David M. Scollard

Increased CXC Ligand 10 Levels and Gene Expression in Type 1 Leprosy Reactions

ABSTRACT Type 1 reaction (T1R) is a systemic inflammatory syndrome causing substantial morbidity in leprosy. T1R results from spontaneously enhanced cellular immunity in borderline types of leprosy, but there are no established laboratory markers for the reaction. Preliminary studies have identified elevated circulating CXC ligand 10 (CXCL10) during T1R. Correlation of CXCL10 with clinical T1R was studied in repeated serum specimens obtained before, during, and after T1R. CXCL10 gene expression was assessed in biopsy specimens taken before and during T1R, and sections were stained for the cytokine using monoclonal antibodies. Sequential serum specimens revealed elevation of circulating CXCL10 associated with episodes of T1R (P = 0.0001) but no evidence of an earlier, predictive change in the level of the chemokine. Reverse transcriptase (RT)-PCR revealed elevated expression of CXCL10 transcripts during T1R, but not in patients who did not have T1R. No significant correlation between CXCL10 and gamma interferon (IFN-γ) mRNA levels was observed. Immunohistochemical staining of the skin biopsy specimens suggested an overall increase in CXCL10 but did not identify a particular strongly staining population of leukocytes. Increased CXCL10 in lesions and serum is characteristic of T1R. CXCL10 measurement offers new possibilities for laboratory diagnosis and monitoring of T1R. Studies of the regulation of CXCL10 may provide insight into the mechanisms of T1R and identify potential new drug targets for treatment.

Tuberculosis and Leprosy: Classical Granulomatous Diseases in the Twenty-First Century

Leprosy and tuberculosis are chronic mycobacterial infections that elicit granulomatous inflammation. Both infections are curable, but granulomatous injury to cutaneous structures, including cutaneous nerves in leprosy, may cause permanent damage. Both diseases are major global concerns: tuberculosis for its high prevalence and mortality, and leprosy for its persistent global presence and high rate of neuropathic disability. Cutaneous manifestations of both leprosy and tuberculosis are frequently subtle and challenging in dermatologic practice and often require a careful travel and social history and a high index of suspicion.

Mechanisms of nerve injury in leprosy

All patients with leprosy have some degree of nerve involvement. Perineural inflammation is the histopathologic hallmark of leprosy, and this localization may reflect a vascular route of entry of Mycobacterium leprae into nerves. Once inside nerves, M. leprae are ingested by Schwann cells, with a wide array of consequences. Axonal atrophy may occur early in this process; ultimately, affected nerves undergo segmental demyelination. Knowledge of the mechanisms of nerve injury in leprosy has been greatly limited by the minimal opportunities to study affected nerves in man. The nine-banded armadillo provides the only animal model of the pathogenesis of M. leprae infection. New tools available for this model enable the study and correlation of events occurring in epidermal nerve fibers, dermal nerves, and nerve trunks, including neurophysiologic parameters, bacterial load, and changes in gene transcription in both neural and inflammatory cells. The armadillo model is likely to enhance understanding of the mechanisms of nerve injury in leprosy and offers a means of testing proposed interventions.

Skin blister immunocytology A new method to quantify cellular kinetics in vivo

A method is described using tuberculin purified protein derivative (PPD) as a model to follow the in vivo cellular immune response. This combines induction of a local response, formation of skin blisters, and staining of the cells appearing in the sterile exudate over time, using standard cytopreparatory and immunoperoxidase techniques. Skin blisters were induced over sites previously injected intradermally with PPD or control saline using suction over a template on the forearm. The cells which appeared in the exudate at 24, 36, 48 and 72 h were collected on small cellulose filters which were divided into several parts. The cells on the filter segments were then stained using a biotin-avidin immunoperoxidase method with a panel of monoclonal antibodies, and with enzyme histochemical techniques. This allowed quantitative estimation over an extended period of total numbers of each cell type responding as a local expression of cellular immunity. The kinetics of an early, non-specific inflammatory response could be distinguished from the later immune response using total cell counts. Maximal cell counts correlated well with PPD induced induration at 48 h, showing an overwhelming predominance of mononuclear cells. Over 72 h, the number of non-specific esterase (NSE) positive cells (macrophage) declined while Leu4 positive cells (T cells) increased. OKT4 positive cells (T helper) outnumbered OKT8 positive cells (T suppressor) as the response developed. This method enables the direct quantitative assessment of cell populations arriving at the site of an immune response using a simple inexpensive technique which is painless, non-invasive and non-scarring.

Evaluation of multi-drug therapy for leprosy in the United States using daily rifampin.

Objectives: To evaluate the occurrence of relapse of multibacillary leprosy after multi-drug treatment including daily rifampin. Methods: A retrospective review was performed utilizing data from the National Hansens Disease Program (NHDP) on patients with leprosy treated and followed from 1988-1997 who received multi-drug therapy including daily rifampin. The occurrence of relapse in this cohort was measured, and demographic data and various clinical variables were also gathered. Results: Ultimately, 158 cases fulfilled the eligibility criteria. 77% of cases were multibacillary patients and were treated with 2 or 3 drug protocols at rates of 36% and 35% before and after 1992, respectively. Only one case of relapse was found, and this patient underwent 2-drug therapy versus 3-drug therapy. Conclusion: These data are remarkable for the absence of relapse with daily rifampin, as contrasted with the published experience using the WHO protocol with monthly rifampin.

Hansen's disease (leprosy) complicated by secondary mycobacterial infection

A patient with Hansens disease received corticosteroids for a type 1 leprosy reaction and subsequently developed a new cutaneous lesion at the original biopsy site from which Mycobacterium fortuitum was cultured. A review of the literature found only two other cases of coinfection with atypical mycobacteria and Mycobacterium leprae, although there are many reports of pulmonary tuberculosis in patients with leprosy. This case highlights the diagnostic difficulties encountered when a patient has two different mycobacterial infections of the skin. The published experience emphasizes that such coinfection is remarkably uncommon in leprosy, despite the frequent use of high doses of corticosteroids for leprosy reactions.

Primary Multidrug-Resistant Leprosy, United States

To the Editor: Since the initiation of multidrug therapy for leprosy (Hansen disease) in the 1980s by using rifampin, dapsone, and clofazimine, resistance to rifampin and dapsone has been observed worldwide and is still prevalent (1,2). Because few alternative effective antileprosy drugs exist, resistance to these first-line drugs could seriously affect leprosy control programs. We report a documented case of primary multidrug-resistant (MDR) leprosy in the United States. A man from American Samoa migrated to Hawaii at age 25 years and, at age 41 years, first sought care for generalized erythematous papules and plaques. A skin biopsy showed borderline lepromatous (BL) leprosy (Figure, panel A). He had no prior history of leprosy and no prior treatment. He was treated for 44 months with a daily regimen of dapsone (100 mg), clofazimine (100 mg), and rifampin (600 mg). He appeared to comply with this regimen, and the lesions slowly resolved. He remained free of any new lesions until 4 years after completing treatment, when multiple brown hyperpigmented patches appeared on his lower legs. A skin biopsy showed only hemosiderin deposition but no organisms. Figure Acid-fast organisms from biopsy specimens of a man with leprosy, United States. Fite-stained sections show numerous acid-fast bacilli in the initial skin biopsy specimen (A) and in the biopsy specimen taken at relapse, 6 years after completion of treatment ... At 51 years of age, 6 years after completing treatment, the man again sought care for a 2-week history of multiple generalized erythematous papules and plaques on his face, trunk, and extremities. Some lesions were pruritic but nontender. A skin biopsy showed chronic inflammatory infiltrates with numerous acid-fast bacilli (Figure, panel B). Clinically considered to have relapsed BL leprosy, he was again treated daily with dapsone (100 mg), clofazimine (50 mg), and rifampin (600 mg). After 1 month of this regimen, no clinical improvement was observed. Real-time PCR using the Mycobacterium leprae–specific repetitive element assay (3) confirmed the presence of M. leprae in biopsy specimens taken at the initial diagnosis and at relapse. Molecular genotyping of these samples with a panel of single-nucleotide polymorphism (SNP) and variable number of tandem repeat (VNTR) markers (4) showed that both biopsy specimens harbored M. leprae with the identical SNP subtype 3I and VNTR profile. PCR/DNA sequencing of the drug resistance–determining regions of M. leprae from these samples showed mutations within codon 53 of the folP1 gene (ACC→GCC) and in codon 425 of the rpoB gene (TCG→TTG). These mutations have been characterized to induce high-level resistance to dapsone and rifampin, respectively (5,6). Careful evaluation of electropherograms of these drug resistance–determining regions showed only the resistant alleles in both strains. These data indicated that this patient had been infected with MDR M. leprae before his initial treatment for leprosy. Therefore, when he was initially treated with leprosy multidrug therapy, he was essentially given clofazimine monotherapy. This treatment appears to have resulted in a slow, temporary clinical improvement. After relapse, he was placed on a daily regimen of clofazimine (100 mg), clarithromycin XL (500 mg), and minocycline (100 mg). The lesions clinically improved within 2 weeks, and the patient no longer noted any pruritus or tenderness in the lesions. This report documents a case of primary MDR leprosy in the United States. In evaluating several previous biopsy samples from other patients in Hawaii, we have not seen any rifampin-resistant or MDR isolates. Health officials in American Samoa, the patient’s country of origin, indicated that they were not aware of drug-resistant M. leprae among their patients (D. Scollard, pers. comm.). The patient reported no family history of leprosy, and no other contact could be identified. The origin of the MDR M. leprae in this case cannot be definitively determined. Drug-resistant leprosy, including dapsone- and rifampin-resistant and MDR leprosy, has been reported in other parts of the world, usually in association with relapse after insufficient therapy (1,2). Relapses in leprosy are not usually seen until many years after completion of treatment (7,8). In the United States, among patients treated for 2 years with a multidrug protocol involving daily rifampin, no relapses were observed after 10–15 years’ follow-up (9). Most new or worsening skin lesions clinically suspected to be relapses are actually leprosy reactions (10), which affect 30%–50% of patients. In the patient reported here, leprosy relapsed with MDR M. leprae 6 years after completion of treatment. The emergence of drug resistance poses a serious threat to leprosy control programs that rely on a secondary intervention, such as chemotherapy, because a leprosy vaccine is not available. Clinicians should be aware that persons who have acquired leprosy in other countries could have infection resulting from drug-resistant M. leprae. When resistance is suspected, biopsy samples should be analyzed by using molecular assays that enable rapid identification of mutations associated with drug resistance directly from paraffin-embedded biopsy specimens. For patients in the United States, this analysis is available through the National Hansen’s Disease (Leprosy) Program (www.hrsa.gov/hansensdisease/diagnosis/index.html), and for US patients, the program provides the 3-drug regimen for leprosy free of charge. When needed, minocycline, clarithromycin, and ofloxacin are provided as alternatives to treat leprosy.

Electron microscope appearance of lepromatous footpads of nude mice [corrected].

Footpad lesions of 3 nude mice infected by Mycobacterium leprae were studied at 9, 12, and 14 months after inoculation with light and electron microscope. The lesions were somewhat similar to those found in nodules in polar lepromatous leprosy. Striated muscles rather than nerves were the preferred site of the growth of M. leprae. Yet, M. leprae were identified in Schwann cells and endothelial cells, singly and in clumps. M. leprae filled macrophages, and free M. leprae were found in large numbers in the endoneurium without producing any significant demyelination.

Comparing the sensitivity of auramine-rhodamine fluorescence to polymerase chain reaction in the detection of Mycobacterium leprae in Fite-negative tissue sections

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