Detection of Mycobacterium leprae DNA by Polymerase Chain Reaction in the Blood of Individuals, Eight Years after Completion of Anti-leprosy Therapy

Vol. 96(8): 1129-1133, November 2001

Adalberto Rezende Santos, Vivian Balassiano**, Maria Leide W Oliveira**, Marcia Aparecida da Silva Pereira*, Patricia Barros Santos*, Wim Maurits Degrave*, Philip Noel Suffys*/⁺

Setor de Hanseníase, Departamento de Medicina Tropical *Laboratório de Biologia Molecular e Diagnóstico de Doenças Infecciosas, Departamento de Bioquímica e Biologia Molecular, Instituto Oswaldo Cruz-Fiocruz, Av. Brasil 4365, 21045-900 Rio de Janeiro, RJ, Brasil **Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil

Thirty eight patients with indeterminate leprosy (HI), at least 4 to 6 years after discharge from multibacillary (MB) or paucibacillary (PB) schemes of anti leprosy multidrug therapy (MDT), were submitted to traditional diagnostic procedures for leprosy and to polymerase chain reaction (PCR) analysis of different clinical samples for detection of Mycobacterium leprae DNA. No significant difference was observed for any of the parameters analyzed between PB or MB schemes of treatment and no indications were found for more efficient outcome of HI using the MB scheme. Remarkably, 18 (54.5%) of the individuals were PCR positive in at least one of the samples: positivity of PCR was highest in blood samples and four individuals were PCR positive in blood and some other sample. Upon comparison of PCR results with clinical and histopathological parameters, no correlation was found between PCR-positivity and eventual relapse. This is the first report on detection of M. leprae DNA in PB patients, more than half a decade after completion of MDT, suggesting that live bacilli are present and circulating much longer than expected, although reinfection of the individuals can not be excluded. Overall, we feel that because of the high sensitivity of the assay, extreme care should be taken about association of PCR results, efficacy of treatment and disease status.

Key words: Mycobacterium leprae - PCR - diagnosis - therapy - post-treatment - paucibacillary leprosy

Leprosy is a chronic infectious disease caused by Mycobacterium leprae and is still a public health problem in many developing countries, including Brazil with 88,029 registered cases and a prevalence of 5.51 per 10.000 at the end of 1997 (Brazilian Ministry of Health 1998). According to the clinical spectrum proposed by Ridley and Jopling (1966), leprosy is characterized by two polar forms: a paucibacillary tuberculoid (TT) and a multibacillary lepromatous lepromatous (LL) form, and by the intermediate forms borderline tuberculoid (BT), borderline borderline (BB) and borderline lepromatous (BL) leprosy. Indeterminate (HI) leprosy is an early and instable stage of the disease and, depending on the host resistance, migrates through the clinical spectrum to one of the definitive forms or goes to self healing. Patients are treated with two or three different drugs (multidrug therapy; MDT) during a period of six months to one year, depending on the clinical form of the disease.

For correct implementation of MDT, the World Health Organization (WHO) classifies leprosy either as multibacillary (MB) or paucibacillary (PB), according to the Bacteriological Index (BI; WHO 1982). In Brazil, MDT was implemented in 1986 and because bacterioscopy was not applied at all basic health units at that time, the Brazilian National Program for Leprosy Control recommended the use of the Mitsuda test as a parameter for establishing the treatment regimen in cases of HI leprosy (Ministério da Saúde 1986). Patients with negative response to Mitsuda were included in the MB group but in some reference centers such patients were treated according to the recommendation of WHO. Leprosy has a long incubation period, a wide spectrum of clinical manifestations and the causative agent M. leprae can not be grown in vitro. This turns detection of subclinical infection and of early stages of the disease, principally the PB forms difficult. Furthermore, evaluation of efficacy of anti leprosy chemotherapy is complicated and the only reliable method to determine bacilli viability is the mouse foot pad technique (Shepard 1960), which is expensive, time consuming and can be used only for MB samples (Baohong 1987). In consequence, evaluation of MDT is mostly based on determination of the frequency of relapse and on the detection of M. leprae DNA by PCR (THELEP 1987, Boerrigter et al. 1991, Williams et al. 1992, Jamil et al. 1993).

Because of Brazil's particular situation where cases with HI leprosy had been treated both with PB and MB MDT schemes, it was possible to evaluate efficiency of these treatment schemes in PB patients, in a retrospective study on individuals that had completed treatment, by comparing PCR-mediated detection of M. leprae DNA with clinical, histopathological and laboratorial data.

MATERIALS AND METHODS

RESULTS

DISCUSSION

ACKNOWLEDGEMENTS

To the staff of the Leprosy Outpatient Unit for sample collection and to Edson CA Albuquerque for Bacteriological Index determination

REFERENCES

Table I | Table II

This work was supported by CNPq and by CBAB.

⁺Corresponding author. Fax: +55-21-2270.9997. E-mail: psuffys@gene.dbbm.fiocruz.br

Recieved 23 February 2001

Accepted 19 June 2001