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313-PA10 Identification of mycobacteria isolates using polymerase chain reaction
Abstracts
zation. The A60WB test was then used to evaluate the incidence of false negative PPD tests in a group of 49 tuberculosis (TB) exposed health care workers exposed to > 30 TB cases per year. Clerical workers (n = 32) were evaluated as controls. TB exposed workers showed a significantly higher incidence of PPD negative skin tests among immunized individuals (A60WB-positive 98%, PPD-positive 76%, P < 0 . 0 0 5 ) while no differences were found in non-exposed workers (A60WB 28%, PPD 25%; P > 0.7). The data indicate that tuberculin skin testing with PPD may underestimate TB contagion in populations at risk.
288-PA10 Anti-PPD and anti-DAT serum antibody markers for the diagnosis of tuberculosis (TB) in the HIV-seropositive (HIV ÷) Amicosante, M. 1, Cuboni, A. 2, Ancora, A. 1, Monno, L. 3, Girardi, E. 4, Angarano, G. 3, Saltini, C. 1 1Dept Medical Sciences, Sect. Respiratory Medicine, University of Modena; 20spedale Civile, Sesto S. Giovanni, Milano; 31nfectious Diseases Institute, University of Bari; 4Centro di Riferimento AIDS, Azienda Ospedaliera Forlanini, S. Camillo, Spallanzani, Roma, Italy
Previous serological studies have shown that M. tuberculosis (M.tb)-specific antibody markers may diagnose anti-M.tb immunity, hence, latent infection and TB risk, in HIV +, PPD-, recall antigen-anergic subjects. This study was designed to evaluate the combined use of two serological markers, the IgM ELISA against PPD (PPD-IgM) (1) and the IgG ELISA against 2,3diacyltrehalose (DAT-IgG) (2) for the diagnosis of active TB and of TB risk in HIV ÷ subjects. A cohort of 102 HIV + individuals (age 29 + 8 years, 80% males; 46% PPD+; Multitest score 1.5 + 1.2; CD4 335 +- 214 cells/ram 3) was evaluated at enrolment and after 12 months. In addition, a sample of 14 HIV ÷ subjects with active TB (age 29 + 5; 86% males; 33% PPD+; 100% culture+) was evaluated a year before and at the time of TB diagnosis. Among cohort subjects, 14 (14%) were PPD-IgM + at enrolment [3 (21%) DAT-IgG*] and 8/64 (12%) were PPD-IgM + after one year [6 (75%) DATIgGq. Of the three subjects who developed TB two were culture + PPD-IgM ÷ and DAT-IgG + (Mantel-Hansel, P < 0.001). In the active TB patient sample, 9 (64%) were PPD-IgM ÷, 8 (57%) DAT-IgG + and 11 (79%) PPDIgM ÷ and/or DAT-IgG ÷. One year before diagnosis, 7 (50%) were PPD-IgM ÷, 5 (36%) DAT-IgG+ and 9 (64%) PPD-IgM ÷ and/or DAT-IgG ÷. The data indicate that the two antibody markers may be highly sensitive in the diagnosis of active TB and of TB risk in HIV ÷ subjects.
295-PA10 Reliability of ELISA in diagnosis of pulmonary tuberculosis EL-Feky, A. 1, Abou Shehatta, M. 1, Khaled, A. 1, EL-Morsi, A. l, Agha, S.2, Viljanen, M 3. Departments of Thoracic Medicine 1, Clinical Pathology 2, Mansoura University, Egypt and National Public Health Institute 3, Turku, Finland
Data on ELISA as a method of serodiagnosis in tuberculosis are contradictory. Therefore its role needs to be
19
reassessed. In this work we studied 61 Egyptian cases with clinical suspicion of pulmonary tuberculosis, 39 males and 22 females with age ranging between 16 and 59 years. After clinical and radiological reevaluation they were categorized into 2 groups: group A high probability of pulmonary tuberculosis (44 cases) and group B low probability of pulmonary tuberculosis (17 cases). For all cases sputum examination for Mycobacterium tb by direct smear, culture, PCR and by Amplicor were carfled out. The sera were tested using ELISA for detection of IgG and IgA antibodies against Mycobacterium tuberculosis. The results were as follows: Direct smear Group A 47.7% Group B 11.7%
Culture
PCR
Amplicor ELISA
31.8% 0%
54.4% 5.8%
47.4% 5.8%
56.8% 41%
These values show that in group A ELISA has sensitivity comparable to PCR. When combining results of smear with that of ELISA in the same group the sensitivity increased to reach 61.3%. However, 2.4% of cases were positive by ELISA but not by other tests, i.e false positive (data not shown). On the other hand in group B although ELISA was positive in 41%, only 11.7% was diagnosed by sputum of PCR, i.e 29.3% of low probability cases were seropositive with no detectable mycobacterium by other tests (data not shown). In conclusion when clinical suspicion is nigh ELISA may be of value in diagnosis of pulmonary tuberculosis, but with low clinical suspicion, the results should be evaluated cautiously and bacteriological confirmation is needed.
313-PAl0 Identification of mycobacteria isolates using polymerase chain reaction Bahrmand, A., Bakaev, B., Madani, H., Saifi, M. Pasteur Institute of lran, Tehran, lran
Tuberculosis (TB) and diseases caused by nontuberculous mycobacteria (NTM) reveal similar clinical manifestations including roentgenography evidence. The protocol of TB treatment differs from that of other mycobacterioses; however, clinical methods commonly used for diagnosis lack the necessary sensitivity and speed. To obviate these difficulties and to identify organisms involved, bacteriological procedures and nested PCR assay with primers specific for M. boris tuberculosis were applied to 329 specimens. The nested PCR assay has demonstrated the highest capability to discover pathogens in sputum: 274 specimens were TB positive by PCR, 224 by culture, and 174 by BACTEC assay. Of the 55 TB negatives, 21 specimens were found to contain NTM. Primers specific for M. fortuitum and M. kansasii were used to discriminate between these and other NTM. The proposed PCR assays having high sensitivity and specificity can support
20
Tubercle and Lung Disease: Supplement 2
a rapid diagnosis and monitoring efficiency of drug therapy in laboratories where routine work with labelled probes is not acceptable.
firmed by positive cultures in BACTEC 460 TB system, 14 active sarcoidosis (SA) 19 lung cancer (LC) and 10 healthy subjects (control). All patients were investigated before starting therapy.
336-PA10 Granulocyte chemiluminescence activity in tuberculosis (TB) contacts
Control TB IgG (U/ml) < 1 3.1 IgA (U/ml) 6.3 + 1.4 12.0 IgM* 0.14 + 0.02 0.25 Total cell No x 106 3.7 + 0.7 11.9 IgM * - normalized values
Zabuska, K., Niemirowska, H., Ponahajba, A., Skopinska, E. Tuberculosis and Lung Diseases Institute, Warsaw, Poland
The phagocytosis process constitutes a important defence mechanism in TB; granulocytes (GL) are an integral part of the immunological system. In our previous studies using the CL technique suppressed phagocytic activity of GL in full venous blood of 30 TB contacts was shown (14600 _+ 1740 cpm/103 neutrophile) in response to chemotactic pepfide N-FORMYL-MET-LEUPHE (FMLP) in comparison to reactivity of GL in healthy blood donors (50600 + 2260 cpm) (p < 0.001). All the contacts showed the exudative type Mantoux reaction (diameter > 20 ram) and presented normal chest X-ray. The present studies have shown that the CL impairment is at least partially reversible by TB chemoprophylaxis. In 10 out of 11 TB contacts the increase in granulocyte CL was observed after chemoprophylaxis (in that 5 persons returned to normal values) and in 1 person no change was observed. The reason for impaired granulocyte CL in full venous blood of TB contacts was sought. In the first stage of the studies CL of purified neutrophils isolated from peripheral blood of 18 TB contacts (age 23-43 yrs) was studied using LKB 1252 Luminometer. The neutrophiles' CL after FMLP stimulation was 22.19 + 3.09 mV and that of 22 healthy volunteers (age 23-48yrs) was 44.27 + 4.21 mV; P<0.001. The results obtained suggest a defect of studied granulocyte function in TB contacts. In the next stage of studies, the pilot studies, it was shown that sera of five TB contacts inhibited in 34-86% neutrophiles' CL of healthy volunteers. Our work represents a novel approach to studying TB contacts. The granulocyte activity assessment in TB contacts using CL technique may, in future, serve as a quick and inexpensive screening test for determining groups with an increased risk of developing the disease.
353-PA10 Evaluation of the specificity of Elisa using antigen A60 for diagnosis of tuberculosis in bronehoalveolar lavage fluid (BALF) Grubek-Jaworska, H., Droszcz, P., Zwolska-Kwiek, Z., Piro~y~ski, P., Walkiewicz, R., Droszcz, W., Walajtys-Rode, E. Clinic of Pneumonology, Warsaw Medical School and Institute of Tuberculosis and Lung Diseases, Warsaw, Poland
The investigation was undertaken to assess the effectiveness of ELISA using A60 antigen assay in ascertaining diagnosis in patients with tuberculosis. IgG, IgM and IgA antibodies against A60 antigen of Mycobacteria were measured in undiluted BALF using ANDA TB (Biologicals, Strasbourg, France) tests in 66 subjects including 23 active pulmonary tuberculosis (TB), con-
+ 0.7 _+ 2.4 + 0.05
SA 4.9 _+ 1.5 12.9 + 3.4 0.26 + 0.04
LC 3.2 + 0.8 16.2 + 2.9 0.23 + 0.05
+_ 1.8
8.7 + 1.9
8.8 + 2.8
No significant differences were found in the levels of IgG, IgM and IgA antibodies against A60 antigen in BALF between investigated groups of patients with TB, SA and LC, despite the marked differences with control group. This implies that assay of antibodies against A60 in BALF is not useful in differentiation of patients with active pulmonary TB from active sarcoidosis and lung cancer.
354-PA10 The value of Nitroblue Tetrazolium reduction test by monocytes isolated from peripheral blood of patients with tuberculosis Dubaniewicz, A. Dept. of Lung Diseases and Tuberculosis, Medical University of Gdaftsk, Poland
The aim of this study was to evaluate the abilities of bactericidal reduction of monocytes isolated from peripheral blood in four tested groups: healthy adults (24 persons); adults with active tuberculosis before antituberculosis treatment (33 patients); adults with tuberculosis after 2 months treatment (12 patients); nonactive tuberculosis (26 persons 14 years after treatment and healthy now). We estimated the reduction abilities of NBT by stimulated BCG and non-stimulated monocytes (by Park method) isolated from peripheral blood (by Boyum methods). We found that in the group with active tuberculosis before treatment the values of NBT reduction by non-stimulated (39,3+/- 14%) and stimulated BCG Monocytes (44,5+/-13%) remained at the same level but significantly higher than the values in the healthy group (non-stimulated-26,1+/-6%; stimulated 33,8+/-10%). 2 months treatment did not alter the ability of NBT reduction in both groups (38,6+/-9%; 44,3+/-7%). In non-active tuberculosis the reduction ability by stimulated monocytes was significantly higher than resulted by non-stimulated cells (34,8+/-5%; 29,4+/-8%). The obtained results suggested that: 1. In the active tuberculosis the values of NBT reduction by non-stimulated and stimulated BCG monocytes remained at the same level. This outcome may be a sign of decrease of energetic reserve due to antigenic Mycobacterium tuberculosis stimulation in organisms with tuberculosis. 2. Monocytes isolated from peripheral blood of healthy people and persons with non-active tuberculosis had greater abilities of bactericidal activation after stimulation with BCG. This outcome may be a sign of
zation. The A60WB test was then used to evaluate the incidence of false negative PPD tests in a group of 49 tuberculosis (TB) exposed health care workers exposed to > 30 TB cases per year. Clerical workers (n = 32) were evaluated as controls. TB exposed workers showed a significantly higher incidence of PPD negative skin tests among immunized individuals (A60WB-positive 98%, PPD-positive 76%, P < 0 . 0 0 5 ) while no differences were found in non-exposed workers (A60WB 28%, PPD 25%; P > 0.7). The data indicate that tuberculin skin testing with PPD may underestimate TB contagion in populations at risk.
288-PA10 Anti-PPD and anti-DAT serum antibody markers for the diagnosis of tuberculosis (TB) in the HIV-seropositive (HIV ÷) Amicosante, M. 1, Cuboni, A. 2, Ancora, A. 1, Monno, L. 3, Girardi, E. 4, Angarano, G. 3, Saltini, C. 1 1Dept Medical Sciences, Sect. Respiratory Medicine, University of Modena; 20spedale Civile, Sesto S. Giovanni, Milano; 31nfectious Diseases Institute, University of Bari; 4Centro di Riferimento AIDS, Azienda Ospedaliera Forlanini, S. Camillo, Spallanzani, Roma, Italy
Previous serological studies have shown that M. tuberculosis (M.tb)-specific antibody markers may diagnose anti-M.tb immunity, hence, latent infection and TB risk, in HIV +, PPD-, recall antigen-anergic subjects. This study was designed to evaluate the combined use of two serological markers, the IgM ELISA against PPD (PPD-IgM) (1) and the IgG ELISA against 2,3diacyltrehalose (DAT-IgG) (2) for the diagnosis of active TB and of TB risk in HIV ÷ subjects. A cohort of 102 HIV + individuals (age 29 + 8 years, 80% males; 46% PPD+; Multitest score 1.5 + 1.2; CD4 335 +- 214 cells/ram 3) was evaluated at enrolment and after 12 months. In addition, a sample of 14 HIV ÷ subjects with active TB (age 29 + 5; 86% males; 33% PPD+; 100% culture+) was evaluated a year before and at the time of TB diagnosis. Among cohort subjects, 14 (14%) were PPD-IgM + at enrolment [3 (21%) DAT-IgG*] and 8/64 (12%) were PPD-IgM + after one year [6 (75%) DATIgGq. Of the three subjects who developed TB two were culture + PPD-IgM ÷ and DAT-IgG + (Mantel-Hansel, P < 0.001). In the active TB patient sample, 9 (64%) were PPD-IgM ÷, 8 (57%) DAT-IgG + and 11 (79%) PPDIgM ÷ and/or DAT-IgG ÷. One year before diagnosis, 7 (50%) were PPD-IgM ÷, 5 (36%) DAT-IgG+ and 9 (64%) PPD-IgM ÷ and/or DAT-IgG ÷. The data indicate that the two antibody markers may be highly sensitive in the diagnosis of active TB and of TB risk in HIV ÷ subjects.
295-PA10 Reliability of ELISA in diagnosis of pulmonary tuberculosis EL-Feky, A. 1, Abou Shehatta, M. 1, Khaled, A. 1, EL-Morsi, A. l, Agha, S.2, Viljanen, M 3. Departments of Thoracic Medicine 1, Clinical Pathology 2, Mansoura University, Egypt and National Public Health Institute 3, Turku, Finland
Data on ELISA as a method of serodiagnosis in tuberculosis are contradictory. Therefore its role needs to be
19
reassessed. In this work we studied 61 Egyptian cases with clinical suspicion of pulmonary tuberculosis, 39 males and 22 females with age ranging between 16 and 59 years. After clinical and radiological reevaluation they were categorized into 2 groups: group A high probability of pulmonary tuberculosis (44 cases) and group B low probability of pulmonary tuberculosis (17 cases). For all cases sputum examination for Mycobacterium tb by direct smear, culture, PCR and by Amplicor were carfled out. The sera were tested using ELISA for detection of IgG and IgA antibodies against Mycobacterium tuberculosis. The results were as follows: Direct smear Group A 47.7% Group B 11.7%
Culture
PCR
Amplicor ELISA
31.8% 0%
54.4% 5.8%
47.4% 5.8%
56.8% 41%
These values show that in group A ELISA has sensitivity comparable to PCR. When combining results of smear with that of ELISA in the same group the sensitivity increased to reach 61.3%. However, 2.4% of cases were positive by ELISA but not by other tests, i.e false positive (data not shown). On the other hand in group B although ELISA was positive in 41%, only 11.7% was diagnosed by sputum of PCR, i.e 29.3% of low probability cases were seropositive with no detectable mycobacterium by other tests (data not shown). In conclusion when clinical suspicion is nigh ELISA may be of value in diagnosis of pulmonary tuberculosis, but with low clinical suspicion, the results should be evaluated cautiously and bacteriological confirmation is needed.
313-PAl0 Identification of mycobacteria isolates using polymerase chain reaction Bahrmand, A., Bakaev, B., Madani, H., Saifi, M. Pasteur Institute of lran, Tehran, lran
Tuberculosis (TB) and diseases caused by nontuberculous mycobacteria (NTM) reveal similar clinical manifestations including roentgenography evidence. The protocol of TB treatment differs from that of other mycobacterioses; however, clinical methods commonly used for diagnosis lack the necessary sensitivity and speed. To obviate these difficulties and to identify organisms involved, bacteriological procedures and nested PCR assay with primers specific for M. boris tuberculosis were applied to 329 specimens. The nested PCR assay has demonstrated the highest capability to discover pathogens in sputum: 274 specimens were TB positive by PCR, 224 by culture, and 174 by BACTEC assay. Of the 55 TB negatives, 21 specimens were found to contain NTM. Primers specific for M. fortuitum and M. kansasii were used to discriminate between these and other NTM. The proposed PCR assays having high sensitivity and specificity can support
20
Tubercle and Lung Disease: Supplement 2
a rapid diagnosis and monitoring efficiency of drug therapy in laboratories where routine work with labelled probes is not acceptable.
firmed by positive cultures in BACTEC 460 TB system, 14 active sarcoidosis (SA) 19 lung cancer (LC) and 10 healthy subjects (control). All patients were investigated before starting therapy.
336-PA10 Granulocyte chemiluminescence activity in tuberculosis (TB) contacts
Control TB IgG (U/ml) < 1 3.1 IgA (U/ml) 6.3 + 1.4 12.0 IgM* 0.14 + 0.02 0.25 Total cell No x 106 3.7 + 0.7 11.9 IgM * - normalized values
Zabuska, K., Niemirowska, H., Ponahajba, A., Skopinska, E. Tuberculosis and Lung Diseases Institute, Warsaw, Poland
The phagocytosis process constitutes a important defence mechanism in TB; granulocytes (GL) are an integral part of the immunological system. In our previous studies using the CL technique suppressed phagocytic activity of GL in full venous blood of 30 TB contacts was shown (14600 _+ 1740 cpm/103 neutrophile) in response to chemotactic pepfide N-FORMYL-MET-LEUPHE (FMLP) in comparison to reactivity of GL in healthy blood donors (50600 + 2260 cpm) (p < 0.001). All the contacts showed the exudative type Mantoux reaction (diameter > 20 ram) and presented normal chest X-ray. The present studies have shown that the CL impairment is at least partially reversible by TB chemoprophylaxis. In 10 out of 11 TB contacts the increase in granulocyte CL was observed after chemoprophylaxis (in that 5 persons returned to normal values) and in 1 person no change was observed. The reason for impaired granulocyte CL in full venous blood of TB contacts was sought. In the first stage of the studies CL of purified neutrophils isolated from peripheral blood of 18 TB contacts (age 23-43 yrs) was studied using LKB 1252 Luminometer. The neutrophiles' CL after FMLP stimulation was 22.19 + 3.09 mV and that of 22 healthy volunteers (age 23-48yrs) was 44.27 + 4.21 mV; P<0.001. The results obtained suggest a defect of studied granulocyte function in TB contacts. In the next stage of studies, the pilot studies, it was shown that sera of five TB contacts inhibited in 34-86% neutrophiles' CL of healthy volunteers. Our work represents a novel approach to studying TB contacts. The granulocyte activity assessment in TB contacts using CL technique may, in future, serve as a quick and inexpensive screening test for determining groups with an increased risk of developing the disease.
353-PA10 Evaluation of the specificity of Elisa using antigen A60 for diagnosis of tuberculosis in bronehoalveolar lavage fluid (BALF) Grubek-Jaworska, H., Droszcz, P., Zwolska-Kwiek, Z., Piro~y~ski, P., Walkiewicz, R., Droszcz, W., Walajtys-Rode, E. Clinic of Pneumonology, Warsaw Medical School and Institute of Tuberculosis and Lung Diseases, Warsaw, Poland
The investigation was undertaken to assess the effectiveness of ELISA using A60 antigen assay in ascertaining diagnosis in patients with tuberculosis. IgG, IgM and IgA antibodies against A60 antigen of Mycobacteria were measured in undiluted BALF using ANDA TB (Biologicals, Strasbourg, France) tests in 66 subjects including 23 active pulmonary tuberculosis (TB), con-
+ 0.7 _+ 2.4 + 0.05
SA 4.9 _+ 1.5 12.9 + 3.4 0.26 + 0.04
LC 3.2 + 0.8 16.2 + 2.9 0.23 + 0.05
+_ 1.8
8.7 + 1.9
8.8 + 2.8
No significant differences were found in the levels of IgG, IgM and IgA antibodies against A60 antigen in BALF between investigated groups of patients with TB, SA and LC, despite the marked differences with control group. This implies that assay of antibodies against A60 in BALF is not useful in differentiation of patients with active pulmonary TB from active sarcoidosis and lung cancer.
354-PA10 The value of Nitroblue Tetrazolium reduction test by monocytes isolated from peripheral blood of patients with tuberculosis Dubaniewicz, A. Dept. of Lung Diseases and Tuberculosis, Medical University of Gdaftsk, Poland
The aim of this study was to evaluate the abilities of bactericidal reduction of monocytes isolated from peripheral blood in four tested groups: healthy adults (24 persons); adults with active tuberculosis before antituberculosis treatment (33 patients); adults with tuberculosis after 2 months treatment (12 patients); nonactive tuberculosis (26 persons 14 years after treatment and healthy now). We estimated the reduction abilities of NBT by stimulated BCG and non-stimulated monocytes (by Park method) isolated from peripheral blood (by Boyum methods). We found that in the group with active tuberculosis before treatment the values of NBT reduction by non-stimulated (39,3+/- 14%) and stimulated BCG Monocytes (44,5+/-13%) remained at the same level but significantly higher than the values in the healthy group (non-stimulated-26,1+/-6%; stimulated 33,8+/-10%). 2 months treatment did not alter the ability of NBT reduction in both groups (38,6+/-9%; 44,3+/-7%). In non-active tuberculosis the reduction ability by stimulated monocytes was significantly higher than resulted by non-stimulated cells (34,8+/-5%; 29,4+/-8%). The obtained results suggested that: 1. In the active tuberculosis the values of NBT reduction by non-stimulated and stimulated BCG monocytes remained at the same level. This outcome may be a sign of decrease of energetic reserve due to antigenic Mycobacterium tuberculosis stimulation in organisms with tuberculosis. 2. Monocytes isolated from peripheral blood of healthy people and persons with non-active tuberculosis had greater abilities of bactericidal activation after stimulation with BCG. This outcome may be a sign of