Serodiagnosis of Childhood Tuberculosis by ELISA
http://www.100md.com
《美国医学杂志》
Jamnalal Bajaj Tropical Disease Research Centre and Department of Biochemistry, Mahatma Gandhi Institute of Medical Sciences, Sevagram, Wardha, Maharashtra, India
Abstract
Abstract. Objective: Diagnosis of childhood tuberculosis remains an enigma despite many recent technological developments. The present study has been taken up with the aim to assess the diagnostic potential of mycobacterium tuberculosis excretory-secretory ES-31 antigen and affinity purified anti ES-31 antibodies in the serodiagnosis of different spectrum of childhood tuberculosis. Methods: Mycobacterium tuberculosis H37Ra excretory-secretory antigen (ES-31) and affinity purified goat anti ES-31 antibodies were used in stick penicillinase ELISA for IgG antibody detection and stick Sandwich penicillinase ELISA for detection of circulating free and immune complexed antigen in the sera of 230 children. Results: Analysis of tubercular antibody, circulating free and immune complexed antigen (CIC-Ag) was done in both pulmonary and extrapulmonary form of childhood tuberculosis and overall sensitivity of 81.4% with a specificity of 93% was achieved for detection of antitubercular IgG antibodies. Of the five cases of pulmonary tuberculosis showing absence of IgG antibody, 3 showed the presence of CIC-Ag and one was found positive for both free and CIC-Ag. Similarly out of 8 cases of extrapulmonary childhood tuberculosis missed by IgG detection 5 were found to be positive for CIC-Ag and 1 showed the positive reaction for both free and immune complexed antigens. Conclusion: IgG antibody to excretory-secretory antigen ES-31 is found to be having good specificity with acceptable sensitivity in detecting different forms of childhood tuberculosis. Further detection of circulating free and / or immunecomplexed antigen can be used as an adjunct tool in the diagnosis of childhood tuberculosis. [Indian J Pediatr 2005; 72 (5) : -387]
Keywords: Immunodiagnosis; Childhood tuberculosis; ELISA; ES - 31
It has been a long journey for scientific community from March 24, 1882, when Robert Koch presented his widely acclaimed paper disclosing the etiological organism of tuberculosis first at a meeting of the physiological society of Berlin to June 11, 1998 when S.T. Cole and his team came out with deciphering the biology of mycobacterial tuberculosis from complete genome sequence. During this period millions of innocent children have died from tuberculosis, a chronic and cruel disease caused by tubercle bacillus and remains an important determinant of morbidity and mortality worldwide. The optimism shown with the advent of chemotherapy in the feasibility of control of tuberculosis has been shattered because of worldwide re-emergence of this disease which includes a large number of pediatric population. Every year, an estimated 3 million cases of TB and 4,50,000 associated deaths occur worldwide in children.
Early and precise diagnosis of childhood tuberculosis is necessary both in order to prevent mortality and morbidity and unjustified chemotherapy. Clinical manifestations being nonspecific, particularly in early stage and extra-pulmonary forms of tuberculosis, it remains easily misdiagnosed, under-diagnosed or paradoxically over treated. A history of recent exposure to a case of active tuberculosis, tuberculin test, chest radiographs and physical examination are often the only support for the diagnosis. The recovery of tubercle bacilli which would establish the diagnosis with certainty, is difficult in children. Various workers in the past have evaluated the utility of different techniques for early diagnosis of childhood TB. Attempts have been made to improve sensitivity and speed of detection of tubercle bacilli by techniques such as radiometric detection of bacterial growth, BACTEC, fluorescent antibody test, gas chromatography, DNA hybridization, PCR and RIA.
However, all of these methods have problems of either sensitivity or specificity or too technically demanding1. ELISA has been found to be a potentially valuable and simple technique[2] Many studies have been undertaken to study its usefulness in adults and children using either crude bacillary antigens[3], tuberculin purified protein derivatives[4] or purified mycobacterial antigens[5] But ironically we are still far from coming out with a simple, reliable, cost effective test with reasonably good sensitivity and specificity which could be used widely in diagnosing different forms of childhood tuberculosis.
So for the future of serodiagnosis of TB, new approaches are required, like looking at antibody responses to actively secreted antigen by living mycobacteria[6] rather than by more usually studied cytoplasmic antigens liberated by sonication or other mechanical measures. The continued release of excretory secretory antigens results in adjuvant effect to evoke good humoral immune response in the host. In our laboratory excretory secretory antigens obtained by in vitro maintenance of tubercular bacilli H37Ra strain have been explored extensively in the diagnosis of tuberculosis.[7-13] Many studies have been undertaken to evaluate the diagnostic potential of ELISA in isolated forms of childhood tuberculosis.
But the studies involving different spectrum of childhood tuberculosis (pulmonary and extrapulmonary) along with inclusion of children with diseases mimicking tuberculosis and healthy children as controls are very few in literature. So this study has been undertaken to evaluate the role of ES-31 antigen and affinity purified anti ES-31 antibodies in the serological diagnosis of different forms of childhood tuberculosis.
MATERIAL AND METHODS
Patients and controls
A total of 230 serum samples were collected from pediatric population, being investigated for tuberculosis, of Wardha district, attending Kasturba Hospital, Mahatma Gandhi Institute of Medical Sciences, Sevagram and Civil Hospital, Wardha (India). These also include serum samples from 55 age and sex matched healthy subjects.
Serum sample were collected from all the subjects at the start of investigations for tuberculosis before any treatment began. 70 of the 175 patients were subsequently found to have tuberculosis. Out of these 70 cases of tuberculosis 30 patients were diagnosed as having pulmonary tuberculosis and 40 cases with extrapulmonary tuberculosis. The sites of extrapulmonary disease in these 40 children were meninges (20), pleura (10), and lymphnodes (10). Nine children with EPTB were also found to have associated pulmonary tuberculosis and out of 30 cases of pulmonary tuberculosis, the gastric aspirates of 5 children were found to be AFB smear and culture positive and 3 of the 30 patients had a history of treated tuberculosis.
105 children were diagnosed as having a disorder other than tuberculosis, 25 had bronchopneumonia, 20 protein energy malnutrition, 5 bronchial asthma, 10 pyogenic meningitis, 10 P.U.O, 4 seizures, 6 encephalitis, 8 nontubercular pleural effusion, 4 empyema, 3 CCF, 8 Non specific lymphadenitis and 2 lymphoma.
Case selection and diagnosis of childhood tuberculosis
Tuberculosis was suspected on history of close contact with a case of active tuberculosis, clinical symptoms and signs suggestive of tuberculosis, histocytological studies, Montoux reactivity, ESR, Gastric aspirates for AFB, chest skiagrams, computerized tomography (CT) scan of head showing basal exudates in case of tubercular meningitis. The biochemistry and cytology of the CSF and pleural fluids showing typical exudative nature with variable number of lymphocytic infiltration and elevated proteins and response to antitubercular treatment were also taken to consideration. Clinician's high degree of suspicion of tuberculosis, depending upon clinical sign and symptoms, characteristic CSF and pleural fluid findings, histopathology alongwith neuroimaging studies played an important role for diagnosing tuberculosis.
Isolation of antigen and antibody
M. tb H37Ra excretory secretory antigen Tb ES- 31, and affinity purified goat anti ES-31 antibodies were isolated as described by Nair et al[9]
ELISA
Stick Indirect Penicillinase ELISA for tuberclous IgG antibody detection and stick sandwich - penicillinase ELISA for detection of circulating free and immune complexed antigen (CIC-Ag) in sera were carried out as described by Nair et al[10] While detecting CIC-Ag, serum samples were pretreated with glycine HCL buffer (0.1M, pH 2.8) followed by heating at 650C for 15 minutes[12]
RESULTS
0The analytical results of 230 sera for detection of mycobacterium tuberculosis IgG antibodies using M. tb ES-31 antigen and circulating free and immune complexed antigen using affinity purified anti ES-31 antibodies are summarized in Table 1 and 2.
At cut off 1:600 dilution for positive reaction for antibody detection 83.3% (25/30) of childhood pulmonary TB sera and 80% (32/40) of children with extrapulmonary tuberculosis showed positive reaction whereas 6.6.% (4/60) disease and 6% (1/15) healthy controls for pulmonary tuberculosis and 8.8.% (4/45) disease and 5% (2/40) healthy controls for EPTB showed cross-reaction with tuberculous antigen giving a specificity of 93.3% and 92.9% respectively. Out of 5 cases found negative for IgG antibody 3 showed the presence of circulating immune complex antigen whereas one was found positive for both circulating free as well as immune complexed antigen.
When affinity purified goat anti ES-31 antibodies were employed in sandwich penicillinase ELISA for free antigen, the positivity obtained was 73.3% (22/30) and 72.5% (29/40) in pulmonary and extrapulmonary tuberculosis with specificities of 96% and 95.2% respectively whereas the assay for circulating immune complexed antigen detection yielded 86.6% (26/30) and 85% (34/40) positivity with corresponding specificity of 90.6% and 89.4% in pulmonary and extrapulmonary forms of childhood tuberculosis respectively. Out of 8 cases of EPTB showing negative reaction for IgG antibody, 5 cases showed positive reaction for CIC antigen whereas one showed positive reaction for both circulating free and CIC antigen.
DISCUSSION
0The usefulness of serodignosis of tuberculosis using excretory-secretory H37Ra ES antigen has been well emphasized in the previous studies from this laboratory.[7-13] However, studies involving different spectrum of childhood tuberculosis and co-relating the antibody and free and CIC antigen in the sera of children suffering from different forms of tuberculosis are quite scanty in literature. Therefore, this study has been undertaken to evaluate the usefulness of M.tb ES-31 antigen and affinity purified anti ES-31 antibodies to detect tubercular IgG antibodies and free as well as CIC antigen to possibly pick up the maximum number of true positive childhood TB cases.
The overall sensitivity and specificity of the present study assay for detection of IgG antibody in the sera of children with tuberculosis was 81.4% and 93% respectively. Patel et al[14] has reported a low sensitivity of 65% with a specificity of 92% with A-60 antigen. Contradictory results have been achieved by Delacourt[15] and S. Gupta[16] using A-60 antigen, whereas Delacourt has reported a sensitivity of 68% with a specificity of 98% for detecting IgG, antibodies to A-60 antigen, S. Gupta has reported the positivity of only 32.7% and 36.6% for IgG antibody in pulmonary and extrapulmnary tuberculosis respectively. Bothamley et al[17] reported a specificity of 95% with a sensitivity of only 44% in histologically and microbiologically proven cases in pediatric TB using SACT assay. Alde et al[18] reported a sensitivity of 85% and specificity of 100% using antigen 5 but have included a large number of AFB positive cases in their study. 21 AFB positive cases out of a total of 40 cases of childhood TB which is not the true clinical picture observed while treating pediatric cases and also inclusion of a low number of control subjects (n=19) limits the interpretation of his study. Children rarely produce sputum for smear examination and gastric aspirates have shown a very low positivity for AFB. In the present study the authors could get only five AFB positive cases out of 30 cases of pulmonary TB. All these five cases were found to be positive by this immunoassay.
A sensitivity of 83.3% with comparable specificity of 93.3% for IgG antibody detection was achieved in the cases of pulmonary tuberculosis which is better than reported by other workers in pauci bacillary and smear negative pulmonary tuberculosis.[16] In the serological diagnosis of pleural effusion false positivity had been a problem.[19] But with the use of purified ES-31 antigen we could observe a sensitivity of 70% with 92% specificity. In the cases of tuberclar lymphadenopathy 8 out of 10 were positive for antitubercular antibody giving a sensitivity and specificity of 80% and 95% respectively. In the detection of TBM cases 85% has shown positivity with specificity of 93.3% for tubercular IgG antibody in the sera samples. One out of 10 was positive for antibody and one for IC-Ag in pyogenic meningitis sera. Previous work in this laboratory using this Excretory-Secretory antigen by Parthak Prodhan[20], has shown that BCG vaccinated children do not show positive reaction for ES antigen antibody and hence BCG vaccinated were not treated as separate group. In general the authors find almost all children are with BCG scar showing that they have been BCG vaccinated and did not react to ES-31 antigen. Further, we did explore for any possible crossreactivity of ES-31 antigen with antibody raised against Staphylococcus aureus (Sigma). No crossreactivity was observed (unpublished observations). The children positive for antibody, IC-Ag may possibly be having subclinical Tb. Follow up of the case may confirm if any in future. In contrast Patel et al[14] has reported 50% positivity in TBM cases using A-60 antigen. Most of the previous studies have evaluated the utility of ELISA in CSF samples for detection of antituberclar antibody and antigen in diagnosis of TBM.[21] Studies on detecting M.tb antibodies and antigen using sera of patients suffering from TBM are very less in number and the present study has shown encouraging results.
The concomitant presence of M.tb ES-31 free antigen in 72.5% and CIC antigen in 85% of sera samples of childhood TB cases were detected. The IgG antibody detection assay to ES-31 antigen has been found to be better in all with 83.3% sensitivity and 93.3% specificity. In 5 cases of pulmonary TB and 6 cases EPTB IgG antibody could not be detected, but cases could be confirmed by the presence of circulating free and / or CIC antigen. Indeed antibodies may have been present in sera from these patients but remained undetected because of being entrapped in circulating immune complexes. Similar observations have been made by other workers.[22]
Since most of the control patients had disease provisionally diagnosed as tuberculosis, the results achieved indicate the specificity of the test in discriminating active tuberculosis from other diseases that mimmick it.
T he present study has shown the potential value of detecting IgG antibody to M.tb ES-31 antigen for diagnosis of different spectrum of childhood tuberculosis, where one does not have many other diagnostic modalities to choose from. The results indicate that the test detects only a few healthy controls and children with other diseases mimicking tuberculosis, thus giving it a high degree of specificity. Further, the assay of circulating free and immune complexed antigen can be used an adjunct tool for rapid confirmation of childhood tubercular cases.
Acknowledgements
This study was supported by grants from Kasturba Health Society and CSIR. We thank Shri. Dhirubhai Mehta; President, KHS for his keen interest and encouragement for this study. Our thanks are due to Dr. (Mrs) P. Chaturvedi, Prof. & Head, Prof. K.Y. Vilhekar & Dr. Manish Jain of Pediatrics Dept. MGIMS, Sevagram and Dr. Kalsait, District Tuberculosis Officer, Wardha for extending cooperation in follow-up of patients. Technical Assistance of Mrs. S. Ingole is appreciated
References
1. Grange JM, Laszlo A. Serodiagnostic tests for tuberculosis: a need for assessment for their operational predictive accuracy and acceptability. Bull WHO 1990; 68 : 571-576.
2. Daniel TM, Debanne SM. The serodiagnosis of tuberculosis and other mycobacterial diseases by enzyme-linked immunosorbant assay. Am Rev Respir Dis 1987; 135 : 1137 - 1151.
3. Kardjito T, Handoyo I. Diagnosis of active tuberculosis by immunological methods: The effect of tuberculin reactivity and previous BCG vaccination on the antibody levels determined by ELISA. Tubercle 1982 ; 63 : 269-274.
4. Redin RC, Zeiss CR, Phair JP. Antibodies to purified protein derivative in different immunoglobulin classes in the diagnosis of tuberculosis in man. Int Arch Allergy Appl Immunol 1983; 70: 25-29.
5. Chan SL, Reggiardo Z, Daniel TM, Girling DJ, Mitchison DA. Serodiagnosis of tuberculosis using an ELISA with antigen 5 and a haemagglutination assay with glycolipid antigen. Am Rev Respir Dis 1990; 142: 385 - 390.
6. Grange JM. The rapid diagnosis of paucibacillary tuberculosis. Tubercle 1989; 70: 1- 4.
7. Kumar S, Chenthamarakshan V, Reddy MVR, Narang P, Gupta OP, Harinath BC. Detection of tuberculous IgG antibody using M.tb H37Ra excretory - secretory antigen and tuberculin purified protein derivative. Ind J Exp Biol 1994; 32: 163 - 167.
8. Lodam AN, Reddy MVR, Harinath BC. Immunodiagnosis of pulmonary tuberculosis by concomitant detection of antigen and antibody to excretory-secretory protein of Mycobacterium tuberculosis H37Ra . J Biosci 1998; 1: 19 - 23.
9. Nair ER, Banerjee S, Kumar S, Reddy MVR, Harinath BC. : Isolation of Mycobacterium tuberculosis 31 kDa antigen protein of diagnostic interest from culture filtrate using anti ES-31 antibody by affinity chromatography. Ind J Clin Biochem 2001; 16: 132-135.
10. Nair ER, Banerjee S, Kumar S, Reddy MVR, Harinath BC. Purification and characterization of 31 kDa mycobacterial excretory-secretory antigenic protein with a diagnostic potential in pulmonary tuberculosis. Scand J Infect Dis 2000;32 : 551-556.
11. Banerjee S, Nair ER, Kumar S, Reddy MVR, Harinath BC. Assay of tubercular antibody, circulating free and immune complexed antigen in the diagnosis of pulmonary tuberculosis. Ind J Clin Biochem 2001; 16 (2) : 203 - 206.
12. Prasad GBKS, Kharat I, Harinath BC. Detection of antimicrofilarial ES antigen - antibody in immune complexes in bancroftian filariasis by enzyme immunoassays. Trans Roy Soc Trop Med Hyg 1983; 77 (6): 771 - 772.
13. Bhaskar A, Pradhan P, Chaturvedi P et al. Immunodiagnosis of childhood pulmonary and extrapulmonary TB using M.tb ES antigen by penicilinase ELISA. Annals Trop Pediatrics 1994;14 : 25-30.
14. Patil AB, Rawat MS, Grover S. ELISA for diagnosis of tuberculosis. Indian Pediatr 1988; 27 : 585 - 589.
15. Delacourt C, Gobin J, Gaillard JL, Blic DJ, Veron M, Scheinmann P. Value of ELISA using antigen 60 for the diag-nosis of tuberculosis in children. Chest 1993; 104(2) : 393- 398.
16. Gupta S, Bhatia R, Datta KK. Serological diagnosis of childhood tuberculosis by estimation of mycobacterial antigen 60 - specific immunoglobulins in the serum. Tubercle and Lung Disease 1997; 78 (1): 21-27.
17. Bothamley G, Udani P, Rudd R, Pastenstein P, Ivanyi J. Humoral response to defined epitopes of tubercule bacilli in adult pulmonary and child tuberculosis. Eur J Clin Microbiol Infect Dis 1988; 7(5) : 637-645.
18. Alde SLM, Pinasco HM, Pelossi RR, Budani HF, Plasma Betran OH, Gonzaleg - Montaner LJ. Evaluation of an enzyme linked immunosorbant assay (ELISA) using an IgG antibody to Mycobacterium Tuberculosis antigen 5 in the diagnosis of acute tuberculosis in children. Am Rev Resir Dis 1989; 139 : 748-751.
19. Dhand R, Gunguli NK, Vaishnavi C, Gilhatra R, Malik SK. False positive assay of Mycobacterium tuberculosis antigens in pleural fluid. Med Microbiol 1998; 26: 241-243.
20. Prodhan P. ELISA using ES-antigen of mycobacterium tuberculosis in Immunodiagnosis of childhood tuberculosis. Thesis submitted for Doctor of Medicine (Pediatrics), Nagpur University, 1992-93.
21. Patel SA, Gourie Devi M, Anand AR, Vijaya AN et al. Significance of mycobacterial immune complexes (IgG) in the diagnosis of tuberculous meningitis. Tubercle Lung Dis 1996; 77 : 164-167.
22. Khomenko AG, Bayensky AV, Chenousova NL, Kulikovskaya NV et al. Serodiagnois of tuberculosis: detection of mycobacterial antibodies and antigens. Tuber Lung Dis 1996; 77 : 510-515(Bhatia AS, Gupta Sonika, )
Abstract
Abstract. Objective: Diagnosis of childhood tuberculosis remains an enigma despite many recent technological developments. The present study has been taken up with the aim to assess the diagnostic potential of mycobacterium tuberculosis excretory-secretory ES-31 antigen and affinity purified anti ES-31 antibodies in the serodiagnosis of different spectrum of childhood tuberculosis. Methods: Mycobacterium tuberculosis H37Ra excretory-secretory antigen (ES-31) and affinity purified goat anti ES-31 antibodies were used in stick penicillinase ELISA for IgG antibody detection and stick Sandwich penicillinase ELISA for detection of circulating free and immune complexed antigen in the sera of 230 children. Results: Analysis of tubercular antibody, circulating free and immune complexed antigen (CIC-Ag) was done in both pulmonary and extrapulmonary form of childhood tuberculosis and overall sensitivity of 81.4% with a specificity of 93% was achieved for detection of antitubercular IgG antibodies. Of the five cases of pulmonary tuberculosis showing absence of IgG antibody, 3 showed the presence of CIC-Ag and one was found positive for both free and CIC-Ag. Similarly out of 8 cases of extrapulmonary childhood tuberculosis missed by IgG detection 5 were found to be positive for CIC-Ag and 1 showed the positive reaction for both free and immune complexed antigens. Conclusion: IgG antibody to excretory-secretory antigen ES-31 is found to be having good specificity with acceptable sensitivity in detecting different forms of childhood tuberculosis. Further detection of circulating free and / or immunecomplexed antigen can be used as an adjunct tool in the diagnosis of childhood tuberculosis. [Indian J Pediatr 2005; 72 (5) : -387]
Keywords: Immunodiagnosis; Childhood tuberculosis; ELISA; ES - 31
It has been a long journey for scientific community from March 24, 1882, when Robert Koch presented his widely acclaimed paper disclosing the etiological organism of tuberculosis first at a meeting of the physiological society of Berlin to June 11, 1998 when S.T. Cole and his team came out with deciphering the biology of mycobacterial tuberculosis from complete genome sequence. During this period millions of innocent children have died from tuberculosis, a chronic and cruel disease caused by tubercle bacillus and remains an important determinant of morbidity and mortality worldwide. The optimism shown with the advent of chemotherapy in the feasibility of control of tuberculosis has been shattered because of worldwide re-emergence of this disease which includes a large number of pediatric population. Every year, an estimated 3 million cases of TB and 4,50,000 associated deaths occur worldwide in children.
Early and precise diagnosis of childhood tuberculosis is necessary both in order to prevent mortality and morbidity and unjustified chemotherapy. Clinical manifestations being nonspecific, particularly in early stage and extra-pulmonary forms of tuberculosis, it remains easily misdiagnosed, under-diagnosed or paradoxically over treated. A history of recent exposure to a case of active tuberculosis, tuberculin test, chest radiographs and physical examination are often the only support for the diagnosis. The recovery of tubercle bacilli which would establish the diagnosis with certainty, is difficult in children. Various workers in the past have evaluated the utility of different techniques for early diagnosis of childhood TB. Attempts have been made to improve sensitivity and speed of detection of tubercle bacilli by techniques such as radiometric detection of bacterial growth, BACTEC, fluorescent antibody test, gas chromatography, DNA hybridization, PCR and RIA.
However, all of these methods have problems of either sensitivity or specificity or too technically demanding1. ELISA has been found to be a potentially valuable and simple technique[2] Many studies have been undertaken to study its usefulness in adults and children using either crude bacillary antigens[3], tuberculin purified protein derivatives[4] or purified mycobacterial antigens[5] But ironically we are still far from coming out with a simple, reliable, cost effective test with reasonably good sensitivity and specificity which could be used widely in diagnosing different forms of childhood tuberculosis.
So for the future of serodiagnosis of TB, new approaches are required, like looking at antibody responses to actively secreted antigen by living mycobacteria[6] rather than by more usually studied cytoplasmic antigens liberated by sonication or other mechanical measures. The continued release of excretory secretory antigens results in adjuvant effect to evoke good humoral immune response in the host. In our laboratory excretory secretory antigens obtained by in vitro maintenance of tubercular bacilli H37Ra strain have been explored extensively in the diagnosis of tuberculosis.[7-13] Many studies have been undertaken to evaluate the diagnostic potential of ELISA in isolated forms of childhood tuberculosis.
But the studies involving different spectrum of childhood tuberculosis (pulmonary and extrapulmonary) along with inclusion of children with diseases mimicking tuberculosis and healthy children as controls are very few in literature. So this study has been undertaken to evaluate the role of ES-31 antigen and affinity purified anti ES-31 antibodies in the serological diagnosis of different forms of childhood tuberculosis.
MATERIAL AND METHODS
Patients and controls
A total of 230 serum samples were collected from pediatric population, being investigated for tuberculosis, of Wardha district, attending Kasturba Hospital, Mahatma Gandhi Institute of Medical Sciences, Sevagram and Civil Hospital, Wardha (India). These also include serum samples from 55 age and sex matched healthy subjects.
Serum sample were collected from all the subjects at the start of investigations for tuberculosis before any treatment began. 70 of the 175 patients were subsequently found to have tuberculosis. Out of these 70 cases of tuberculosis 30 patients were diagnosed as having pulmonary tuberculosis and 40 cases with extrapulmonary tuberculosis. The sites of extrapulmonary disease in these 40 children were meninges (20), pleura (10), and lymphnodes (10). Nine children with EPTB were also found to have associated pulmonary tuberculosis and out of 30 cases of pulmonary tuberculosis, the gastric aspirates of 5 children were found to be AFB smear and culture positive and 3 of the 30 patients had a history of treated tuberculosis.
105 children were diagnosed as having a disorder other than tuberculosis, 25 had bronchopneumonia, 20 protein energy malnutrition, 5 bronchial asthma, 10 pyogenic meningitis, 10 P.U.O, 4 seizures, 6 encephalitis, 8 nontubercular pleural effusion, 4 empyema, 3 CCF, 8 Non specific lymphadenitis and 2 lymphoma.
Case selection and diagnosis of childhood tuberculosis
Tuberculosis was suspected on history of close contact with a case of active tuberculosis, clinical symptoms and signs suggestive of tuberculosis, histocytological studies, Montoux reactivity, ESR, Gastric aspirates for AFB, chest skiagrams, computerized tomography (CT) scan of head showing basal exudates in case of tubercular meningitis. The biochemistry and cytology of the CSF and pleural fluids showing typical exudative nature with variable number of lymphocytic infiltration and elevated proteins and response to antitubercular treatment were also taken to consideration. Clinician's high degree of suspicion of tuberculosis, depending upon clinical sign and symptoms, characteristic CSF and pleural fluid findings, histopathology alongwith neuroimaging studies played an important role for diagnosing tuberculosis.
Isolation of antigen and antibody
M. tb H37Ra excretory secretory antigen Tb ES- 31, and affinity purified goat anti ES-31 antibodies were isolated as described by Nair et al[9]
ELISA
Stick Indirect Penicillinase ELISA for tuberclous IgG antibody detection and stick sandwich - penicillinase ELISA for detection of circulating free and immune complexed antigen (CIC-Ag) in sera were carried out as described by Nair et al[10] While detecting CIC-Ag, serum samples were pretreated with glycine HCL buffer (0.1M, pH 2.8) followed by heating at 650C for 15 minutes[12]
RESULTS
0The analytical results of 230 sera for detection of mycobacterium tuberculosis IgG antibodies using M. tb ES-31 antigen and circulating free and immune complexed antigen using affinity purified anti ES-31 antibodies are summarized in Table 1 and 2.
At cut off 1:600 dilution for positive reaction for antibody detection 83.3% (25/30) of childhood pulmonary TB sera and 80% (32/40) of children with extrapulmonary tuberculosis showed positive reaction whereas 6.6.% (4/60) disease and 6% (1/15) healthy controls for pulmonary tuberculosis and 8.8.% (4/45) disease and 5% (2/40) healthy controls for EPTB showed cross-reaction with tuberculous antigen giving a specificity of 93.3% and 92.9% respectively. Out of 5 cases found negative for IgG antibody 3 showed the presence of circulating immune complex antigen whereas one was found positive for both circulating free as well as immune complexed antigen.
When affinity purified goat anti ES-31 antibodies were employed in sandwich penicillinase ELISA for free antigen, the positivity obtained was 73.3% (22/30) and 72.5% (29/40) in pulmonary and extrapulmonary tuberculosis with specificities of 96% and 95.2% respectively whereas the assay for circulating immune complexed antigen detection yielded 86.6% (26/30) and 85% (34/40) positivity with corresponding specificity of 90.6% and 89.4% in pulmonary and extrapulmonary forms of childhood tuberculosis respectively. Out of 8 cases of EPTB showing negative reaction for IgG antibody, 5 cases showed positive reaction for CIC antigen whereas one showed positive reaction for both circulating free and CIC antigen.
DISCUSSION
0The usefulness of serodignosis of tuberculosis using excretory-secretory H37Ra ES antigen has been well emphasized in the previous studies from this laboratory.[7-13] However, studies involving different spectrum of childhood tuberculosis and co-relating the antibody and free and CIC antigen in the sera of children suffering from different forms of tuberculosis are quite scanty in literature. Therefore, this study has been undertaken to evaluate the usefulness of M.tb ES-31 antigen and affinity purified anti ES-31 antibodies to detect tubercular IgG antibodies and free as well as CIC antigen to possibly pick up the maximum number of true positive childhood TB cases.
The overall sensitivity and specificity of the present study assay for detection of IgG antibody in the sera of children with tuberculosis was 81.4% and 93% respectively. Patel et al[14] has reported a low sensitivity of 65% with a specificity of 92% with A-60 antigen. Contradictory results have been achieved by Delacourt[15] and S. Gupta[16] using A-60 antigen, whereas Delacourt has reported a sensitivity of 68% with a specificity of 98% for detecting IgG, antibodies to A-60 antigen, S. Gupta has reported the positivity of only 32.7% and 36.6% for IgG antibody in pulmonary and extrapulmnary tuberculosis respectively. Bothamley et al[17] reported a specificity of 95% with a sensitivity of only 44% in histologically and microbiologically proven cases in pediatric TB using SACT assay. Alde et al[18] reported a sensitivity of 85% and specificity of 100% using antigen 5 but have included a large number of AFB positive cases in their study. 21 AFB positive cases out of a total of 40 cases of childhood TB which is not the true clinical picture observed while treating pediatric cases and also inclusion of a low number of control subjects (n=19) limits the interpretation of his study. Children rarely produce sputum for smear examination and gastric aspirates have shown a very low positivity for AFB. In the present study the authors could get only five AFB positive cases out of 30 cases of pulmonary TB. All these five cases were found to be positive by this immunoassay.
A sensitivity of 83.3% with comparable specificity of 93.3% for IgG antibody detection was achieved in the cases of pulmonary tuberculosis which is better than reported by other workers in pauci bacillary and smear negative pulmonary tuberculosis.[16] In the serological diagnosis of pleural effusion false positivity had been a problem.[19] But with the use of purified ES-31 antigen we could observe a sensitivity of 70% with 92% specificity. In the cases of tuberclar lymphadenopathy 8 out of 10 were positive for antitubercular antibody giving a sensitivity and specificity of 80% and 95% respectively. In the detection of TBM cases 85% has shown positivity with specificity of 93.3% for tubercular IgG antibody in the sera samples. One out of 10 was positive for antibody and one for IC-Ag in pyogenic meningitis sera. Previous work in this laboratory using this Excretory-Secretory antigen by Parthak Prodhan[20], has shown that BCG vaccinated children do not show positive reaction for ES antigen antibody and hence BCG vaccinated were not treated as separate group. In general the authors find almost all children are with BCG scar showing that they have been BCG vaccinated and did not react to ES-31 antigen. Further, we did explore for any possible crossreactivity of ES-31 antigen with antibody raised against Staphylococcus aureus (Sigma). No crossreactivity was observed (unpublished observations). The children positive for antibody, IC-Ag may possibly be having subclinical Tb. Follow up of the case may confirm if any in future. In contrast Patel et al[14] has reported 50% positivity in TBM cases using A-60 antigen. Most of the previous studies have evaluated the utility of ELISA in CSF samples for detection of antituberclar antibody and antigen in diagnosis of TBM.[21] Studies on detecting M.tb antibodies and antigen using sera of patients suffering from TBM are very less in number and the present study has shown encouraging results.
The concomitant presence of M.tb ES-31 free antigen in 72.5% and CIC antigen in 85% of sera samples of childhood TB cases were detected. The IgG antibody detection assay to ES-31 antigen has been found to be better in all with 83.3% sensitivity and 93.3% specificity. In 5 cases of pulmonary TB and 6 cases EPTB IgG antibody could not be detected, but cases could be confirmed by the presence of circulating free and / or CIC antigen. Indeed antibodies may have been present in sera from these patients but remained undetected because of being entrapped in circulating immune complexes. Similar observations have been made by other workers.[22]
Since most of the control patients had disease provisionally diagnosed as tuberculosis, the results achieved indicate the specificity of the test in discriminating active tuberculosis from other diseases that mimmick it.
T he present study has shown the potential value of detecting IgG antibody to M.tb ES-31 antigen for diagnosis of different spectrum of childhood tuberculosis, where one does not have many other diagnostic modalities to choose from. The results indicate that the test detects only a few healthy controls and children with other diseases mimicking tuberculosis, thus giving it a high degree of specificity. Further, the assay of circulating free and immune complexed antigen can be used an adjunct tool for rapid confirmation of childhood tubercular cases.
Acknowledgements
This study was supported by grants from Kasturba Health Society and CSIR. We thank Shri. Dhirubhai Mehta; President, KHS for his keen interest and encouragement for this study. Our thanks are due to Dr. (Mrs) P. Chaturvedi, Prof. & Head, Prof. K.Y. Vilhekar & Dr. Manish Jain of Pediatrics Dept. MGIMS, Sevagram and Dr. Kalsait, District Tuberculosis Officer, Wardha for extending cooperation in follow-up of patients. Technical Assistance of Mrs. S. Ingole is appreciated
References
1. Grange JM, Laszlo A. Serodiagnostic tests for tuberculosis: a need for assessment for their operational predictive accuracy and acceptability. Bull WHO 1990; 68 : 571-576.
2. Daniel TM, Debanne SM. The serodiagnosis of tuberculosis and other mycobacterial diseases by enzyme-linked immunosorbant assay. Am Rev Respir Dis 1987; 135 : 1137 - 1151.
3. Kardjito T, Handoyo I. Diagnosis of active tuberculosis by immunological methods: The effect of tuberculin reactivity and previous BCG vaccination on the antibody levels determined by ELISA. Tubercle 1982 ; 63 : 269-274.
4. Redin RC, Zeiss CR, Phair JP. Antibodies to purified protein derivative in different immunoglobulin classes in the diagnosis of tuberculosis in man. Int Arch Allergy Appl Immunol 1983; 70: 25-29.
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