Bacillus Calmette Guerin (BCG) as a Diagnostic Tool in Adult Pulmonary Tuberculosis*

Bacillus Calmette Guerin (BCG) as a Diagnostic Tool in Adult Pulmonary Tuberculosis* Chuen-Yin W. Dy, M.D.,** Luis T. Go, M.D.*** Anne Du-Yabut, M.D.,*** Eugenio P. Reyes, M.D.*** and Melecia Antonio-Velmonte, M.D.**** (*PSMID-UNILAB JuniorAwardee, 1992, **UP-FGH Fellow, Infectious Disease Section, Department of Medicine, ***Resident, Department of Medicine, UP-PGH and ****Chief, Infectious Disease Section, Department of Medicine, UP-PGH) ABSTRACT BCG was administered concomitantly with PPD to 414 adult subjects who were divided into 4 groups. The differences in the induration diameter post-bcg were statistically significant between those with evidence of pulmonary tuberculosis and those who were free of the disease. The duration of the lesion was also found to be significantly different, although the initiation of BCG induration was the same for subjects with active disease and those who were exposed to tuberculosis. PPD showed inconsistent results among the different populations. Overall, BCG had high sensitivity and specificity compared to PPD. We conclude that BCG is more useful than PPD as a diagnostic tool in pulmonary tuberculosis among adults. [Phil J Microbiol Infect Dis 1993; 22(1):31-40] Key words: pulmonary tuberculosis, BCG, PPD INTRODUCTION Despite advances in medical science, tuberculosis remains as one of the leading causes of morbidity and mortality in developing countries. In areas where tuberculosis is less prevalent, failure to include it in the differential diagnosis may lead to unnecessary delay in the correct diagnosis and possibly to a tragic outcome. On the other hand, in areas with high prevalence of the disease, indiscriminate diagnosis of tuberculosishasresultedin failureto detect early the more severe or fatal ailments like bronchogenic carcinoma. 1 Early case finding is therefore necessary for proper treatment and to reduce the likelihood of qomplications. The diagnostic armamentarium available to the clinician in the work-up for tuberculosis includes tuberculin skin test, chest x-ray, smear and culture of secretions and tissues, and biopsy of lymph node and other organs. Tuberculosis, however, is a disease with protean manifestations and often presents diagnostic problems. The PPD skin test is considered specific but lacks sensitivity, 2 while chest x-ray findings may be inconclusive. Furthermore, definite diagnosis requires the demonstration of the tubercle bacilli on culture from specimens taken from the host, which in numerous occasions hadbeen difficult. This failure to demonstrate the tubercle bacilli in culture could partly be due to the inability by some patients to spill enough bacilli in the sputum and to inappropriate specimen collection. Culture for acid-fast bacilli (AFB) usually requires long periods of incubation and demands dedication and skill on the part of the technician. Also, facilities and expertise for performing these studies must be available. However this is no talways the case especially in the remote provinces. It has been reported that the recovery rate is so dismal even in some medical centers in Manila. 2 Egmore in 1989 stated that that BCG vaccination induration could be used as a screening test for tuberculosis control. This was based on the observation by Public health officials who were performing BCG vaccination without prior tuberculin testing as early as 1936. An "accelerated" response to BCG was observed. This paved the way for the use of acceleratedbcg reaction as an indication of tuberculous infection and disease. 3 Presently, BCG is being utilized not only as an immunizing agent but also as a diagnostic tool in ascertaining tuberculosis infection in the pediatric age group. Both local and international studies have shown that BCG has a high sensitivity and specificity of up to 100 percent among

well-nourished and malnourished children. In contrast, PPD has rather low sensitivity (19 to 41 percent among nourished and 79 percent among malnourished children) but high specificity (up to 100 percent). The consensus is that the BCG test is superior to the Mantoux test. However, its usefulness in the adult population has not been fully assessed. OBJECTIVES General Objective: To determine the usefulness of BCG as a diagnostic tool in adult pulmonary tuberculosis. Specific Objectives: 1. To determine the relationship of BCG reaction with sputum positivity 2. To evaluate the reaction to BCG in patients who present with sputum negative for AFB and a clinical response to anti-tuberculosis treatment 3. To evaluate the reaction to BCG in subjects free of the disease 4. To compare the two intradermal tests, namely the Mantoux and the BCG, with regards to sensitivity and specificity in the diagnosis of adult pulmonary tuberculosis with sputum examination as the gold standard. MATERIALS AND METHODS Subjects All subjects at least 18 years of age who fulfilled the inclusion criteria were enteredin the study. The study population was divided into four groups. Group A subjects were composed of patients confirmed to have pulmonary tuberculosis by sputum examination, i.e. positive AFB smear or culture. Group B were those subjects with signs and symptoms of tuberculosis but whose sputum was AFB and/or culture negative, Group C comprised hospital personnel with varying degrees of exposure to tuberculosis. Group D were made up of subjects with no known exposure to tuberculosis. Groups A, B and D were composed of patients in the out-patient service and those admitted in the wards, while group C was composed of physicians, nurses, and technicians of the Philippine General Hospital. A complete history and physical examination was performed on each subject. Inclusion criteria for Group A: 1. Subjects with signsand symptoms suggestive of TB: a. cough b. anorexia c. weightless d. afternoon fever e. night sweats f. hemoptysis 2. Chest x-ray findings suspicious of tuberculosis 3. Sputum AFB smear and/or culture positive for tubercle bacilli Inclusion criteria for Group B: 1.Chest x-ray findingsof tuberculosis 2. Symptomatic as in Group A 3. Improvement on anti-tuberculosis chemotherapy 4.negativeAFB smear andculture

Inclusion criteria for Group C: 1. Hospital personnel in contact with patients 2. Laboratory technicians handling specimens suspected to contain tubercle bacilli 3. Asymptomatic 4. Normal chest x-ray. Inclusion criteria for Group D: 1. No known exposure to tuberculosis 2. Asymptematic 3. Normal chest x-ray Exclusion criteria for all groups: Materials 1. Patients on steroid therapy 2. Those on immunosuppressive therapy 3. Pregnant subjects 4. Patients on more than 2 weeks of anti-tuberculosis medications 5. Patients known or suspected to have immunodeficiency states. Tuberculin: Purified protein derivative (PPD) RT23 with Tween 80 as preservative, prepared by Statens Serum Institute in Copenhagen and equivalent to 5 TU with lot number 92-54, expiry April 1993. BCG Vaccine: Freeze dried vaccine containing live attenuated strain (Pasteur 1173 P2) of Mycobacterium bovis manufactured by Statens Serum Institute of Copenhagen with lot number 92-202, expiry August 1992. Syringe use: Tuberculin syringe with short (one-half inch) bluntly beveled, gauge 26 steel needle to provide a properly calibrated leak free system with assurance of slow injection of test material. Handling of BCG and PPD: The containers of the biologicals were protected against damage from heat by keeping them under efficient cold-chain storage and were opened only immediately before use. Methods The following procedures were done on each subject: A BCG test and Mantoux test were simultaneously performed. The injection sites were prepared by cleaning with alcohol and allowing it to dry. The Mantoux test was done by injecting 0.1 ml PPD into the flexor (volar) surface of the left forearm at the junction of the upper third and lower two-thirds. The injection was made just beneath the surface of the skin with the needle bevel upward. A discrete pale elevation of the skin 6 to 10 millimeters in diameter was produced. On the right arm, BCG test was done by injecting 0.1 ml of BCG vaccine in the middle or lower deltoid region intradermally and very slowly to avoid damage to tissue until a well-defined wheal was produced. Each patient was followed up after 24, 48, 72 hours and weekly until lesions were healed.

Measurement of Test site PPD: Presence or absence of induration, the size of which was determined by palpation. The diameter of this induration was measured perpendicularly to the long axis of the forearm and recorded in millimeters. BCG: Induration was determined by palpation the greatest diameter ofwhich is measured in millimeter. Measurement of both PPD and BCG was done twice by the same investigator and the average size was the one recorded. By definition, a positive Mantoux test is the presence of an induration of 8 mm or more on the 48th or 72nd hour post-injection, when the induration is most evident. A positive BCG test, on the other hand, is an induration of 10 mm or more within 72 hours, Analysis of Data Contingency tables were constructed. Test results of PPD and BCG were recorded and analysed for each group of subjects. One-way analysis of variance was done to test the null hypothesis. If significant, multiple comparisons were done to pinpoint which among the four groups differ. A p-value of less than 0.05 was considered statistically significant. Sensitivity, specificity, positive and negative predictive values of PPD and BCG were calculated. RESULTS A total of 468 subjects were recruited. However, 54 subjects failed to return for followup, leaving 414 subjects for evaluation. Their distribution into the 4 groups is shown in Table 1. The male to female ratio was 1 to 1.2; the youngest subject was 18 years old while the oldest was 72 years of age. The mean age of the subjects of each group is shown in Table 2. Table 3 summarizes the PPD induration, the initiation of BCG reaction, the size of the BCG induration, and the total duration of BCG reaction among the 4 groups. The interval between the time of BCG inoculation to the appearance of the lesion was the same for groups A, B, and C (P>0.05). The difference between these groups and group D was significant, Figure 1, p <0.001. Table 1. Sex distribution of patients Group Male Female Total A B C D 52 60 33 43 45 48 77 56 97 108 110 99 Total 188 226 414 Table 2. Mean age of subjects Group Mean Age (Range) A B C D 43.6 47.5 30.2 27.4 (26-70) (20-72) (21-60) (18-51) Table 3. BCG and PPD reaction among the 4 groups Group Initiation of BCG induration(days) Mean BCG induration (mm) Total BCG duration (days) Mean PPD induration (mm) A 1.74 ± 0.68 21.53 ± 2.51 34.29 ± 7.48 9.26 ± 3.86 B 2.05 ± 1.44 19.98 ± 2.29 34.00 ± 7.28 8.55 ± 4.98 C 1.81 ± 0.52 7.56 ± 2.64 42.42 ± 10.50 8.51 ± 3.64 D 10.86 ±!4.80 5.67 ± 1.74 40.31 ± 13.14 3.70 ± 1.90 The induration size was significantly different between those with evidence of tuberculosis (group A and B) and those without the disease (group C and D) as depicted in figure

2, p<0.05. As shown in the same figure, there was no statistical difference between groups A and B as well as between groups C and D with regards to induration size, p>0.05. Figure 1. Initiation of BCG induration Figure 2. Size of BCG induration The course of BCG reaction, figure 3, took from 21 days to 73 days. Again, there was a significant difference between those having active disease and those free of the disease, p<0.05. In contrast, no difference was found between groups A and B and between groups C and D, p>0.05. There was no significant difference between groups A, B and C with regards to PPD induration size, though the difference between group D and the other 3 groups was significant, figure 4, p<0.05. In group A, 94 out of 97 subjects developed an induration post-bcg ranging from 15 mm to 30 mm in diameter. Three subjects failed to develop a reaction. Only 63 subjects had positive PPD, Table 4, kappa = - 0.175. In group B, 100 subjects had a reaction to BCG. The induration ranged from 16 to 27 millimeters in diameter. Eight subjects had no induration. PPD test was positive in 57 subjects, Table 5, kappa = - 0.143.

In group C, seven subjects had BCG induration of 10 to 12 millimeters in diameter. PPD was positive in 105 subjects, Table 6, kappa = - 0.75. Figure 3. Duration of BCG reaction Table 4. Group A test results PPD BCG (+) 61 33 94 BCG (-) 2 1 3 Total 63 34 97 Table 6. Group C test results PPD BCG (+) 7 0 7 BCG (-) 98 5 103 Total 105 5 110 Table 5. Group B test results PPD BCG (+) 54 46 100 BCG (-) 3 5 8 Total 57 51 108 Table 7. Group D test results PPD BCG (+) 0 0 0 BCG (-) 6 93 99 Total 6 93 99 Figure 4. Size of PPD induration In group D, no significant induration was observed in the BCG site of all subjects. PPD was positive in 6 subjects, Table 7, kappa = - 0.03.

Figure 5 is the graphical representative of the BCG induration size among the 4 groups. The difference between subjects free of tuberculosis and those with active infection is apparent. Subjects from groups C and D have induration size of 12 mm or less with the majority having an induration size around 5 mm. In contrast, the subjects from groups A and B have induration size more than 15 mm with the majority having an induration size around 20 mm. Figure 6 is the graphical representation of PPD induration among the 4 groups. No apparent difference is depicted among the groups aside from a low positive reaction of subjects from group D. Figure 5. BCG induration among the 4 Groups Figure 6. PPD induration among the 4 Groups Sensitivity, specificity, positive and negative predictive value of BCG and PPD were calculated from the contingency tables, Tables 8 and 9. Sensitivity and specificity of BCG was found to be 94.63 percent and 96.65 percent, respectively; while that of PPD was 58.54 percent and 46.89 percent, respectively. The positive predictive value and negative predictive value for BCG were 96.52 percent and 94.84 percent, respectively; while those of PPD were 51.95 percent and 53.55 percent, respectively. All but 9 BCG indurations, whether significant or not, were accompanied by surrounding erythema. Three of the 9 subjects came from group C, while the other 6 came from group D. A

total of 147 subjects experienced pruritus of varying degree over the BCG induration site. This was more frequent in group C (98 subjects) and least frequent in group D (8 subjects). It occurred in 24 subjects in group A and 17 subjects in group B. Tenderness over the BCG site developed in 80 subjects: 18 from group A, 21 from group B, 38 from group C, and 3 from group D. Abscess developed in 1 subject each from groups A and B, and in 5 subjectsfrom group C. Table 8. Correlation between BCG test and pulmonary tuberculosis Tuberculosis BCG (+) 194 7 201 BCG (-) 11 202 213 Total 205 209 414 Table 9. Correlation between PPD and pulmonary tuberculosis Tuberculosis PPD (+) 120 111 231 PPD (-) 85 98 183 Total 205 209 414 DISCUSSION The reaction to PPD is an indispensable conventional tool for detecting tuberculous infection. 4,5 However, in areas with high prevalence of tuberculosis, the validity of the PPD test is often questioned. In addition, the reaction is reported to vary with several factors like age, nutritional status, and frequency of tuberculin skin testing. 2 In a study done in Jamaica, 83 percent of adults without tuberculosis had positive PPD test. 6 Moreover, PPD has a 10 to 20 percent false positive rate and a 1 to 10 percent false negative rate. False positive reactions are acceptable in a screening test whereas false negative tests are not. 5 The BCG test is based on an altered state of tissue reactivity to tuberculin. The immune reaction that occurs in the hypersensitive person following exposure to tuberculo-protein can probably account for the tissue destructions. 4 The theoretical basis for this could be explained by the following: T-lymphocytes are stimulated to secrete lymphokines which activate macrophages to inhibit the bacilli. Gamma interferon, the best characterized of the macrophage-activating lymphokines, may be important in the immunopathology. 8 When a human macrophage is activated by exposure to gamma interferon in vitro it develops an increased potential for massive release of tumor necrosis factor (TNF). 9,10 Likewise, such exposure to gamma interferon produces functional and phenotypic changes that includes the generation of an enzyme (1-hydroxylase) which enables the macrophage to convert circulating inactive form of vitamin D3 into the active derivative, cholecalciferol. Recently, it has been demonstrated that exposure of macrophage to activated vitamin D3 also primes for TNF release. 7 Histologically, the lesion is characterized by fibrin deposition and capillary damage. 7 Beck observed that there is early migration of large, numbers of lymphocytes and monocytes over a short period of time, which could result in an increase in local metabolic demand and hypoxia. The accompanying hyperemic reactions and a slower central blood flow may be suggestive of microcirculatory stasis and incipient ischemia. The predominance of activated macrophages indicates that a high rate of cytokine secretion could be responsible for tissue damage. 11 Normally, BCG injected intradermally producesa wheal, which disappears in about half an hour. A small red nodule will appear after 2 to 3 weeks, which increases in size for about a week. This nodule develops into a small superficial abscess, which then ulcerates around the 4th to 6th week. The ulcer rapidly crusts and heals spontaneously. After 8 to 12 weeks from the vaccination date, the process becomes complete having a depressed whitish and smooth scar measuring about 3 to 5 mm in diameter. 2,4 In studies done in children, an accelerated reaction to BCG was observed among those with pulmonary tuberculosis. According to definition, this is the evolution of an induration of about 5 to 10 mm or more in diameter within 48 to 72 hours, pustules by 5 to 7 days, and healing by 2 to 3 weeks. 3,4 Our study did not show an accelerated course of BCG reaction in adult patients. However, the subjects with active disease and those

who were constantly being exposed to tuberculosis showed early reaction, within 24 to 72 hours post-bcg inoculation, with the course of the reaction being significantly shorter in the former. The immunogenicity and reactogenicity of the vaccine varies according to the BCG strain used. Pasteur 1173 P2, Copenhagen 1331, Glaxo 1077, Tokyo 172, and Moreau (Brazil, Cuba) are the currently available strains. Some strains, such as Pasteur 1173 P2 and Copenhagen 1331, are stronger than others. 12,13 BCG is one of the safest vaccines and its correct use does not have increased risk in previously infected persons. Most local and regional undesirable reactions are of little consequence and slowly evolve toward resolution and cure, usually without treatment. 13 The only contraindication to its administration is thc presence of immunodeficiency states. 2 Complications of BCG inoculation include the development of undulating ulcer, hypertrophic scar and keloid. Other complications such as scrofuloderma, eczematous lesions, and osteitis occur very rarely. 2 Not considered as complications are small abscesses at the site of vaccination which heals within 2 months or suppurative adenitis of moderate degree. 4 The complications to vaccination could be due to the inoculation of a vaccine containing too high a number of attenuated BCG organisms. It likewise could follow a subcutaneous or too deep an injection, or inoculation of a larger volume of vaccine. 12,13 The training level of the staff performing the procedure clearly has a bearing on the incidence of complications. 12 Proper technique and handling of the vaccine, if observed, will be a good measure to prevent complications. It is likewise important that the biological product be standardized before clinical use to avoid excessively low antigen concentration resulting in tests of low sensitivity, or exceedingly high antigen concentration with the risk of unacceptably high frequency of adverse effects. 11 Seven subjects in our study developed cold abscess for more than 2 months. Four of them were treated with incision and drainage, while the others healed spontaneously. All pustular lesions were managed by local care. No systemic medication was required for the pruritus, tenderness, pustule and abscess. Teulieres et al found that there is a correlation between the concentration of the BCG vaccine and the incidence of undesirable side effects. In his study of 291 infants, the administration of either the usual dose or one half that dose elicited the same immune response but with a significant reduction of side effects in the latter. 12 Since our study utilized an arbitrary 0.1 millimeter of BCG vaccine this dose could probably still be reduced. Further trials can be performed to determine the optimal dose that will elicit the maximum response with minimal side effects. CONCLUSION From the results of the study, we can make the following conclusions: BCG is a useful diagnostic aid in adult pulmonary tuberculosis; unlike pediatric cases, no accelerated response is demonstrated; BCG induration appears within 72 hours in active TB cases and those strongly exposed to TB; mean BCG induration size is 12 mm or less in normal subjects and 15 mm or more in active TB cases; duration of BCG reaction differs by approximately one week in the two populations; BCG test is more sensitive and more specific than the Mantoux test; no serious adverse reaction is observed; and the dose used in this study which is arbitrary may still be reduced to minimize adverse reactions. REFERENCES 1. Sollano JD, Navarro C. Acid-fast bacilli isolation at the Santo Thomas University Hospital. Santo Thomas J Med 1982; 31(1):62-70. 2. The 1st National Consensus in Tuberculosis Committee Reports. Chest Diseases 1989; 16(3 suppl): 13-20. 3. Taleon AT, et al. BCG vaccination as an alternative diagnostic tool in childhood tuberculosis. Phil J Microbiol Infect Dis 1990; 19(1):16-19.

4. Reyes-Rivera CT. Bacillus Calmette Guerin: A vaccine as well as a diagnostic tool for tuberculosis. Santo Thomas J Med 1984; 33:385-389. 5. Reports of the Committee on Infectious Diseases. American Academy of Pediatric 22nd Ed. 1991; 489-492 6. Bain BC, et al. Significance of the tuberculin skin test in pulmonary tuberculosis in Jamaica. West Indies Med J 1987; 36:231-235. 7. Rook GAW. Role of activated macrophages in the immunopathology of tuberculosis. Brit Med Bull 1988; 44(3):611-623. 8. Rook GAW, et al. Vitamin D3, gamma interferon, and control of proliferation of M. tuberculosis by human monocytes. Immunology 1986; 57:159-160. 9. Nedwin GE, et al. Effect of interleukin 2, interferon gamma and mitogens on the production of tumor necrosis factor Alpha or Beta. J Immunology 1985; 135:2492-2497. 10. Rook GAW, et al. The role of gamma interferon, Vit D3 metabolites and tumor necrosis factor in the pathogenesis of tuberculosis. Immunology 1987; 62:229-234. 11. Beck JS, et al. Histometric studies on biopsies of tuberculin skin test showing evidence of ischemia and necrosis. J Path 1989; 159: 317-322, 12. Luelmo F. BCG vaccination. Am Roy Resp Dis 1982; 125(3 pt 2):70-72. 13. Tuelieres L, et al. Comparative trial of administration of half and quarter dose of intradermal pasteur BCG on 291 infants from birth to 1 year in French Guyana. Vaccine 1991; 9: 521-523.