Of a total of 11532 Venezuelan patients with American cutaneous leishmaniasis (ACL) receiving immunotherapy with a combined vaccine containing heat-killed Leishmania promastigotes and bacille Calmette-Guerin (BCG) during the period 1990-99, we evaluated 5341 from 4 widely separated geographical states. Clinical healing varied from 91.2 to 98.7%, with an average of 95.7%. Adverse reactions were mild and limited to those associated with BCG vaccination alone. Immunotherapy failures in 143 patients included 54.5% with typical localized ulcers and 45.5% with non-mucosal intermediate cutaneous leishmaniasis (ICL). Less than 2% of the patients in this study had lesions suggestive of ICL. The disproportionately large number of immunotherapy failures in the ICL group suggests that it should not be used as monotherapy in this group. Weaker reactivity to purified protein derivative in immunotherapy failures, while not statistically significant in the small group reported here, suggests the possibility that these patients develop a relatively torpid immune response. The high percentage of clinical cures achieved with immunotherapy, associated with few secondary effects and low cost, support the use of immunotherapy in the routine treatment of localized ACL.

Possible drug resistance in Mycobacterium leprae strains from Venezuela and three other South American countries was surveyed by molecular methods. None of the 230 strains from new leprosy cases exhibited drug resistance-associated mutations. However, two of the three strains from relapsed cases contained dapsone resistance mutations, and one strain also harbored a rifampin resistance mutation. Single nucleotide polymorphism analysis of these strains revealed five subtypes: 3I (73.8%), 4P (11.6%), 1D (6.9%), 4N (6%), and 4O (1.7%).The success of leprosy control programs relies heavily upon multidrug therapy (MDT) (rifampin, dapsone, and clofazimine). Therefore, it is important that drug resistance trends be monitored periodically and Mycobacterium leprae strains genotyped in order to understand transmission patterns and genetic diversity. In the present study, strains of M. leprae from Bolivia, Brazil, Uruguay, and Venezuela were genotyped using single nucleotide polymorphism (SNP) analysis (17, 18), and their drug resistance-determining regions (DRDRs) in the rpoB, folP1, and gyrA genes were sequenced. In parallel, a real-time PCR assay that targets the most frequent sites of drug resistance in rpoB and folP1 was established.A total of 233 M. leprae strains were obtained from patient biopsy specimens or from slit skin smears; the majority of the patients were from Venezuela (n ϭ 197), and the remainder were from Bolivia (n ϭ 10), Brazil (n ϭ 24), and Uruguay (n ϭ 2). Reference DNA from drug-susceptible strains TN, Br4923, NHDP63, and Thai53 was used as a control for the sequencing and the real-time PCR-based TaqMan assay. A known rifampinresistant strain, 85054 (French West Indies), with an rpoB mutation (Ser425Leu) (8), was included as a positive control. This strain also has a dapsone resistance mutation (Pro55Leu) in folP1.DNA extraction from skin biopsy specimens/slit skin samples was carried out by the "freeze-boiling" method as described previously (24), followed by PCR amplification and DNA sequencing (18). Primers used for analyzing the drug resistance-associated loci are shown in Table 1. The sequences, obtained from an ABI 3130xl genetic analyzer (Applied Biosystems, Life Technologies Corporation, CA), were aligned against the sequences of the TN reference strain using CodonCode Aligner software (Dedham, MA) to identify polymorphisms. SNP genotyping was done as described previously (17, 18). The frequency of SNP7614 (presence of "T" at this position in subtype 3I strains) (18) was also determined.TaqMan probe assays using real-time PCR were performed in triplicate. Each 20-l reaction mixture contained 100 nM probe, 200 nM primers (Table 2) in 1ϫ TaqMan PCR master mix (Applied Biosystems, CA), and 2 l of DNA. PCR mixtures were subjected to thermal cycling for 2 min at 50°C and 10 min at 95°C followed by 40 cycles of 15 s at 95°C and 1 min at 60°C with a 7900HT Fast real-time PCR system (Applied Biosystems, CA). An increase in fluorescence indicates perfect complementarity between template and probe. ...

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The clinico-pathologic spectrum ofleprosy in its diverse fo rms is the expression of the immunologic response of the human host to infection by My cobacterium leprae.In this spectrum, the ' lepromatous pole is characterized by a total lack of cell-mediated effector immunological responses; this lack is also seen to a lesser degree in borderline lepromatous (BL) as well as in other fo rms, which present a variable degree of deficiency of this type of immunity as they are located along the spectrum towards the tuberculoid pole of the disease.There is substantial evidence that the immunological defect which reaches its maximum expression in lepromatous patients is also present in other population groups, including a small portion of the healthy population. This evidence includes the fo llowing observations: (a) A significant proportion of patients with the indeterminate form of leprosy show immunological behaviour similar to that of the lepromatous patient, characterized by a lack of cell-mediated manifestations toward M. leprae. Without adequate treatment, this indeterminate form of leprosy shows a tendency to progress to lepromatous leprosy (LL). We have seen this phenomenon of progression towards the LL pole in indeterminate patients who, after many years of treatment, have stopped their medication due to various reasons and suddenly re-appear with LL or BL disease. On the other hand, indeterminate lesions in Mitsuda-positive patients show a tendency to regress spontaneously, or to evolve towards the highly resistant fo rms of the disease.' (b) Studies of the Mitsuda reaction in the general population, both in endemic and non-endemic areas, show that a small proportion of the general population is persistently Mitsuda-negative. In 1978 we studied the persist ence of this negativity towards lepromin in the population of a non-endemic area of Venezuela and found that 1·25% of the general population remained