Skin Test Negative Individuals Research Articles

APAAACI clinical pathway on direct provocation testing for penicillin allergy delabeling

Background: Allergy to penicillin is commonly reported in many countries and is an overwhelming global public health concern. Penicillin allergy labels can lead to the use of less effective antibiotics and can be associated with antimicrobial resistance. Appropriate assessment of suspected penicillin allergy (often including skin testing, followed by drug provocation testing [DPT] performed by allergists) can prevent the unnecessary restriction of penicillin or delabelling. Many countries in the Asia Pacific (AP) have very limited access to allergy services, and there are significant disparities in the methods of evaluating penicillin allergy. Therefore, a clinical pathway for the management of penicillin allergy is essential. Objectives: To develop a risk-stratified clinical pathway for delabeling penicillin allergy, taking into account the distinct epidemiology, patient/sensitization profiles, and disparities of allergy services or facilities within the AP. Methods: A risk-stratified penicillin allergy delabeling clinical pathway was formulated by the Drug Allergy Committee of the Asia Pacific Association of Allergy, Asthma and Clinical Immunology. and members of the Penicillin Allergy Disparities survey in AP each representing one country/region of the AP. The clinical pathway was tested based on a database of anonymized patients who were sequentially referred for and completed penicillin allergy evaluation in Hong Kong. Results: The clinical pathway was piloted employing a “hub-and-spoke” approach to foster multidisciplinary collaboration between allergists and nonallergists. A simulation run of the algorithm on a retrospective Hong Kong cohort of 439 patients was performed. Overall, 367 (84%) of patients were suitable for direct DPT and reduced the need for skin testing or specialist’s care for 357 (97%) skin test-negative individuals. Out of the skin test-negative patients, 345 (94%) patients had a negative DPT. Conclusions: This risk-stratification strategy for direct oral DPT can reduce the need for unnecessary skin testing in patients with low-risk penicillin allergy histories. The hub and spoke model of care may be considered for further piloting and validation in other AP populations that lack adequately trained allergists.

Allergic Rhinitis with Negative Skin Tests

Recently, several studies have revealed a subset of patients who have positive nasal provocation to allergens despite having a negative skin prick test. It has been hypothesized that these patients have localized allergic rhinitis. However, the prevalence varies greatly, ranging from 0% to 100% of skin test-negative individuals. This wide range in prevalence is likely related to differences in methodology, including differences in allergen manufacturers, concentrations, and numbers of allergens tested and, perhaps most importantly, criteria for a positive nasal challenge. Despite the evidence to date, many challenges exist with regard to the concept of localized nasal allergy. Further studies will be required to further define the immunopathology, prevalence, practical diagnostic tests, and management.

Toll-like receptor 4 polymorphisms predispose to cutaneous leishmaniasis

The clinical spectrum of cutaneous leishmaniasis (CL) is extremely variable. Studies in experimental leishmaniasis have revealed a role for TLR4 in control of infection. In the present study the associations between TLR4 mutations (Asp299Gly and Thr399Ile) with outcome of CL have been investigated. Genotyping for Asp299Gly and Thr399Ile was performed in patients with chronic (N = 22) and acute (N = 61) CL, asymptomatic (N = 45) and healthy leishmanin skin test negative individuals (N = 75). The results showed the frequency of the Asp299Gly genotype was increased in patients with chronic disease (OR 25.3, 95% CI 5.2–115.6, P < 0.001) and patients with acute disease (OR 8.03, 95% CI 1.7–37.7, P = 0.006) compared to LST negative subjects. Thr399Ileu genotype was significantly over represented among patients with chronic disease (27.3%, P < 0.001), patients with acute disease (13.1%, P = 0.016), and asymptomatic donors (15.6%, P = 0.008) in comparison with LST negative normal group (1.3%). Both variants were found together more frequently in patients with chronic disease compared to the patients with acute disease (P = 0.045), and asymptomatic donors (P = 0.045). The results provide evidence that polymorphisms of TLR4 gene may lead to the increased susceptibility to and severity of infection by Leishmania major. The concomitant carriage of both mutations increases the susceptibility of individuals to CL.

An in vitro model of the leukocyte interactions associated with granuloma formation in Mycobacterium tuberculosis infection

The principal defense of the human host against a Mycobacterium tuberculosis infection is the formation of granulomas, organized collections of activated macrophages, including epithelioid and multinucleated giant cells, surrounded by lymphocytes. This granuloma can sequester and contain the bacteria preventing active disease, and if the granuloma is maintained, these bacteria may remain latent for a person's lifetime. Secretion of a variety of chemoattractant cytokines following phagocytosis of the bacilli by the macrophage is critical not only to the formation of the granuloma but also to its maintenance. To investigate this process of early granuloma formation, we developed an in vitro model composed entirely of human cells. Combining blood lymphocytes and autologous macrophages from healthy purified protein derivative skin test-negative individuals and mycobacteria resulted in the formation of small, rounded aggregate structures. Microscopic examination found macrophage-specific CD68(+) epithelioid macrophages and small round CD3(+) lymphocytes that in complex resembled small granulomas seen in clinical pathology specimens. Acid-fast staining bacteria were observed between and possibly within the cells composing the granulomas. Supernatants from the infected cells collected at 24 and 48 h and 5 and 9 days after infection were analyzed by a multiplexed cytokine bead-based assay using the Luminex 100 and were found to contain interleukin (IL)-6, IL-8, interferon-gamma and tumor necrosis factor-alpha, cytokines known to be involved in human granuloma formation, in quantities from two-fold to 7000-fold higher than supernatants from uninfected control cells. In addition, chemotaxis assays demonstrated that the same supernatants attracted significantly more human peripheral blood mononuclear cells than those of uninfected cells (P<0.001). This model may provide insight into the earliest stages of granuloma formation in those newly infected.

Surrogate markers of immunity to Leishmania major in leishmanin skin test negative individuals from an endemic area re-visited

Background In the screening of vaccine candidates it is important to select candidates that evoke immune responses associated with protection. Valid surrogate markers against human leishmaniasis are still lacking. Methods A controlled injection of live Leishmania known as leishmanization, (LZ), was used to evaluate vaccine (alum-precipitated autoclaved Leishmania major with BCG) efficacy and more accurately define surrogate markers of immunity to leishmaniasis in humans. Cellular immune responses to this artificial infection were monitored in the volunteers prior to and 9 months post infection. Comparisons were made between those who developed a lesion after infection and those who did not. Results In the volunteers monitored there was no significant difference in LST, IFNγ production, or source of IFNγ between those who developed a lesion and those who did not after LZ, with the exception that ulcer development was associated with an enhanced number of IFNγ secreting CD4 + CD45RA − (memory) T cells. Discussion Ulcer development following LZ was lower than anticipated by a pilot study (47% versus 78%) using the same stabilate several years earlier. While this may be an effect of low viability/virulence of the LZ inocula, alternative explanations are also possible. The IFNγ responses in the study subjects were significantly lower compared to volunteers with previous history of cutaneous leishmaniasis. The findings raise the possibility that the selection of LST-negative volunteers in an endemic area may bias the study towards potentially non/low L. major-reactive volunteers.

House dust endotoxin exposure mitigates asthma severity in a tropical environment

Rationale Studies have shown exposure to bacterial endotoxin to be associated with asthma severity. Asthma prevalence and morbidity has been increasing disproportionately in tropical, developing countries, where sources of domestic endotoxin, especially humidity, animals in close proximity to humans, and high allergen levels are extreme. To date, no studies have characterized domestic endotoxin exposure in the tropics nor examined the impact of this exposure on asthma. Methods To investigate the relationship between exposure to house dust endotoxin (HDE) and asthma severity in an Afro-Caribbean asthmatic population from Barbados, we measured HDE concentrations in the homes of 87 asthmatic sib-pairs. Asthma severity was assessed using a validated questionnaire that yields a quasi-quantitative Asthma Severity Score. Household characteristics considered to reflect overall household hygiene were assessed and tested for association with HDE concentrations. Results We observed HDE concentrations to be high relative to reports in urban settings in non-tropical environments (mean living room composite=21,061 EU/m 2, range=2649-185,468 EU/m 2). Several household characteristics were positively associated with HDE levels, including carpeting (p=0.01), decreased frequency of vacuuming (p=0.003), and cats in the home (p=0.001). Increasing endotoxin content was associated with decreasing asthma severity scores among skin test negative individuals only (p=0.01). Conclusions HDE levels were high in this tropical environment, but comparable to levels observed in farming populations. Despite early and significant exposure to endotoxin, asthma prevalence is high in this population. The inverse relationship between HDE levels and asthma severity supports the idea that microbial load may be protective and highlights the complex role of HDE in disease expression.

T-Cell Responses to CD1-Presented Lipid Antigens in Humans with Mycobacterium tuberculosis Infection

CD1-restricted presentation of lipid or glycolipid antigens derived from Mycobacterium tuberculosis has been demonstrated by in vitro experiments using cultured T-cell lines. In the present work, the frequency of T-cell responses to natural mycobacterial lipids was analyzed in ex vivo studies of peripheral blood lymphocytes from human patients with pulmonary tuberculosis, from asymptomatic individuals with known contact with M. tuberculosis documented by conversion of their tuberculin skin tests, and from healthy tuberculin skin test-negative individuals or individuals vaccinated with Mycobacterium bovis BCG. Proliferation and gamma interferon enzyme-linked immunospot assays using peripheral blood lymphocytes and autologous CD1(+) immature dendritic cells revealed that T cells from asymptomatic M. tuberculosis-infected donors responded with significantly greater magnitude and frequency to mycobacterial lipid antigen preparations than lymphocytes from uninfected healthy donors. By use of these methods, lipid-antigen-specific proliferative responses were minimally detectable or absent in blood samples from patients with active tuberculosis prior to chemotherapy but became detectable in blood samples drawn 2 weeks after the start of treatment. Lipid antigen-reactive T cells were detected predominantly in the CD4-enriched T-cell fractions of circulating lymphocytes, and anti-CD1 antibody blocking experiments confirmed the CD1 restriction of these T-cell responses. Our results provide further support for the hypothesis that lipid antigens serve as targets of the recall response to M. tuberculosis, and they indicate that CD1-restricted T cells responding to these antigens comprise a significant portion of the circulating pool of M. tuberculosis-reactive T cells in healthy individuals with previous exposure to M. tuberculosis.

Penicillin skin testing in advance of need: Multiyear follow-up in 568 test result-negative subjects exposed to oral penicillins

Background: There are few published data on adverse drug reactions and/or resensitization associated with oral penicillin use in penicillin allergy history-positive/penicillin skin test-negative individuals during routine clinical care with multiyear follow-up. Objectives: We sought to provide long-term follow-up data on the type, severity, and frequency of adverse reactions associated with oral penicillin use in individuals who have histories of penicillin “allergy” yet also have negative results on penicillin skin tests done in advance of need. We also aimed to repeat testing on individuals with penicillin-associated adverse reactions. Methods: Medical records were reviewed for penicillin use and associated adverse reactions in all 568 penicillin skin test-negative individuals who had received at least 1 course of oral penicillin after testing but before December 31, 2001, during routine care. These individuals were drawn from a group of 1246 penicillin skin test-negative individuals seen initially between November 16, 1994, and August 13, 2001. Results: The mean length of follow-up was 4.26 ± 1.64 years (range, 0.39-7.12 years). The mean penicillin exposure was 3.94 ± 3.91 courses (range, 1-22 courses). Only 65 (11.4%) of 568 subjects had any penicillin-associated reactions, and 6 subjects had 2 reactions each. A reaction occurred in 27 subjects (4.8%) with their first penicillin reexposure. There were 71 (3.2%) reactions with 2236 total penicillin courses. There were no serious reactions. Repeated testing was done in 33 subjects older than age 18 years. Only 1 subject was positive on repeated penicillin skin testing. Conclusion: Penicillin use after negative penicillin skin testing done in advance of need is safe, and resensitization is rare. (J Allergy Clin Immunol 2003;111:1111-5.)

Repeat penicillin skin testing after penicillin use associated adverse drug reactions in penicillin skin test negative individuals

Repeat penicillin skin testing after penicillin use associated adverse drug reactions in penicillin skin test negative individuals

Human T‐cell responses to Trichophyton tonsurans: inhibition using the serum free medium Aim V

Proteins of the fungus Trichophyton tonsurans have been shown to give strong T cell proliferative responses in vitro using lymphocytes from individuals with immediate or delayed skin tests. Furthermore, Trichophyton-specific T-cell lines produce distinct patterns of cytokine production depending upon the skin-test reactivity of the host. However, skin-test negative individuals generally give limited responses. A recent study has demonstrated dust mite specific proliferation with lymphocytes from atopic and non-atopic subjects using the serum free medium Aim V. Compare the T-cell reactivity to Trichophyton and dust mite antigens in Aim V and media containing 10% pooled AB serum. Peripheral blood mononuclear cells (PBMC) were separated from subjects with different skin-test reactivities and cultured either in media with serum or serum free media. Proliferation to two Trichophyton extracts was decreased in serum free media among subjects with either immediate or delayed hypersensitivity. Trichophyton skin-test negative subjects gave poor proliferative responses in both culture conditions. A similar decrease in proliferation in serum free media was observed in cultures with PHA and tetanus toxoid. In contrast, the majority of individuals showed increased proliferation to dust mite antigens when cultured in serum free media. Cultures in serum free medium produced less IFNgamma, IL-4, or IL-5 compared with cultures in AB medium. In vitro T-cell responses to the dermatophyte fungus T. tonsurans are inhibited by the serum free medium Aim V. This inhibition is seen equally with cells from individuals with delayed and immediate hypersensitivity. Furthermore, the results do not support the view that AB serum is masking T-cell responses in skin-test negative individuals.

The Relationship of Nonspecific Airway Responsiveness and Atopy to the Rate of Decline of Lung function: The Normative Aging Study

Nonspecific airway hyperresponsiveness and atopy have been postulated to be risk factors that predispose individuals to the development of chronic obstructive pulmonary disease. The purpose of the present study was to examine the relationship of methacholine airway responsiveness and markers of atopy to rate of decline in lung function among 790 men, age 40 to 79 yr, followed in the Normative Aging Study. Airway responsiveness was assessed at the end of follow-up, which ranged from 14 to 24 yr. Multiple linear regression analysis indicated that greater methacholine airway responsiveness was associated with more rapid annual FEV1 decline. Stratified analysis suggested that this association was stronger among skin test-negative, current smokers. No significant relationship was found between serum IgE level and annual FEV1 decline. A weak but statistically significant correlation between eosinophil count and mean annual FEV1 decline was noted among skin test-negative individuals (r = -0.08; p = 0.03). Results from the present study do not show a clear association between atopy and decline in FEV1. These data, however, do indicate an independent association between heightened methacholine responsiveness on decline in FEV1 in adult males.

Transient Increase of IgG Fc Receptor‐Bearing T Lymphocytes following Positive PPD Skin Testing

In tuberculin-sensitive individuals, IgG Fc receptor (FcR)-bearing lymphocytes in the peripheral blood increased transiently following PPD-tuberculin skin test. This rise in circulating FcR-bearing cells appeared to peak about 36--48 h after the intradermal inoculation of PPD and seemed to occur largely in the T cell population. Skin test-negative individuals showed no significant changes in their circulating FcR-bearing cells following PPD inoculation. Peripheral blood lymphocytes from PPD-sensitive individuals were fractionated into non-T cell and T cell-enriched populations by E rosette sedimentation technique. FcR-bearing cells in the T cell-enriched population were eliminated by EA rosette sedimentation: i.e. FcR-negative T cells. Then, equal numbers (1 X 10(5) cells each) of non-T cells and unfractionated or FcR-negative T cells were recombined in culture. Prior to PPD inoculation, there was no significant difference between these two cell mixtures in the in vitro cellular response to PPD or mitogens. When these cell populations were obtained 36--48 h after PPD inoculation, however, the combination of non-T cells and FcR-negative T cells responded to PPD much better than the combination of non-T cells and unfractionated T cells, whereas the mitogen-induced cellular proliferation of these two cell mixtures did not differ from each other.

Lymphocyte Multiplication in Vitro Induced by Mitogens and Antigens

A simple technique requiring only 0.2 ml whole blood for measuring the response of lymphocytes in cultures to each of various mitogens and antigens has been elaborated. The response is quantified by comparing the number of lymphocytes with and without a stimulating agent. The increment of cell numbers is given by a cell multiplication index. In healthy subjects PHA induced almost a doubling of the cell numbers in 3 days, i.e. an index of 1.90 +/- 0.38. After 7 days the indexes for PHA, PWM and Con A were 7.25 +/- 4.12, 2.72 +/- 0.65 and 1.81 +/- 0.31, respectively. PPD and Candida-extract induced cell multiplication in skin positive individuals, with indexes ranging from 1.12 to 3.05. In contrast, patients with various severe immune deficiencies showed decreased responses to at least one mitogen, depending on the type of the deficiency. Likewise, skin test negative individuals had no or faint in vitro response to the antigens. The method, which correlated well with the response by a conventional method for incorporation of tritiated thymidine, has a high degree of precision and sensitivity, and should be applicable for routine use.

Soluble antigens of mycelia and spherules in the in vitro detection of immunity to Coccidioides immitis.

The effect of a Coccidioides immitis spherule-derived antigen (spherulin) upon in vitro thymidine uptake by sensitized and nonsensitized lymphocytes was studied, and optimal conditions for the assay were determined. The mitogenic portion of the antigen was shown to be nondialyzable, as was true for a mycelial-derived antigen (coccidioidin). Concentrations of 20 mug of nondialyzable spherulin solids and 17 mug of nondialyzable coccidioidin solids per ml of culture were shown to be suitable for differentiating skin test-positive from skin test-negative individuals. Comparison with mycelial filtrate suggested spherulin may be a more potent mitogen for sensitized lymphocytes.

ASSOCIATION OF HOUSE-DUST AND GRASS-POLLEN ALLERGIES WITH SPECIFIC IgA ANTIBODY DEFICIENCY

Adults classified as skin-test positive to the house-dust mite Dermatophagoides farinæ had significantly more IgE antibody and significantly less IgA antibody to this allergen than did individuals who were skin-test negative. Those classified as skin-test positive to timothy-grass pollen had significantly more IgE anti-grass-pollen antibody than did skin-test negative individuals but showed only a non-significant trend towards less IgA antibody. However, when the data were expressed in terms of month of sampling, the atopic individuals were found to have significantly less IgA antibody during the pollen season. These findings support the view that overstimulation of the IgE system in atopy results from a defective IgA response.

Inhibition of Leukocyte Migration: An Evaluation of this in Vitro Assay of Delayed Hypersensitivity in Man to a Soluble Antigen

Abstract An in vitro assay of delayed hypersensitivity in man, modified from the method of Søborg and Bendixen, was evaluated. This assay is based on the inhibition of migration of human peripheral leukocytes in the presence of specific soluble antigen. Twelve of 13 individuals who were skin test positive to tuberculin showed inhibition of leukocyte migration when incubated with 100 µg/ml of soluble purified protein derivative. Only 1 of 12 skin test negative individuals showed a similar inhibition of leukocyte migration. Analysis of the migrating populations showed that the migration of mononuclear cells and not of neutrophils was inhibited.

Coccidioidin Delayed Hypersensitivity: Skin Test and In Vitro Lymphocyte Reactivities

Summary Delayed skin test reactivity and in vitro antigen-induced lymphocyte proliferation were compared in three groups of subjects. Addition of coccidioidin to lymphocyte cultures led to an increased rate of isotope uptake into DNA in cells of strongly coccidioidin skin test positive subjects but not in cells of weakly coccidioidin skin test positive or of coccidioidin skin test-negative individuals. Coccidioidin-induced lymphocyte proliferation was antigen-dose dependent. There was no evidence of in vitro cross-reactivity between coccidioidin and tuberculin in lymphocyte cultures.

Histoplasmosis: To Skin Test or Not to Skin Test?

SummaryA single skin test with commercially-available histoplasmin did not stimulate significant antibody response in the sera of 187 persons when measured by the yeast phase complement-fixation, collodion agglutination or histoplasmin latex agglutination tests. Similarly, no effect was noted with the histoplasmin complement-fixation test in the sera of 97 skin-test negative individuals; in contrast 26.7% of skin-test positive persons developed significant titer increases.