Use of recombinant purified protein derivative (PPD) antigens as specific skin test for tuberculosis.

Background & objectives: Purified protein derivative (PPD) is currently the only available skin test reagent used worldwide for the diagnosis of tuberculosis (TB). The aim of this study was to develop a Mycobacterium tuberculosis specific skin test reagent, without false positive results due to Bacillus Calmette-Guerin (BCG) vaccination using recombinant antigens. Methods: Proteins in PPD IC-65 were analyzed by tandem mass spectrometry and compared to proteins in M. tuberculosis culture filtrate; 54 proteins were found in common. Top candidates MPT64, ESAT 6, and CFP 10 were overexpressed in Escherichia coli expression strains and purified as recombinant proteins. To formulate optimal immunodiagnostic PPD cocktails, the antigens were evaluated by skin testing guinea pigs sensitized with M. tuberculosis H37Rv and BCG. Results: For single antigens and a cocktail mixture of these antigens, best results were obtained using 3 μg/0.1 ml, equivalent to 105 TU (tuberculin units). Each animal was simultaneously tested with PPD IC-65, 2 TU/0.1 ml, as reference. Reactivity of the multi-antigen cocktail was greater than that of any single antigen. The skin test results were between 34.3 and 76.6 per cent the level of reactivity compared to that of the reference when single antigens were tested and 124 per cent the level of reactivity compared to the reference for the multi-antigen cocktail. Interpretation & conclusions: Our results showed that this specific cocktail could represent a potential candidate for a new skin diagnostic test for TB.

Conversion of tuberculin skin test in adults

Tuberculosis is a major public health hazard. The tuberculin skin test is one of the diagnostic tools in this regard. Since BCG vaccination is performed during infancy in Iran, a positive tuberculin skin test [PPD] may confuse the physician. For this reason we performed this study on adults. A descriptive study was performed on 433 soldiers between 2006 and 2007. Demographic data like age, level of education, family history of tuberculosis, place of residence, cigarette smoking, and chronic cough for more than 3 weeks, were collected from each patient. All patients had a history of BCG vaccination during infancy and its scar was detected in all of them. Purified protein derivative [PPD] test was performed. A 0.1 ml of 5TU PPD solution was injected intradermally into the volar face of the forearm and after 72h transverse diameter of the induration was measured in millimeters with a transparent ruler. Induration size greater than l0mm was considered a positive reaction. All patients were followed for one year and participated again in the second phase of PPD injection after one year. Data were analyzed by using SPSS software ver.13. Paired t-test and Chi-square test were used for statistical evaluation. All soldiers were male with a mean age of 23.2 +/- 1.8 yrs. Twenty-three cases [5.3%] had positive tuberculin skin tests after one year. The highest level of education was high school diploma in 288 soldiers [66.5%]. There was no significant correlation between the educational background and positivity of the tuberculin skin test [p = 0.219]. Twenty-two cases [5%] had a history of cigarette smoking which was significantly related to positive tuberculin skin tests [p=0.001]. There was chronic cough in 44 [10.6%] soldiers which did not have any significant correlation with tuberculin skin test results [p = 0.6]. This study showed that the prevalence of new cases of tuberculosis was more than 5% per year. Therefore, performing tuberculin skin test in BCG vaccinated adults is important

A lyophilized formulation to extend the shelf-life of tuberculin PPD.

This study was aimed to develop a dry purified protein devirative (PPD) preparation to extend the shelf-life of tuberculin PPD. Five percent sucrose (S), 6.5% mannitol (M), 2.5% trehalose (T) or 0.3% Hemaccel (H) was added to each formulation. In vivo and in vitro analyses were carried out to determine the efficacy of the lyophilized products. In the in vivo test, the delayed type hypersensitivity (DTH) responses of the lyophilized preparations were compared to the liquid preparation (CL) after injection into BCG vaccinated guinea pigs. The preparations of H, M, T, and S generated DTH responses of 100, 90, 89, and 60%, as compared to the response of CL, respectively. There was no loss of tuberculin activity in the H formula. A statistically significant difference in activity was found between S and CL (p < 0.05). The cellular test for IFN-gamma secretions was performed using the whole blood of human subjects screened for DTH response to tuberculin PPD Mantoux tests. The detection of IFN-gamma secretions was done using ELISA and the efficacy was expressed in terms of percentage of IFN-gamma responses to the tuberculin antigens. The results of CL, H, M, T and S were 3.28, 10.40, 0.84, 1.52 and 1.29%, compared to mitogen stimulation, respectively. The lyophilized H, M and T formulations and the liquid CL were studied for their shelf-life stabiliy. Accelerated degradation was done by storing the samples at higher temperatures of 37 degrees C and 56 degrees C for 3, 6, 9 and 12 months. All the tuberculin PPD solutions were injected into BCG vaccinated guinea pigs at the end of each storage period and the activity of each solution was evaluated. The formulation with the Hemaccel as excipient gave a superior response than the others at the normal storage temperature of 40 micro C for 12 months. Therefore, Hemaccel provides protection for PPD activity. This supports the potential for the development of lyophilized tuberculin PPD with the addition of 0.3% Hemaccel to extend shelf life.

Serodiagnosis of extra pulmonary tuberculosis using A-60 antigen.

The diagnosis of extrapulmonary tuberculosis including neurotuberculosis is difficult because of the low yield of culture positivity for Mycobacterium tuberculosis (M. tb). Serodiagnosis has emerged as a useful aid to the diagnosis of extrapulmonary tuberculosis. The utility and efficacy of detection of antimycobacterial antibodies to A-60 antigen in serum and/or cerebrospinal fluid (CSF) was analysed in 100 patients-neurotuberculosis-72, abdominal tuberculosis-12 and others-16. The overall positivity rate for the test was 75%. The positivity rate of the test in serum and/or CSF was 79.2% (57 of 72) in neurotuberculosis and 62.5% (10 of 16) for other forms of extrapulmonary tuberculosis. The positivity rate for antimycobacterial antibodies was higher for patients with tubercular meningitis -94.7%. To conclude, testing for antimycobacterial antibodies to A-60 antigen is a useful adjunct in the diagnosis of extrapulmonary tuberculosis especially neurotuberculosis.

Reaction to tuberculin testing in Saudi Arabia.

An epidemiological study of tuberculosis infection in the Kingdom of Saudi Arabia was conducted, between January 1987 to February 1990. A proportional to population size sampling method was used for the whole country, and 1933 subjects were screened. A pre- designed questionnaire was used to collect details of BCG scar, age, sex, residence area, nationality, education, occupation and tuberculosis test. The relative importance of these factors was assessed by using Logistic Regression Analysis. The extent to which these factors affected the severity of tuberculosis was also examined. A number of statistically significant association were found between positive tuberculin test (> 10mm) and age (p < 0.0001), sex (p = 0.018), nationality (p = 0.009), residence area (p = 0.05) and occupation (p = 0.0003).