Mycobacterium leprae DNA Associated with Type 1 Reactions in Single Lesion Paucibacillary Leprosy Treated with Single Dose Rifampin, Ofloxacin, and Minocycline Ana Lucia O. M. Sousa, Mariane M. A. Stefani,* Gisner A. S. Pereira, Mauricio B. Costa, Paula F. Rebello, Maria Katia Gomes, Kazue Narahashi, Thomas P. Gillis, James L. Krahenbuhl, and Celina M. T. Martelli Tropical Pathology and Public Health Institute, Federal University of Goias, Goiania, Goias, Brazil; Alfredo da Matta Foundation–WHO Reference Leprosy Laboratory, Manaus, Amazonas, Brazil; Federal University of Rio de Janeiro, Rio de Janeiro, Brazil; Secretariat of Health, Porto Velho, Rondonia, Brazil; National Hansen’s Disease Programs, Baton Rouge, Louisiana Abstract. Leprosy affects skin and peripheral nerves, and acute inflammatory type 1 reactions (reversal reaction) can cause neurologic impairment and disabilities. Single skin lesion paucibacillary leprosy volunteers (N  135) recruited in three Brazilian endemic regions, treated with single-dose rifampin, ofloxacin, and minocycline (ROM), were monitored for 3 years. Poor outcome was defined as type 1 reactions with or without neuritis. IgM anti-phenolic glycolipid I, histopathology, Mitsuda test, and Mycobacterium leprae DNA polymerase chain reaction (ML-PCR) were performed at baseline.  2 test, Kaplan-Meir curves, and Cox proportional hazards were applied. The majority of volunteers were adults with a mean age of 30.5 ± 15.4 years; 44.4% were ML-PCR positive. During follow-up, 14.8% of the patients had a poor clinical outcome, classified as a type 1 reaction. Older age ( 40 years), ML-PCR positivity, and lesion size > 5cm were associated with increased risk. In multivariate analysis, age ( 40 years) and ML-PCR positivity remained baseline predictors of type 1 reaction among monolesion leprosy patients. INTRODUCTION As reported by the World Health Organization (WHO) at the beginning of 2006, the registered global prevalence of leprosy was 219,826, with Brazil reporting the second largest number of both registered cases and newly detected cases during 2005. Newly detected cases in Brazil have shown only a modest decline over the last 5 years, indicating that active transmission of Mycobacterium leprae is still occurring despite more than two decades of effective multidrug therapy (MDT). 1 Acute inflammatory episodes before, during, or after MDT, known as type 1 (reversal reaction) and type 2 (erythema nodosum leprosum [ENL]) leprosy reactions, can lead to permanent disabilities and handicaps. The clinico- pathologic characteristics of tuberculoid and lepromatous forms of leprosy and their associated reactions depend on TH 1 - and TH 2 -type immunity in skin lesions, respectively. 2–4 To determine MDT regimens for treating patients, leprosy can be classified as paucibacillary (PB), defined by few local- ized skin and neurologic lesions with low bacterial load, and multibacillary (MB), characterized by high bacterial load and systemic clinical features. In 1997, the WHO proposed a single dose ROM regimen (rifampicin, ofloxacin, minocy- cline) to treat single skin lesion leprosy based on a clinical trial performed in India. 5 Leprosy diagnosis is largely clinically based and would ben- efit from reliable, specific, and sensitive laboratory tests ca- pable of diagnosing early paucibacillary forms of the disease. 6 Anti-phenolic glycolipid I (PGLI) serology and acid-fast ba- cilli (AFB) staining techniques have low sensitivity for early PB leprosy diagnosis and, therefore, have been restricted to research applications. 7–10 Detection of M. leprae DNA by polymerase chain reaction (PCR) in tissue samples has been shown to increase the sensitivity and specificity of early PB leprosy diagnosis 11 ; however, its usefulness for prognosis re- mains to be shown. Single skin lesion, paucibacillary (SSL-PB) disease is con- sidered one of the earliest clinical forms of leprosy. A multi- centric prospective cohort of SSL-PB patients recruited in three Brazilian endemic settings and treated with one-dose ROM therapy was conducted, and the clinico-epidemiologic, histopathology, and cytokine features have been described previously. 7,8,12 This study describes the main clinico- epidemiologic and serologic features and the presence of M. leprae DNA detected by PCR associated with poor outcome, predominantly type 1 reactions, for a subgroup of this SSL- PB cohort during a 3-year clinical follow-up after ROM therapy. MATERIALS AND METHODS Study population and setting. Leprosy volunteers were re- cruited among outpatients attending health care units from three Brazilian endemic regions: north, Amazonas and Ron- donia States; central-west, Goias State; southeast, Rio de Ja- neiro State. Written informed consent was obtained from all participants or legal guardians for volunteers < 18 years old. This study was approved by the National Ethical Review Board (CONEP–Brazilian Ministry of Health). From November 1997 through December 1998, a total of 259 consecutive cases of single skin lesion leprosy patients (SSL-PB) were enrolled and treated with single-dose ROM therapy. Because of logistics, in two recruitment sites, the collection and storage of specimens for ML-DNA PCR started later. Therefore, for 135 volunteers, skin biopsies were available for ML-PCR, and for this reason, the results herein presented refer to a subgroup of patients. All volunteers had a neuro-dermatological examination performed by dermatologists, with expertise in leprosy, to select eligible SSL-PB patients. Lesion size and the presence of a bacillus Clamette-Gue ´ rin (BCG) scar were reported for all patients at baseline as previously reported. 7,8 Inclusion and exclusion criteria. Inclusion criteria were newly detected untreated single skin lesion leprosy patients without nerve involvement and classified as paucibacillary ac- * Address correspondence to Mariane M. A. Stefani, Tropical Pa- thology and Public Health Institute, Universidade Federal de Goias Rua 235 esq. c/1ª Avenida, S/N, Setor Universitario, Goiania, Goias CEP 74605-050, Brazil. E-mail: mstefani@iptsp.ufg.br Am. J. Trop. Med. Hyg., 77(5), 2007, pp. 829–833 Copyright © 2007 by The American Society of Tropical Medicine and Hygiene 829