Anti-PPD Immunoglobulin G in Tuberculosis Patients

Transcription

1 Anti-PPD Immunoglobulin G in Tuberculosis Patients Amorn Leelarasamee, M.D.* Jim Tulloch, M.D. Ashley Stevenson, M.Sc...,, f1inlifj Anti-PPD 61111h.lflaualA ~ llah'ihu1wl'hl v v (I'U1 ~(l1i'fti1. Jim Tulloch, Ashley Stevenson Division oj Clinical Epidemiology, The Royal Newcastle Hospial, University oj Newcastle, Australia. y """'... v 11'U'111 T:1f1l1f1l'l1fJllCl::t11mU VCl']J'I'( ~ 2527; 2: Abstract Sera from patients with active tuberculosis, treated tuberculosis, non-tuberculous pulmonary diseases and from healthy subjects before and 3 months after BCGvaccination were assayed for immunoglobulin G antibody against purified protein derivative by an enzyme-linked immunosorbent assay using polystyrene microtitre plate. Marked increase in immunoglobulin G antibody activity in patients with active tuberculosis was seen (P = 0.006) but there were some degrees of overlap between the values for tuberculosis group and those for non-tuberculous pulmonary diseases and healthy subjects. Level of immunoglobulin G antibody activity increased with age in healthy Papua New Guineans (P = 0.044). Activity of immunoglobulin G antibody before and three months after BCG vaccination in seven nurse students was not significantly different (P = 0.133). This assay has potential as a rapid diagnostic tool for detection of active tuberculosis. (key words: PPD, immunoglobulin G, Tuberculosis, BCGvaccination) From the Royal Newcastle Hospital; University ofnewcastle, Australia. "Present address: Clinical Epidemiology Unit, Department of Medicine, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok Thailand 63

2 ::lin"; 1 "-'vvvn"; 2 " 64 U J-< tu.n. -:JJ.O INTRODUCTION It has been estimated that, in developing countries, 4-5 million highly infectious cases of smear-positive tuberculosis occur each year, with an equal number of less infectious cases, including those positive by culture only and culture-negative cases, the latter being the most frequent form of pulmonary disease in children. Thus, each year about 10 million persons still develop tuberculosis and at least 3 millions die from this disease. 1 Case-finding activites in many developing countries depend principally on the examination of patients presenting themselves with relevant symptoms to a health facility," Tuberculosis suspects are identified and arranged for the bacteriological examination of their sputum or other clinical specimens. Some may submit inadequate specimens, or none at all, since three consecutive days are required to collect specimens. There are special difficulties in making examinations for tubercle bacilli as it requires skill and time for the task to be performed efficiently. Rapid presumptive diagnosis can be made if acid-fast bacilli are seen microscopically though one has to be cautious that the property of acid-fast staining is not exclusive to Mycobacterium tuberculosis. It is most useful in identifying open cases who excrete numerous bacilli in specimens. Specimen culture, if available, is mandatory for a definite diagnosis but, unlike the situation with other common pathogenic bacteria, considerable time is required before the final report of the culture can be made. The tuberculin skin test loses its utility for the diagnosis of active case in area where prevalence of the disease is high and BCG vaccination has been practiced. With respect to the rapidity and sensitivity, laboratory methods for identification of active cases which are currently available, are not so satisfactory from the clinician's point of view. Serologic tests have been widely used in the diagnosis of infectious diseases. Since the second decade of this century, many investigators have worked to develop a serologic test for the diagnosis of tuberculosis.' Antibodies to mycobacterial antigens have been identified in patients with tuberculosis using a variety of techniques. Among these are the hemagglutination test, kaolin agglutination test, precipitation test using immunodiffusion, and the latex agglutination test."? No method has been shown to be sufficiently sensitive. Thus, distinguishing among those patients with healed tuberculosis, healthy tuberculin reactors and the patients vaccinated with BCG vaccine remains an unresolved problem. Bates in his recent publication considers those serological tests mentioned not so useful for the diagnosis of active tuberculosis." Currently, ELISA is one of the most efficient procedures to reveal antibodies against certain antigens. Its rapidity and simplicity make it a potentially valuable alternative method of diagnosis or confirmation of tuberculosis. Using this technique, various groups have recently reported the usefulness of the test and claimed it to be very sensitive and speciflc."?" Its capability inseparating active from inactive tuberculosis render the test more useful for the diagnosis of active tuberculosis than the tuberculin skin test in endemic areas where most of the healthy adult population is tuberculin-positive. Hopefully, such a test would help solve some of the above mentioned problems. Since the prevalence of tuberculosis in Australia is expected to be quite low, a test with high sensitivity, as reported by Zeiss et al," will be adopted, with slight modification, in this study. It is an attempt to elucidate various aspects of applicability of the ELISA in people living in Australia and Papua New Guinea by measuring and comparing levels of IgG antibody to purified protein derivative antigen (PPD) in persons with various stages of tuberculosis and in healthy individuals. PATIENTS AND METHODS 1. Definition of study groups: a. Tuberculosis Patients: Those persons suffering from bacteriologically proven tuberculosis of any organ. At the time of study they were either on treatment, or not, and were further divided into two subgroups: patients with or without clinical disease at the time of specimen collection. b. Non-Tuberculosis Patients: Persons as similar as possible to those with active TB, with non-tuberculous destructive or chronic pulmonary diseases or chronic systemic disease. Patients in this group must have definite diagnosis at the time of study or very soon after. c. Healthy Persons: Those without apparent dillease in the preceding three months. Subjects in this group received intradermally five tuberculin units of PPD in 0.1 ml of solution stabilized with Tween 80 and were subdivided according to the skin test result. (The tuberculin skin test was considered positive if an area of induration of 10 mm in diameter or greater was present 72 hours after the innoculation. The test was recorded as negative or intermediate if the diameter of the induration was equal to, or less than, 4 mm or 5-9 mm, respectively, Individuals who had a negative reaction were given BCG vaccination at the time of reading the skin test. Due to the shortage of time and the low prevalence of tuberculosis in the Newcastle area where the study was conducted, suspected tuberculosis patients were not included in this study.

3 , l J Infect Dis Antimicrob Agents Vol. 1 No.2 Apr. - Jun '" 2. Recruitment of subjects two years previously. All specimen was kept in small Twenty-five healthy student nurses at Royal aliquots at -70 C. Newcastle Hospital volunteered to have blood collected at the time of routine tuberculin skin testing. 3. ELISA Reagents and methods Four were found to be tuberculin positive and four Purified protein derivative (PPD) of Tuberculin had an intermediate reaction. Only seven of the extracted from human strain of M. tuberculosis was seventeen nurses who were tuberculin-negative and purchased from the Commonwealth Serum Labora had BCG vaccination, presented for a second blood tories, Melbourne, Australia in concentrated solution collection three months after vaccination. BCG scar at a strength of 2 mg per ml. Alkaline phosphatase at the site of vaccination was identifiable in all. One conjugated goat anti-human IgG antibodies (gamma blood donor and five healthy subjects at the Royal chain specific) from TAGO. Inc., Burlingame, CA Newcastle Hospital also volunteered to serve as con trols. Their current reactivity to tuberculin was un known. TB patients with clinical disease or complete re Table 1 Mean, standard deviation, maximum and minimum values of covery were recruited from the Royal Newcastle Hos absorbance readings in student nurses before and after BeG pital (5 patients), The Prince of Wales Hospital (11 vaccination. patients) both in New South Wales (TB-NSW) and from Madang General Hospital (18 patients) in Papua Before BCG 3 months after BCG New Guinea (TB-PNG). n=7 n=7 Non-TB patients were recruited from the Royal Mean absorbance reading 0.062* 0.086* Newcastle Hospital (7 patients) and The Prince of Standard deviation Wales Hospital (3 patients) by reviewing the hospital records and sputum culture results to ensure that no Maximum evidence of active tuberculosis was present at the Minimum time of blood collection. *Paired t-test (p=0.133); BMDP was used for computation Sixty-six serum specimens were randomly select *95% confident interval for the mean difference of the absorbance ed from sera obtained in Papua New Guinea from pre readings is and sumably healthy individuals in a malaria field survey **standardized value. Midpoints of absorbance readings years or less 5-9 years years Over 20 years *One asterisk represented one absorbance reading. Two values were excluded from years age group (1.694 and 2.244) and two from those over 20 years (2.302 and 2.358). / Fig. 1 Histogram of absorbance readings by four age groups in 62 healthy Papua New Guineans*

4 :!-lei v ei ~ 66; un 1 ijvvn 2 W.O , USA. Bovine serum albumin in powder and p-nitrophenyl phosphate supplied as 5 mg per tablet were obtained from Sigma Chemical Co., St. Louis, USA. All other chemicals were of analytical grade. Each sample was tested in triplicate. One positive and one negative control were included in each microtitre plate. ELISA technique as reported by Zeisset al" was adopted and modified by adding 40 /lg of' PPD in 0.1 ml into eachwell of polystyrene microtitre plate. The amounts of other reagents were proportionally reduced accordingly. Since the absorbance readings of a single specimen varied from day to day, the assay was standardized by adjusting the subsequent absorbance readings of all specimens to be comparable to the first day. By matching the absorbance readings of the negative and positive controls of each plate to the first plate, a regression equation was derived and all values were converted to the standardized values. RESULTS Of the patients recruited, seven cases from Papua New Guinea (PNG) were excluded from the study since their clinical status did not meet the definition of any of the study groups. Table 2 Age-specific mean ELISA absorbances, measures of dispersion and maxima and minima in healthy Papua New Guineans~ " (..':,"o~;:.~ 4 Yean or 5-9 Yean 10-19Yean over 20 Years less n = 10 n=9 n= 17 n= 26 Mean absorbance reading J'. r I j Standard. deviation ' Standard error of mean Maximum Minimum *Two upper extreme values were excluded from each of years and over 20 years before analysis (see footnote a).l ': " Table 3 Distribution by age, sex and clinical status of subjects in each study group. TB-NSW n= 16 TB-PNG n= 18 Non-TB n = 10 Healthy n=31 Age Mean ± S.D ± ± ± ± 15.0 Range Sex Male 12 (75%) 11 (16%) 5 (50%) 6 (19%) Duration of illness (months) Mean ± S.D. 5.2 ± ± ± 2.8 Range (number of patients) Severity of illness at the time of specimen collection Well Mild Severe 5 3 Duration of treatment before specimen collection (months) Mean ± S.D. 3.7 ± ± ± 2.8 Range Unknown

5 J Infect Dis Antimicrob Agents Vol. 1 No.2 Apr. -Jun It should be mentioned here that for the Australian TB patients (TB-NSW) serum samples were obtained, in most cases, after the diagnosis had been established for some months. Only two patients still suffered from clinical disease at the time of specimen collection. The diagnosis in one of these cases was tuberculosis. of the vertebral column; the ELISA absorbance for this case was The other surffered from severe pulmonary tuberculosis since 1960 and currently from psychiatric disease; his absorbance reading was Results of absorbance readings from the study in each groups are summarized in the following tables and figures. Analysis of variance of all groups revealed F = 2.87, tail probability = BMDP was used for computation. Pairwise comparisons of age-specific mean absorbance readings were performed using the separate variance t test with the Bonferroni adjustment for significance levels for multiple comparison. Mean absorbance in the < 5 years age group was significantly less than in both the years and over 20 years age groups (P < 0.05 and 0.01, respectively). Midpoints of absorbance readings ' TB patients in New TB patients in Papua Non TB Healthy South Wales New Guinea patients subjects Fig. 2 Histogram of the absorbance readings of Australian and Papua New Guinean tuberculosis patients and in Australian non-tuberculous cases and healthy subjects. Table 4 Mean ELISA absorbances, measures of dispersion and maxima and minima for Australian and New Guinean TB patients, and Australian non-tb patients and healthy subjects" TB patients TB patients Non-TB Healthy innsw inpng patients subjects n = 16 n= 18 n= 10 n= 31 Mean Standard deviation Standard error of mean Maximum Minimum P<O.OOI when all groups were included in the analysis. When the group of TB patients in Papua New Guinea was excluded from the analysis, P= and in subsequent pairwise comparisons among the groups, the difference of IgG level between Australian TB patients and healthy individuals remained statistically significant (Bonferroni t test P<0.05).

6 ::<I;i 1.11J~1J;i 2 "" 68 un H t t m.fj. -1J.fJ DISCUSSION For the assay to be very useful for case-finding in an endemic area, the absorbance reading should be minimally interfered with by previous BCG vaccination which may stimulate antibody production against PPD. Theoretically, the highest value obtained from a vaccinated person should be far below the diagnostic cut-off point for the test. We chose three months after vaccination as an optimal time to assay antibody levels since most vaccinees show reactivity to tuberculin in skin testing at this time. It was not feasible for us to have periodic blood collections for longterm follow-up of persons in this group. Perhaps due to small sample size, we were unable to show the statistical significance of the difference of the mean absorbance readings obtained before and after BCG vaccination (P = 0.133). Nevertheless, the highest mean value which was (or as the mean plus two standard deviations) in the BCG vaccinated group is far below 0.23 which we feel is the optimal cut-off point in our test for Australians. In fact, the range of absorbance readings in the vaccinated group is very similar to those in the non-tuberculous patients and in healthy persons. This finding agrees with results presented in previous publications.pi" Therefore, we may conclude that BCG vaccination has very little impact if any, on the diagnostic purpose of the test for active case-finding. In Papua New Guinea where tuberculosis is still prevalent, it was anticipated that the level of certain immunoglobulins against tuberculous antigens would increase with age and consequent exposure to tubercle bacilli. In this study, 66 sera collected from presumably healthy Papua New Guineans, and stored at -70 C for two years, were selected according to age group and assayed for IgG antibodies against purified protein derivative of tuberculin. The mean value of IgG antibodies as reflected by absorbance readings was higher in age groups of years and over 20 years compared with those less than 5 years (P < 0.05 and P < 0.01 respectively). This finding suggests that for the ELISA test to be used as a diagnostic test in an endemic area, the cut-off point for detection of active TB case should be adjusted for age. One alternative explanation for higher levels of IgG antibodies against PPD in adults may lie in non-specific stimulation of anti-ppd IgO production by non-tuberculous infections. A total of four cases were excluded from the analysis because of extremely high absorbance readings which would suggest these cases to be active TB cases. Three out of four had absorbance readings over 2.0. This study would suggest that the incidence of tuberculosis in that area is around %. The usefulness of the ELISA test in the field survey for v.",. ~ v ~ 17UfTJ 1:'jfWi fit ')ff) II ti~fj7fi TiliJ, tiun the purpose of case-finding is of great interest since the assay is rapid and technically and economically feasible. Since tuberculosis is not a major problem in Newcastle and Sydney in Australia, most TB patients in this study had sera collected long after bacteriological diagnosis had been established and hence examination of the sensitivity and specificity of the test was not the main purpose of this study. We have simply examined the levels of IgG antibodies to PPD in TB patients compared to non-tb patients and healthy individuals. As we see from Figure 2 and Table 3, absorbance readings in sera from TB patients in Papua New Guinea yielded very high values when compared even with Australian TB patients. This finding was unexpected and raised some questions which could not be answered in this study. The high value of absorbance readings in Papua New Guinean TB patients may be due to the higher prevalence of tuberculosis and other tropical infectious diseases in that area. Unfortunately we could not obtain contemporary serum specimens from healthy individuals and non-tb cases living in the same area to serve as controls. Anyway, this finding underscores the importance of location, prevalence of tuberculosis or race as major factors in determining the result of the test. The cut off point and method of the test should be modified to suit local needs. With regard to IgG levels in Australian TB patients, non-tb patients and healthy individuals, the difference between mean absorbance readings for the three groups was still significant (P = ). When pairwise comparisons among the three groups were carried out, the difference of mean absorbance reading between TB patients and healthy individuals or non-tb patients was significant at a p value of 0.05 or less, in both cases. The highest absorbance reading in TB patients was obtained from a patient who still suffered from tuberculosis at the time of blood collection despite being treated for two months. Other TB patients yielded absorbance readings between This is not surprising since they had been treated for periods varying up to many months and many had recovered completely at the time of specimen collection. Use of the test to determine the effectiveness of current anti-tuberculous treatment while patients are in the follow-up period warrants explanation. Serum from patients who fail to respond to anti-tuberculous treatment should be included to see if the high absorbance readings are maintained. Due to different methods lqf antibody detection and the study design we were unable to compare our result with others who demonstrated increased antibody titers after anti-tuberculous therapy, usually reaching maximal levels within 90 days.7,2o-22 Since the maximal absorbance readings in Aus

7 J Infect Dis Antimicrob Agents Vol. 1 No.2 Apr. -Jun tralian non-tb patients and healthy persons were and respectively, we feel that if the test is to be used in Australia for diagnosis of active or inactive TB the optimal cut-off point may be around The appropriateness of this value will require further confirmation. Further study would also be required in Papua New Guinea before the full value of the test in that setting can be assessed. ACKNOWLEDGEMENTS We would like to express our gratitude to Pro.fessors R. Clancy and S. Leeder under whose guidance this study was carried out. We are also most grateful to the following persons who assisted in the identification of patients and the collection of serum specimens: Dr. K. Murree Allen and Srs. M. Levi and E. Duffan of Royal Newcastle Hospital; Professor B. Gandevia and Drs. B. Jarvie and P. Robertson at the Prince of Wales Hospital, Sydney; and Drs. M. Davis and P. Watt and Ms. M. Connellan of Madang General Hospital, Papua New Guinea (with the further assistance of the Papua New Guinea Institute of Medical Research). Misses M. Watson of the Newcastle Technical College and M. Marks of the Royal Newcastle Hospital are thanked for their permission to invite trainee. nurses to participate in the study. We are grateful to the nurses who volunteered and also to the many patients who willingly co-operated. Finally we would like to thank Mrs J. Lennan who repeatedly processed the words of this report. REFERENCES 1. Immunological research in tuberculosis: Memorandum from a WHO meeting Bulletin of the World Health Organization 1982; 60: Tuberculosis control (Technical Report Series). Report of a joint IUAT/WHO study group. World Health Organization, Geneva, Middlebrook G, Dubos RJ. Specific serum agglutination of erythrocytes sensitized with extracts of tubercle bacilli.j Exp Med 1948; 88: Reggiardo Z, Aber VR, Mitchison DA, Devi S. 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