Development of LepReact, a defined skin test for paucibacillary leprosy and low-level M. leprae infection.

The persistence of new leprosy cases in endemic areas such as India, Brazil, Bangladesh, and the Philippines has encouraged studies of chemoprophylaxis among contacts of patients. Epidemiological screening tools to enable early detection of infected individuals in endemic populations would be critical to target individuals most in need of intervention. Despite decades of attempts, however, there still are no tests available for the early detection of low-level infection with Mycobacterium leprae. In this report, we describe the development of a leprosy skin test using M. leprae-specific antigens. We selected the chimeric LID-1 fusion protein, formulated to achieve maximum performance at a minimal dose, as a skin test candidate based on its ability to elicit delayed-type hypersensitivity (DTH) reactions in M. leprae immune guinea pigs in a sensitive and specific manner, i.e., with no cross-reactivity observed with other mycobacterial species. Importantly, evaluations in armadillos indicated that intradermal inoculation of formulated LID-1 could distinguish uninfected from M. leprae-infected animals manifesting with symptoms distinctly similar to the PB presentation of patients. Together, our data provide strong proof-of-concept for developing an antigen-specific skin test to detect low-level M. leprae infection. Such a test could, when applied with appropriate use of chemo- and/or immunoprophylaxis, be instrumental in altering the evolution of clinical disease and M. leprae transmission, thus furthering the objective of zero leprosy.

Variables affecting interpretation of skin prick test results.

BACKGROUND: Both performer- and device-dependent variabilities have been reported in sizes of wheal responses to skin prick tests. OBJECTIVE: We aimed to evaluate whether or not variabilities in sizes of wheal responses influence the final interpretation of skin prick tests; in other words, the decision on whether or not there is an allergy to a given antigen. METHODS: Skin prick tests with positive and negative controls and extracts of Dermatophagoides farinae and Dermatophagoides pteronyssinus were done for 69 patients by two different persons, using two different puncturing devices- disposable 22-gauge hypodermic needles and metal lancets. RESULTS: Among four different skin prick tests, the average coefficients of variation in sizes of wheal responses were near to or higher than 20% for all of them. On the other hand, in the final interpretation of results, kappa values indicated substantial or almost perfect agreements between these tests. However, the frequency of establishing allergy to the house dust mites widely ranged in these tests (20.8-35.8% for D. farinae and 20.8-28.3% for D. pteronyssinus). LIMITATIONS: The conduction of the study in a single center and the comparisons of results of only two performers. CONCLUSION: We feel that variabilities in sizes of wheal responses of skin prick test can influence its categorical results.

The challenge of producing skin test antigens with minimal resources suitable for human application against a neglected tropical disease; leprosy.

True incidence of leprosy and its impact on transmission will not be understood until a tool is available to measure pre-symptomatic infection. Diagnosis of leprosy disease is currently based on clinical symptoms, which on average take 3-10 years to manifest. The fact that incidence, as defined by new case detection, equates with prevalence, i.e., registered cases, suggests that the cycle of transmission has not been fully intercepted by implementation of multiple drug therapy. This is supported by a high incidence of childhood leprosy. Epidemiological screening for pre-symptomatic leprosy in large endemic populations is required to facilitate targeted chemoprophylactic interventions. Such a test must be sensitive, specific, simple to administer, cost-effective, and easy to interpret. The intradermal skin test method that measures cell-mediated immunity was explored as the best option. Prior knowledge on skin testing of healthy subjects and leprosy patients with whole or partially fractionated Mycobacterium leprae bacilli, such as Lepromin or the Rees' or Convit' antigens, has established an acceptable safety and potency profile of these antigens. These data, along with immunoreactivity data, laid the foundation for two new leprosy skin test antigens, MLSA-LAM (M. leprae soluble antigen devoid of mycobacterial lipoglycans, primarily lipoarabinomannan) and MLCwA (M. leprae cell wall antigens). In the absence of commercial interest, the challenge was to develop these antigens under current good manufacturing practices in an acceptable local pilot facility and submit an Investigational New Drug to the Food and Drug Administration to allow a first-in-human phase I clinical trial.

Safety and efficacy assessment of two new leprosy skin test antigens: randomized double blind clinical study.

BACKGROUND: New tools are required for the diagnosis of pre-symptomatic leprosy towards further reduction of disease burden and its associated reactions. To address this need, two new skin test antigens were developed to assess safety and efficacy in human trials. METHODS: A Phase I safety trial was first conducted in a non-endemic region for leprosy (U.S.A.). Healthy non-exposed subjects (n = 10) received three titrated doses (2.5 µg, 1.0 µg and 0.1 µg) of MLSA-LAM (n = 5) or MLCwA (n = 5) and control antigens [Rees MLSA (1.0 µg) and saline]. A randomized double blind Phase II safety and efficacy trial followed in an endemic region for leprosy (Nepal), but involved only the 1.0 µg (high dose) and 0.1 µg (low dose) of each antigen; Tuberculin PPD served as a control antigen. This Phase II safety and efficacy trial consisted of three Stages: Stage A and B studies were an expansion of Phase I involving 10 and 90 subjects respectively, and Stage C was then conducted in two parts (high dose and low dose), each enrolling 80 participants: 20 borderline lepromatous/lepromatous (BL/LL) leprosy patients, 20 borderline tuberculoid/tuberculoid (BT/TT) leprosy patients, 20 household contacts of leprosy patients (HC), and 20 tuberculosis (TB) patients. The primary outcome measure for the skin test was delayed type hypersensitivity induration. FINDINGS: In the small Phase I safety trial, reactions were primarily against the 2.5 µg dose of both antigens and Rees control antigen, which were then excluded from subsequent studies. In the Phase II, Stage A/B ramped-up safety study, 26% of subjects (13 of 50) showed induration against the high dose of each antigen, and 4% (2 of 50) reacted to the low dose of MLSA-LAM. Phase II, Stage C safety and initial efficacy trial showed that both antigens at the low dose exhibited low sensitivity at 20% and 25% in BT/TT leprosy patients, but high specificity at 100% and 95% compared to TB patients. The high dose of both antigens showed lower specificity (70% and 60%) and sensitivity (10% and 15%). BL/LL leprosy patients were anergic to the leprosy antigens. INTERPRETATION: MLSA-LAM and MLCwA at both high (1.0 µg) and low (0.1 µg) doses were found to be safe for use in humans without known exposure to leprosy and in target populations. At a sensitivity rate of 20-25% these antigens are not suitable as a skin test for the detection of the early stages of leprosy infection; however, the degree of specificity is impressive given the presence of cross-reactive antigens in these complex native M. leprae preparations. TRIAL REGISTRATION: ClinicalTrials.gov NCT01920750 (Phase I), NCT00128193 (Phase II).

The clinical significance of immunological contact urticaria to processed grains.

Contact urticaria, is characterized by an urticarial wheal-and-flare reaction at the site of contact by an allergen. Immunological contact urticaria, while less common than non-immunological contact urticaria, has more potentially serious consequences, and therefore, its recognition and treatment is important. Immunological contact urticaria is a type I hypersensitivity reaction. Potential complications include organ system involvement other than skin and even anaphylaxis and death. A vast majority of immunological contact urticaria is work-related. We will discuss the definition of immunological contact urticaria, the mechanism of the contact urticarial reaction, contact urticaria in the occupational setting, and the role of grains in contact urticaria. Testing and treatment are also briefly discussed.

Clinical presentation of tuberculoid leprosy in an epidermodysplasia verruciformis patient.

Epidermodysplasia verruciformis (EV) is triggered by a variety of mechanisms that at least partly include genetic background. We present a Brazilian man with a 30-year history of flat, wart-like lesions with clinical, histopathological, and evolutive aspects consistent with papillomavirus (HPV)-associated EV. Histological analysis of the wart lesions showed epidermis with hyperkeratosis, regular acanthosis, hypergranulosis, and cells with abundant basophilic cytoplasm. Moreover, a perivascular lymphocytic infiltrate was found in the superficial dermis, consistent with a viral wart. Type-2-HPV DNA was detected in various fragments of skin-wart lesions using the polymerase chain reaction (PCR). Two years after the EV diagnosis, the patient presented with an anesthetic well-demarcated, erythematous and mildly scaly plaque on his right forearm. A histopathological analysis of this lesion demonstrated the presence of a compact tuberculoid granuloma. Ziehl-Neelsen staining demonstrated the presence of rare acid-fast bacilli and confirmed the tuberculoid leprosy diagnosis. The patient's Mitsuda Intradermal Reaction was positive. To elucidate the possible mechanism involved in this case of EV, we genotyped the HLA genes of this patient. DQB genotyping showed the polymorphic HLA alleles DQB1*0301 and 0501. The patient was treated with a paucibacillary multi-drug therapy scheme, and the disease was cured in six months. This report describes an EV patient with an M. leprae infection, confirming that tuberculoid leprosy patients possess a relatively specific and efficient cell-mediated immunity against the bacillus and, therefore, localized forms of the disease. Moreover, we show the possible involvement of the polymorphic HLA alleles DQB1*0301 and 0501 in EV induction mechanisms.

Clinicoepidemiologic features of chronic urticaria in patients having positive versus negative autologous serum skin test: a study of 100 Indian patients.

BACKGROUND: Chronic urticaria patients who demonstrate autoantibodies against the high-affinity receptor of IgE (FceRI) or IgE itself tend to have a high itch and wheal score, and systemic symptoms may have a significant bearing on their management in terms of super pharmacologic doses of antihistamines needed or use of immunomodulators. Most studies have used histamine release assays rather than autologous serum skin tests (ASSTs) for correlating urticaria severity and histamine releasing activity. METHODS: An ASST was performed in 100 (M:F, 31:69) chronic urticaria patients aged between 14 and 63 (mean, 32.69 ± 13) years with an objective to study the clinicoepidemiologic features like age, sex, age of onset and duration, frequency and distribution of wheals, urticaria severity, angioedema and systemic manifestations in ASST-positive and ASST-negative patients. RESULTS: ASST was positive in 46% of the patients and negative in 54% of the patients, respectively. Both groups showed no statistically significant difference for epidemiological details. However, the ASST-positive patients had a higher mean urticaria activity score, frequent involvement of more body sites, particularly palms and soles, presence of throat angioedema and general constitutional, respiratory or gastrointestinal symptoms in comparison with the ASST-negative patients. CONCLUSIONS: Apparently, ASST-positive patients have more severe clinical manifestations of chronic urticaria. The knowledge will be useful for the treating dermatologists and patients alike in view of its therapeutic implications.

Decreased cutaneous resonance running time in cured leprosy subjects.

BACKGROUND/OBJECTIVES: Leprosy prominently involves both the skin and peripheral neural tissues and some symptoms persist after microbial cure. Because alterations in the dermis also occur in leprosy, we assessed here whether there were changes in cutaneous resonance running time (CRRT), a parameter that is influenced by collagen properties, in cured leprosy subjects. METHODS: A reviscometer was used to measure the CRRT at various directions on the dorsal hand and the flexural forearms of 76 cured leprosy subjects aged 50-85 years and 68 age-matched normal subjects. RESULTS: In comparison to normal subjects, CRRTs on the hands and the forearms were significantly reduced in all directions in cured leprosy, except at the 1-7, 2-8 and 3-9 o'clock directions on the forearms. CRRTs were reduced significantly at both the 4-10 and 5-11 o'clock directions on the forearm in lepromatous (73.33 +/- 4.19 at 4-10 o'clock and 67.44 +/- 2.71 at 5-11 o'clock direction) and borderline lepromatous types (77.58 +/- 5.84 at 4-10 o'clock and 79.85 +/- 6.81 at 5-11 o'clock direction) as compared with normal (143.10 +/- 7.75 at 4-10 o'clock and 125.18 +/- 8.14 at 5-11 o'clock direction). On the hand, CRRTs at all directions, except that at 4-10 o'clock direction, were also significantly reduced in lepromatous and borderline lepromatous types in comparison with normal. Significant differences in CRRT at some directions were found among the various subtypes of leprosy. CONCLUSION: CRRTs were abnormal in the cured leprosy subjects as a whole, but varied with leprosy subtypes, which suggested that the extent of reduction of CRRTs correlates with the severity of immune alteration. These results suggest that CRRT measurements could be a useful approach to quantify the extent of some residual abnormalities in cured leprosy and perhaps could also be used to evaluate the efficacy of treatment.

Adverse reactions to cosmetics and methods of testing.

Untoward reactions to cosmetics, toiletries, and topical applications are the commonest single reason for hospital referrals with allergic contact dermatitis. In most cases, these are only mild or transient and most reactions being irritant rather than allergic in nature. Various adverse effects may occur in the form of acute toxicity, percutaneous absorption, skin irritation, eye irritation, skin sensitization and photosensitization, subchronic toxicity, mutagenicity/genotoxicity, and phototoxicity/photoirritation. The safety assessment of a cosmetic product clearly depends upon how it is used, since it determines the amount of substance which may be ingested, inhaled, or absorbed through the skin or mucous membranes. Concentration of ingredients used in the different products is also important. Various test procedures include in vivo animal models and in vitro models, such as open or closed patch test, in vivo skin irritation test, skin corrosivity potential tests (rat skin transcutaneous electrical resistance test, Episkin test), eye irritation tests (in vivo eye irritancy test and Draize eye irritancy test), mutagenicity/genotoxicity tests (in vitro bacterial reverse mutation test and in vitro mammalian cell chromosome aberration test), and phototoxicity/photoirritation test (3T3 neutral red uptake phototoxicity test). Finished cosmetic products are usually tested in small populations to confirm the skin and mucous membrane compatibility, and to assess their cosmetic acceptability.