Effects of repeated administration of intradermal skin test by Mantoux method on delayed-type hypersensitivity responses in healthy young and elderly subjects.

The elegant and comprehensive review of markers for the evaluation of immunomodulation published in the British Journal of Nutrition (1) highlighted the importance of delayed-type hypersensitivity (DTH) responses as a highly suitable marker of immune function. Because the DTH ‘skin test’ can predict morbidity and mortality, it has been suggested as a clinically meaningful test to evaluate the effect of nutritional as well as other types of interventions on the immune response. Previously Multitest CMI, a procedure that involved the simultaneous administration of seven recall antigens was used to evaluate DTH. One advantage of this test was that it did not result in a significant boosting effect upon repeated administration and thus could be used more than once in the same individual. However, this test is no longer commercially available and to our knowledge there is no plan to introduce a similar product to the market. An alternative approach is the standard Mantoux technique in which several antigens (tetanus toxoid, tuberculin, Candida and Trichophyton) are administered intradermally, and the diameters of skin induration are read at 24 and 48 h. The Mantoux method may be advantageous because of better sensitivity to detect induration in immunosuppressed patients. There is relatively limited information available on the effect of repeated use of this test in healthy subjects. This information is needed since evaluating the effect of nutritional and other interventions to modify the immune response will require repeated administration of the DTH test and a significant boosting effect introduced by the test itself would confound the treatment effect. In order to address this question, we conducted a study to test the magnitude, if any, of the boosting effect due to repeated administration of different antigens by the Mantoux method in young and old healthy subjects. Ten young (aged 20–40 years) and ten old (aged 66–78 years) free-living men were recruited for this study. Since the magnitude of the DTH skin test is different between men and women, only men were recruited in order to reduce gender-associated variation. Exclusion from enrolment in the study included presence of active neoplastic disease; chronic antibiotic use; immunosuppressive drug or glucocorticoid steroid use (prednisone or equivalent .10 mg/d) 1 month prior to or during the study; recent infection (#2 weeks before enrolment); prior positive tuberculin skin test or BCG vaccination; known severe allergy to any kind of food or drug. The Tufts University New England Medical Center Human Investigation Committee approved the study protocol and informed consent. Each age group of volunteers was randomly assigned to receive either two or three intradermal skin tests. Volunteers were advised to refrain from vaccination, drastic changes in usual dietary intake or activity levels, and intake of micronutrient supplements, and to stop use of nonsteroidal anti-inflammatory drugs 72 h prior to administration of the test. Four recall skin test antigens, tuberculin (Tubersol; Aventis Pasteur, Swiftwater, PA, USA), Candida albicans (Candin, Allermed laboratories, San Diego, CA, USA) and Trichophyton species (Trichophyton mentagrophytes in conjunction with T. rubrum; Hollister-Stier Labs, Spokane, WA, USA), tetanus toxoid (tetanus toxoid USP; Aventis Pasteur, Swiftwater, PA), and a negative control (0·9 % normal saline; Bound Tree Medical, Dublin, OH, USA) were injected intradermally in a standard volume of 0·1 ml (except for tetanus toxoid, which was injected in a volume of 0·025 ml (0·2 LF units per dose) intradermally at separate sites on the volar surface of the forearm in a clean area free of hair or acneiform rash. To determine the magnitude of the boosting effect due to repeated administration, the young and old volunteers were divided into two groups. One group (n 5 per age group) received the test antigens and a negative control at 0, 1, and 3 months while the other group (n 5 per age group) received them at 0 and 3 months. The tests were administered and diameters of skin induration were read at 24 and 48 h by the same investigator (T. A.). The diameter of each induration was measured twice using a metric caliper; the two measurements were averaged and the mean score was recorded. A positive skin test was defined as an induration diameter exceeding 5 mm. The number of positive responses to different antigens, diameter of induration in response to each antigen, and a composite score based on the responses to all antigens were calculated. Data are presented as means and standard errors of the mean, and were analysed using repeated measure two-way ANOVA for the effect of age and repeated administration using Systat 10 software (Systat, Evanston, IL, USA). The mean age of the young subjects was 29·4 (SE 1·7) years and of the elderly was 71·9 (SE 1·4) years. None of the subjects showed a positive response to the saline injection. At baseline, the young subjects had higher total diameter of induration compared to that of the older subjects (46·6 (SE 4·0) mm v. British Journal of Nutrition (2008), 99, 1388–1390 doi: 10.1017/S000711450786853X q The Authors 2007