline tuberculoid (BT), borderline lepromatous (BL) and lepromatous (LL). Antibody levels against ceramide were observed in sera of leprosy patients with 37.5% of Ind, 28% of TT, 66% BT, 78% BL and 62% LL patients positive as against 8% endemic normal sera. The mean OD ranged from 0.141 to 0.275 in different groups of leprosy. In contrast, S-100 was detected in 71.4% Ind, 88.8% TT, 76.4% BT, 100% BL and 95.8% LL, while 5% of ENL samples were positive for S-100 antigen. Mean S-100 levels in these different categories of patients were significantly higher Ind--0.45 ng/ml, TT--0.32 ng/ml, BT--0.23 ng/ml, BL--0.23 ng/ml, LL--0.19 ng/ml as compared to that of normal 0.07 ng/ml. In general S-100 seems to be a more sensitive and reliable marker than anticeramide antibodies for nerve damage. Five out of 7 indeterminate cases show increased levels of S-100, showing an extent of nerve damage similar to that of TT and could be a useful marker for assessing nerve damage in indeterminate patients for better management.

Relationship of C3 & C4 levels with filarial immune complexes in bancroftian filariasis.

C3 and C4 levels were determined by radial immuno-diffusion technique and filarial circulating immune-complexes by anti-C3 ELISA in microfilaraemic individuals and patients with clinical filariasis. Decreased levels of C3 and C4 were observed in both groups of filarial patients. Low levels of complement components were associated with low levels of circulating immune complexes.

Efficacy of two different DEC regimens in the treatment of human filarial infection.

Two regimens of diethyl carbamazine (DEC) viz., 14 day and 5 day, were compared for microfilaricidal effect and side effects, in the treatment of bancroftian filariasis. The rate of successful treatment, cure rate and decrease in mf count were found to be significantly high with the 14 days regimen when assessed immediately after treatment. About 40 per cent of subjects on the 14 days regimen and 66 per cent of patients on 5 days regimen experienced side reactions. The severity of side reactions was more in patients on 5 days regimen. When the effect of DEC was assessed one year after treatment with the 14 days regimen and compared with the results immediately after treatment, the rate of successful treatment, cure rate and decrease in mf count were reduced significantly. The 14 days DEC regimen with initial low dose of DEC along with antipyretics may be better accepted in the control programmes of filariasis.