To improve diagnostic reliability, we aimed to evaluate the sensitivity of tuberculosis skin testing to topical zinc supplementation at the test site.
Methods: We performed this study on 100 children between the ages of 6 and 14 years, and plasma zinc levels were analyzed after 10-12 hours of fasting. After tuberculosis skin testing (PPD), we applied 40% zinc oxide cream on one forearm and a placebo on the other forearm.PPD indurations were measured 72 hours later.
Results: In this study, 26% of the children showed increased PPD indurations following local zinc supplementation.There was no correlation between the induration size and serum zinc levels.
Conclusion: We conclude that,topical zinc cream supplementation can enhance the sensitivity of tuberculin reactivity for diagnosing tuberculosis.
Keywords: Topical zinc cream supplementation,tuberculosis skin test,plasma zinc levels
Tuberculosis is a serious illness in developing countries, especially in asymptomatic, immunosuppressed children .It is estimated that the annual risk of tuberculosis infection in children in developing countries is 2-5% (1). The estimated risk of a young child developing tuberculosis upon infection with Mycobacterium tuberculosis, as indicated by a positive tuberculin test, is approximately 10% (2). Nearly 8-20% of the deaths caused by tuberculosis occur in children (3). We can diagnose latent infections by means of a tuberculin skin test (PPD). However, the reliability of this test is limited by false-negative results, especially in malnourished children. Deficiency of zinc, a micronutrient that modulates immune response and supports antibacterial immunity, can cause false-negative results in PPD skin tests (4,5,6).Therefore, we investigated whether the application of a topical zinc supplementation could enhance the sensitivity of the tuberculin skin test for the diagnosis of tuberculosis.
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This study was performed on 100 children between the ages of 6-14 years who were hospitalized in our pediatric clinic between January and December of 2009-2010.After receiving ethical approval and informed written consent, venous blood was collected from the children after 10-12 hours of fasting. Plasma zinc levels were analyzed by atomic absorption spectroscopy. Subjects with severe infections, which could affect the immune system, anergic viral infections, steroid administration, and immune suppression, were excluded.
We injected 0.1ml tuberculin (5U in 0.1 ml Aventis-Pasteur) into the volar surfaces of both forearms. After implementation, we covered the skin-test side of the left arm with zinc oxide dissolved in an aqueous cream of 40% elemental zinc.The skin test side of right arm was covered with 1 ml of placebo cream. After 72 hours, the indurations sizes on both arms were measured by a ball-point pen.
SPSS version 11.5 was used for statistical analysis.The Wilcoxon matched-pairs signed-rank test was performed to compare the PPD indurations. Correlations between the PPD results (with zinc supplementation or placebo) and the plasma zinc levels were analyzed with the Spearman rank correlation test.
Of the 100 children included in this study, 41% were male and 59% were female with a mean age of 9 years. All the patients had tuberculin test scars. The mean serum zinc level of the females was 89.86 mcg/dl, and the mean was 90.15 mcg/dl for the males.The serum zinc levels were lower than normal (<70 mcg/dl) in 12 of the patients. Seventy-six percent of the PPD induration responses were smaller than 5mm with placebo cream, 22% were 6-14 mm and 2% were larger than 15 mm.The induration responses to PPD with the zinc ointment were as follows: 71% were smaller than 5 mm, 21% were 6-14mm, and 8% were larger than 15mm.There was a significant difference in induration size upon the application of the topical zinc cream (p<0.0001) (Figure 1).
The mean age of the children was 8.98±2.76 (min:3, max:14) years. While the mean PPD induration size of the children with the placebo ointment was 2.55±4.45, the mean PPD induration size with the zinc supplementation was 3.58±5.68 mm. The mean PPD induration size with the zinc supplementation was significantly higher than the mean PPD induration size with the placebo (pË‚0.0001) (Table 1). The mean serum zinc level of the children was 90.07±24.71mcg/dl. There was no significant correlation between the plasma zinc levels and the PPD induration sizes with the placebo and zinc supplementation (Table 1).
Figure 1: PPD measurements with placebo and zinc supplementation
In this study, 26 of the patients showed increased PPD induration sizes following local zinc supplementation.Three patients had initial negative PPD responses, which became weakly positive following the topical zinc supplementation. Six patients had initial indurations between 6 and 14 mm, which were measured as positive (>15mm) after applying the local zinc cream (Figure 2).
Figure 2: Increased PPD induration measurements after zinc supplementation
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There was no correlation between the induration sizes and serum zinc levels (p>0.05), and there was no significant difference between serum zinc levels and induration sizes (p>0.05) of the male and female children.
Table 1: Outcomes related to PPD with zinc and the placebo
PPD with placebo
PPD with zinc
Plasma zinc Level
Mean value ±Standard Deviation (SD)
Zinc is essential for the normal development and function of cell-mediated immunity, neutrophils and natural killer (NK) cells. It is needed for DNA synthesis, RNA transcription, cell division and cell activation.Zinc deficiency adversely affects the secretion and function of cytokines, which are the basic messengers of the immune system.Zinc is involved in the maturation and differentiation of T cells.Expression of the interleukin(IL)-2 and IFN-Æ´ (Th1 cytokines) genes is zinc-dependent.IL-2 is involved in the activation of NK and cytolytic T cells.Together, IFN-Æ´ and IL-2 play a major role in the killing of parasites,viruses and bacteria by macrophages and monocytes. Zinc deficiency is associated with impaired phagocytic function,lymphocyte depletion,decreased immunoglobulin production, a reduction in the CD4/CD8 ratio and decreased IL-2 production.Severe zinc deficiency is characterized by depressed immune function, frequent infections, bullous pustular dermatitis, diarrhea and alopecia. Zinc circulates at a concentration of 70 to 120mcg/dl; 60% is bound to albumin, and 30% is tightly bound to macroglobulin (7, 8, 9, 10, 11, 12, 13).
Low plasma zinc levels are usually defined as less than 60 mcg/dl.Some investigators argue that plasma zinc measurements are relatively insensitive, and zinc levels in neutrophils and lymphocytes may be more sensitive.The criterion for zinc deficiency is a decreased zinc level either in lymphocytes (<50 mcg/10 cells) or in granulocytes (<42 mcg/10 cells)(14, 15).Bhargavi et al performed a similar study on 50 healthy adult volunteers. They initially evaluated serum zinc levels. They then placed PPD at the proximal sides of the palmar forearms and placed Candida antigens at the distal sides of the forearms.They placed placebo cream on one forearm and zinc cream on the other arm. The indurations sizes were measured after 24, 48 and 72hours.The indurations sizes were larger in 32% of the arms that received zinc cream (16).In zinc-deficient subjects, topical zinc application enhanced the tuberculosis skin test augmentation. The zinc cream had no effect on subjects with normal zinc levels.The skin tests with zinc applications were significantly more likely to yield positive results when compared to the contralateral control skin tests with the placebo cream application (94% zinc, 76% placebo) (16).This study differed from ours in that they found statistically significant differences in the PPD responses of patients with lower zinc levels, but there were no differences in the patients with normal serum zinc levels. In our study, there was a significant difference in the PPD indurations sizes after applying local zinc supplementation, independent of plasma zinc levels. This study was similar to ours, in that they evaluated the ability of local zinc supplementation to enhance the PPD response through immunomodulation.
Kwok et al investigated the ability of topical zinc supplementation to increase the sensitivity of the tuberculin skin test. Thirty-eight of 58 elderly patients (66%) had negative skin test reactions with the placebo ointment.Fourteen of these negative responders (37%) showed positive reactions with the topical zinc ointment, 12 (32%)had weakly positive reactions and 12 remained negative.They determined that 37% of the patients with negative PPD tested positive after applying the zinc cream ointment ( 17). Although this study was performed with elderly people, it supports our study because it shows that topical zinc supplementation can modulate the immune response.
Cuevas et al investigated the differences in PPD responsiveness in patients younger than 15 years. The patients had received tuberculin skin testing in 2-year intervals. The group receiving the oral zinc supplementation prior to applying the test showed increased positive responsiveness and induration measurements compared to the placebo group (18). This study differed from ours, via their use of oral zinc supplementation. However, the two studies support each other because they conclude that zinc modulates cell-mediated immune responsiveness.
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Castillo-Duran et al evaluated changes in immunity and development upon zinc supplementation in 32 marasmic infants.Half of the subjects was given 2 mg/kg oral zinc for 3 months, and the other half took the placebo. They evaluated PPD responses on the first and 90th day post-treatment. Prior to applying zinc, 1/3 of the patients showed positive PPD results. After the zinc supplementation, 2/3 of the patients revealed positive PPD results.There was no significant difference in the PPD responsiveness in the placebo group (19). This study supports the immunomodulatory effect of zinc supplementation on enhancing PPD reactivity.
Kramer TR et al investigated T lymphocyte proliferation in 140 children aged between 6 and 13 years by evaluating PPD responsiveness after 6 months of 25 mg/day oral zinc supplementation.The group receiving zinc supplementation showed significant increases in PPD responsiveness compared to the placebo group (20). While this study differed from ours, through their use of systemic zinc combined with vitamin A for a longer duration, it also supports our finding that zinc supplementation has an immunomodulatory effect.
Although tuberculin skin testing plays a major role in the diagnosis of latent tuberculosis, nutritional and immune status, chronic illness and environmental factors can decrease its sensitivity.The incidence of tuberculosis has increase during both adulthood and childhood. As a result,the effect of zinc on PPD responsiveness has gained importance as a diagnostic approach. Although systemic zinc supplementation has been preferred in previous studies, we aimed to examine the effect of local zinc cream application, which could provide a simple and inexpensive approach to enhancing PPD sensitivity for the diagnosis of disease.
We believe that, future studies using larger patient cohorts will support the sensitivity of PPD responses to topical zinc supplementation for cases with lower plasma zinc levels.
To enhance tuberculin skin test sensitivity and reduce false-negative results, we recommend using local zinc ointments, without prior screening of plasma zinc levels,after performing a PPD.An optimal diagnostic approach is to use the booster effect of topical zinc following a PPD to enhance tuberculin reactivity.