La quimioterapia de la lepra: una historia interpretatia / No disponible

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Does clofazimine prevent erythema nodosum leprosum (ENL) in leprosy? A retrospective study, comparing the experience of multibacillary patients receiving either 12 or 24 months WHO-MDT.

OBJECTIVE: To compare the occurrence, duration and severity of ENL in leprosy patients treated with either 12 or 24 months of standard multi-drug therapy (MDT). STUDY POPULATION: 296 patients treated with MDT for 2 years, between 1985 and 1992 and followed up as part of a relapse study; and 293 patients, treated between 1998 and 2004, with MDT for 1 year and also followed up as part of a relapse study. The Chi squared test and multiple logistic regression analysis were used to test for statistical significance. RESULTS: ENL was not significantly more common, but it was longer-lasting and more severe in patients receiving only 12 months of MDT, as compared with those receiving 24 months treatment. A high BI at the start of treatment significantly increased the risk of severe ENL by a factor of between 6 and 12, while treatment with 12 instead of 24 months of MDT significantly increased the risk by a factor of between 3 and 10. CONCLUSIONS: This study provides further evidence that a high initial BI is the key risk factor for ENL. It also suggests that the difference between these two cohorts in their experience of ENL as demonstrated in this study, may be related to the different amounts of clofazimine which the two cohorts were given in the early years of their treatment. Further studies are needed to determine whether clofazimine could be used more specifically to reduce the severity of ENL in the small group of patients at high risk for the condition.

The activity of several newer antimicrobials against logarithmically multiplying M. leprae in mice.

INTRODUCTION: Moxifloxacin, rifampicin, rifapentine, linezolid, and PA 824, alone and in combination, have been previously administered, as single doses and five times daily doses, to M. leprae infected mice during lag phase multiplication and were each found to have some bactericidal activity. DESIGN: The fluroquinolones, ofloxacin, moxifloxacin and gatifloxacin, (50 mg/kg, 150 mg/kg and 300 mg/kg) and the rifamycins (5 mg/kg, 10 mg/kg, and 20 mg/kg), rifampicin and rifapentine, were evaluated alone and in combination for bactericidal activity against M. leprae using the mouse footpad model during logarithmic multiplication. Linezolid and PA 824 were similarly evaluated alone and linezolid in combination with rifampicin, minocycline and ofloxacin. RESULTS: The three fluroquinolones and rifamycins were found alone and in combination to be bactericidal at all dosage schedules. PA 824 had no activity against M. leprae, while linezolid at a dose of 25 mg/kg was bacteriostatic, and progressively more bactericidal at doses of 50 mg/kg and 100 mg/kg. No antagonisms were detected between any of these drugs when used in combinations. CONCLUSION: Moxifloxacin, gatifloxacin, rifapentine and linezolid were found bactericidal against rapidly multiplying M. leprae.

Analysis of antibody responses to Mycobacterium leprae phenolic glycolipid I, lipoarabinomannan, and recombinant proteins to define disease subtype-specific antigenic profiles in leprosy.

A simple serodiagnostic test based on the Mycobacterium leprae-specific phenolic glycolipid I(PGL-I), for individuals with leprosy is nearly universally positive in leprosy patients with high bacillary loads but cannot be used as a stand-alone diagnostic test for the entire spectrum of the disease process. For patients with early infection with no detectable acid-fast bacilli in lesions or with low or no antibody titer to PGL-I, as in those at the tuberculoid end of the disease spectrum, this diagnostic approach has limited usefulness. To identify additional M. leprae antigens that might enhance the serological detection of these individuals, we have examined the reactivity patterns of patient sera to PGL-I, lipoarabinomannan (LAM), and six recombinant M. leprae proteins (ML1877, ML0841, ML2028, ML2038, ML0380, and ML0050) by Western blot analysis and enzyme-linked immunosorbent assay (ELISA). Overall, the responses to ML2028 (Ag85B) and ML2038 (bacterioferritin) were consistently high in both multibacillary and paucibacillary groups and weak or absent in endemic controls, while responses to other antigens showed considerable variability, from strongly positive to completely negative. This analysis has given a clearer understanding of some of the differences in the antibody responses between individuals at opposite ends of the disease spectrum, as well as illustrating the heterogeneity of antibody responses toward protein, carbohydrate, and glycolipid antigens within a clinical group. Correlating these response patterns with a particular disease state could allow for a more critical assessment of the form of disease within the leprosy spectrum and could lead to better patient management.

Reactions following completion of 1 and 2 year multidrug therapy (MDT).

We evaluated the incidence, severity, and duration of reactional states in 139 multibacillary (MB) leprosy patients in the first 2 years after the completion of the 1 year regimen of multidrug therapy (MDT) currently recommended by the World Health Organization (WHO) and compared those findings with 295 MB leprosy patients treated with the same regimen previously recommended for 2 years. During the first year after the completion of 1 year MDT, patients experienced 1 or more reactional states 27% of the time, the vast majority being lepra type 1 reactions (reversal reactions, RR), whereas patients who received 2 year MDT experienced a reactional state during that time period only 8% of the time (P < 0.001). Furthermore, during the first year after the completion of therapy, and during the first 2 years, both the number of reactional states and reversal reactions were significantly (P < or = 0.004) more frequent, severe, of longer duration, and more commonly associated with neuritis.

The efficacy of a four-week, ofloxacin-containing regimen compared with standard WHO-MDT in PB leprosy.

OBJECTIVES: To compare the efficacy of a 4-week ofloxacin-containing regimen and the standard WHO-MDT regimen for PB leprosy, in terms of the rate and timing of relapse after treatment completion. DESIGN: 124 PB patients were enrolled in a randomised, double-blind trial. Of these, 66 received the standard 6-month WHO-MDT regimen, whereas 58 received 28 daily supervised doses of rifampicin 600mg + ofloxacin 400 mg, plus 5 months of placebo. Patients were regularly monitored for clinical response and for signs of relapse after treatment completion. RESULTS: Patients enrolled in the ofloxacin group had a mean follow-up of 10.8 years (628 patient-years) with 1 early relapse at 3 years after treatment completion. On relapse, this patient remained smear negative but was reclassified by current WHO criteria (> or =6 skin lesions) as multibacillary (MB). Patients on the WHO-MDT regimen had a mean follow-up of 11.3 years (749 patient-years) with two late relapses at 8 and 12 years, both still classified as PB on relapse. CONCLUSION: In conclusion, both regimens appeared generally efficacious, and, in particular, resulted in few relapses.

A continuation: study and characterisation of Mycobacterium leprae short tandem repeat genotypes and transmission of leprosy in Cebu, Philippines.

OBJECTIVE: To study the stability and allelic diversity of tandem repeat loci in M. leprae in leprosy patients of Cebu, Philippines, and the suitability of multilocus variable number of tandem repeat (VNTR) analysis (MLVA) typing for detecting transmission. METHODS: Seventy newly diagnosed leprosy patients consulting at the Leonard Wood Memorial, Cebu Skin Clinic Total DNA was extracted from slit skin smear (SSS) scrapings of each patient and used for amplification of 13 M. leprae VNTR loci by single locus or multiplex PCR. Number of repeats for each VNTR locus was obtained by DNA sequencing or fragment length analysis methods. Medical, social and geographic details were included in the molecular epidemiology database. RESULTS AND CONCLUSIONS: Multiplex PCR (MP) and fragment length analysis (FLA) methods were found to be more efficient and accurate compared to single short tandem repeat (STR) amplification and DNA sequencing. Intra-patient MLVA patterns from four different samples were conserved in the minisatellites, while differences in one or more of the polymorphic and stutter prone microsatellites was observed, in four of five patients. The 13 loci could differentiate M. leprae strains in Cebu, however, MLVA patterns were stable enough during incubation and transmission between individuals within multi-case families. Thus M. leprae MLVA has potential for strain typing and transmission studies in Cebu.

Rapid killing of M. leprae by moxifloxacin in two patients with lepromatous leprosy.

INTRODUCTION: Previously we reported a 2-month clinical trial of moxifloxacin therapy in eight patients with MB leprosy (7 LL and 1 BL), finding both rapid killing of M. leprae and clinical improvement, without serious side effects or toxicities. Here we report the outcomes in two patients treated with moxifloxacin. DESIGN: Two previously untreated LL patients were treated with a single 400 mg dose of moxifloxacin, no therapy for 7 days and then daily 400 mg moxifloxacin for 48 days. Clinical response, viability of M. leprae in the skin, and side effects/toxicities were carefully monitored. RESULTS: In both patients a single dose of moxifloxacin resulted in significant killing of M. leprae (P < 0.001%). In both patients no viable M. leprae were found after 15 doses of moxifloxacin. Improvement in skin lesions occurred again remarkably rapidly and no untoward effects were noted. CONCLUSION: Loss of viable M. leprae was quite rapid, similar to that found previously only for rifampicin, patients improved rapidly, and moxifloxacin was well tolerated.

Population-based molecular epidemiology of leprosy in Cebu, Philippines.

To address the persisting problem of leprosy in Cebu, Philippines, we compiled a database of more than 200 patients who attend an established referral skin clinic. We described the patient characteristics in conventional demographic parameters and also applied multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) and single nucleotide polymorphism (SNP) typing for Mycobacterium leprae in biopsied skin lesion samples. These combined approaches revealed that transmission is ongoing, with the affected including the young Cebuano population under 40 years of age in both crowded cities and rural areas of the island. The emergence of multicase families (MCF) is indicative of infection unconstrained by standard care measures. For the SNPs, we designed a low-cost PCR-restriction fragment length polymorphism typing method. MLVA in M. leprae was highly discriminatory in this population yet could retain broad groups, as defined by the more stable SNPs, implying temporal marker stability suitable for interpreting population structures and evolution. The majority of isolates belong to an Asian lineage (SNP type 1), and the rest belong to a putative postcolonial lineage (SNP type 3). Specific alleles at two VNTR loci, (GGT)5 and 21-3, were highly associated with SNP type 3 in this population. MLVA identified M. leprae genotype associations for patients with known epidemiological links such as in MCFs and in some villages. These methods provide a molecular database and a rational framework for targeted approaches to search and confirm leprosy transmission in various scenarios.

A comparative clinical trial in multibacillary leprosy with long-term relapse rates of four different multidrug regimens.

As a participant in a multicenter trial, we evaluated the relapse rate in 189 multibacillary (MB) leprosy patients treated with four different regimens and followed-up for as many as 12 years after the initiation of treatment. Treatment regimens included 1 year of WHO MDT (a regimen including dapsone, clofazimine, and rifampin), 2 years of WHO MDT, 1 month of daily rifampin and daily ofloxacin, and 1 year of WHO MDT plus an initial 1 month of daily rifampin and daily ofloxacin. Relapse rates after 9 and 12 years from the initiation of therapy in the three regimens that included WHO MDT were 0-3%, whereas relapses occurred in those treated with the 1-month regimen alone at a significantly greater rate (P < 0.05): 11% at 9 years and 25% at 12 years. Relapses occurred late, beginning at 5 years after the initiation of therapy, and were confined to those patients histopathologically borderline lepromatous and polar lepromatous having a high bacterial burden. Prospects for an alternative effective short-course therapy of leprosy are presented.

The diarylquinoline R207910 is bactericidal against Mycobacterium leprae in mice at low dose and administered intermittently.

The diarylquinoline R207910 is profoundly bactericidal in a murine model of tuberculosis. Previously, R207910 was also found to be bactericidal for Mycobacterium leprae-infected mice during lag phase. Herein we evaluate the bactericidal efficacy of R207910 (1 to 120 mg/kg of body weight) when administered five times weekly, once weekly, and once monthly during logarithmic multiplication of M. leprae organisms. All treatments were found to be bactericidal, suggesting that both low and intermittent dosing with R207910 holds promise for leprosy patients.

A novel method for simple detection of mutations conferring drug resistance in Mycobacterium leprae, based on a DNA microarray, and its applicability in developing countries.

A simple method to detect mutations in the genome of Mycobacterium leprae that confer resistance to key drugs for leprosy was exploited on the basis of a reverse hybridization system. A series of oligonucleotide probes corresponding to each mutation in the folP1, rpoB and gyrA genes for dapsone, rifampicin and ofloxacin resistance, respectively, were selected and fixed on a glass slide as capture probes, to develop a DNA microarray termed the leprosy drug susceptibility-DNA microarray (LDS-DA). Mutations in clinical isolates of M. leprae were successfully identified by the LDS-DA. Feasibility studies were conducted to evaluate the performance of the LDS-DA in two developing countries, Myanmar and the Philippines. The high concordance of the results obtained by this method with the results of nucleotide sequencing strongly supports the applicability of the LDS-DA as a drug susceptibility test in place of sequencing, a time-consuming and costly procedure. This is a rapid and simple method for the simultaneous susceptibility testing of three front-line drugs for leprosy, and solves the problems of previously reported methods.

Powerful bactericidal activity of moxifloxacin in human leprosy.

In a clinical trial of moxifloxacin in eight multibacillary leprosy patients, moxifloxacin proved highly effective. In all trial patients, a single 400-mg dose of moxifloxacin resulted in significant killing (P

Use of protein antigens for early serological diagnosis of leprosy.

Leprosy is a chronic and debilitating human disease caused by infection with the Mycobacterium leprae bacillus. Despite the marked reduction in the number of registered worldwide leprosy cases as a result of the widespread use of multidrug therapy, the number of new cases detected each year remains relatively stable. This indicates that M. leprae is still being transmitted and that, without earlier diagnosis, M. leprae infection will continue to pose a health problem. Current diagnostic techniques, based on the appearance of clinical symptoms or of immunoglobulin M (IgM) antibodies that recognize the bacterial phenolic glycolipid I, are unable to reliably identify early-stage leprosy. In this study we examine the ability of IgG within leprosy patient sera to bind several M. leprae protein antigens. As expected, multibacillary leprosy patients provided stronger responses than paucibacillary leprosy patients. We demonstrate that the geographic locations of the patients can influence the antigens they recognize but that ML0405 and ML2331 are recognized by sera from diverse regions (the Philippines, coastal and central Brazil, and Japan). A fusion construct of these two proteins (designated leprosy IDRI diagnostic 1 [LID-1]) retained the diagnostic activity of the component antigens. Upon testing against a panel of prospective sera from individuals who developed leprosy, we determined that LID-1 was capable of diagnosing leprosy 6 to 8 months before the onset of clinical symptoms. A serological diagnostic test capable of identifying and allowing treatment of early-stage leprosy could reduce transmission, prevent functional disabilities and stigmatizing deformities, and facilitate leprosy eradication.

Methods for the classification of leprosy for treatment purposes.

The World Health Organization advocates 2 leprosy treatment regimens on the basis of disease classification (as multibacillary or paucibacillary) by skin lesion count. This method, which, in the Philippines, results in a high prevalence (78%) of patients with multibacillary leprosy, was directly compared with classification using standard histopathological and microbiological criteria in 264 currently untreated patients with leprosy. Of those whose leprosy was classified as paucibacillary, 38%-51% of patients had multibacillary leprosy according to classic criteria and were thus at risk of undertreatment according to World Health Organization recommendations.

Evaluation of a novel vaccine (HVJ-liposome/HSP65 DNA+IL-12 DNA) against tuberculosis using the cynomolgus monkey model of TB.

We have developed a novel tuberculosis (TB) vaccine; a combination of the DNA vaccines expressing mycobacterial heat shock protein 65 (HSP65) and interleukin 12 (IL-12) delivered by the hemagglutinating virus of Japan (HVJ)-liposome (HSP65+IL-12/HVJ). This vaccine provided remarkable protective efficacy in mouse and guinea pig models compared to the BCG vaccine, on the basis of an induction of the CTL activity and improvement of the histopathological tuberculosis lesions, respectively. Furthermore, we extended our studies to a cynomolgus monkey model, which is currently the best animal model of human tuberculosis. This novel vaccine provided a higher level of the protective efficacy than BCG based upon the assessment of mortality, the ESR, body weight, chest X-ray findings and immune responses. Furthermore, the combination of HSP65+IL-12/HVJ and BCG by the priming-booster method showed a synergistic effect in the TB-infected cynomolgus monkey (100% survival). These data indicate that our novel DNA vaccine might be useful against Mycobacterium tuberculosis for human clinical trials.

The frequency of drug resistance mutations in Mycobacterium leprae isolates in untreated and relapsed leprosy patients from Myanmar, Indonesia and the Philippines.

INTRODUCTION: The magnitude of drug resistance in Mycobacterium leprae to dapsone, rifampicin, and ofloxacin was studied in three Southeast Asian countries with a high prevalence of leprosy. METHODS: M. leprae from the skin of leprosy patients was collected in North Maluku and North Sulawesi in Indonesia, Yangon in Myanmar, and Cebu in the Philippines. Mutations in the drug resistance determining regions in the folP1, rpoB, and gyrA genes, which have been proven to confer resistance, were analysed. In addition, samples from 51 newly diagnosed cases and 13 patients with leprosy relapse in Cebu were submitted for susceptibility testing in the mouse footpad. RESULTS: Of 252 isolates obtained from new cases, 3% were dapsone resistant and 2% were rifampicin resistant. In samples taken from patients with relapsed leprosy (n = 53), significantly more resistance mutations were detected: 15% had dapsone resistance mutations, and 8% had rifampicin resistance mutations. Two patients with relapsed leprosy had mutations for both dapsone and rifampicin resistance. No mutations conferring quinolone resistance were detected. No mutations were detected in the folP1 gene of M. leprae isolates with a low degree of resistance to dapsone. DISCUSSION: Detection of drug-resistant cases by mutation detection in the drug resistance determining region of the genome is a practical method for monitoring resistance. A comparison of the results obtained in this study with previous data obtained prior to the use of multidrug therapy (MDT), does not indicate clearly whether the magnitude of drug resistance has changed. Larger studies of resistance mutations in M. leprae isolated from patients with relapsed leprosy are needed to confirm our results. CONCLUSION: We recommend monitoring the magnitude of drug resistance globally, by testing M. leprae DNA from relapse cases and a representative sample of new cases.

Use of protein microarrays to define the humoral immune response in leprosy patients and identification of disease-state-specific antigenic profiles.

Although the global prevalence of leprosy has decreased over the last few decades due to an effective multidrug regimen, large numbers of new cases are still being reported, raising questions as to the ability to identify patients likely to spread disease and the effects of chemotherapy on the overall incidence of leprosy. This can partially be attributed to the lack of diagnostic markers for different clinical states of the disease and the consequent implementation of differential, optimal drug therapeutic strategies. Accordingly, comparative bioinformatics and Mycobacterium leprae protein microarrays were applied to investigate whether leprosy patients with different clinical forms of the disease can be categorized based on differential humoral immune response patterns. Evaluation of sera from 20 clinically diagnosed leprosy patients using native protein and recombinant protein microarrays revealed unique disease-specific, humoral reactivity patterns. Statistical analysis of the serological patterns yielded distinct groups that correlated with phenolic glycolipid I reactivity and clinical diagnosis, thus demonstrating that leprosy patients, including those diagnosed with the paucibacillary, tuberculoid form of disease, can be classified based on humoral reactivity to a subset of M. leprae protein antigens produced in recombinant form.

ML0405 and ML2331 are antigens of Mycobacterium leprae with potential for diagnosis of leprosy.

Despite the success of multidrug therapy in reducing the number of registered leprosy cases worldwide, evidence suggests that Mycobacterium leprae continues to be transmitted. A serological diagnostic test capable of identifying and allowing treatment of early-stage disease could reduce transmission and prevent the onset of the disability, a common complication of the disease in later stages. Serological diagnosis based on antibody recognition of phenolic glycolipid I (PGL-I) cannot reliably identify individuals with lower bacterial indices (BI). One strategy that might improve this situation is the provision of highly specific serological antigens that may be combined with PGL-I to improve the sensitivity of diagnosis. Using serological expression cloning with a serum pool of untreated lepromatous leprosy (LL) patients, we identified 14 strongly reactive M. leprae proteins, 5 of which were previously unstudied. We present results suggesting that two of these proteins, ML0405 and ML2331, demonstrate the ability to specifically identify LL/borderline lepromatous (BL) patients on the basis of immunoglobulin G (IgG) reactivity. In a household contact study, LL index cases were identified on the basis of this reactivity, while household contacts of these patients demonstrated undetectable reactivity. At a serum dilution of 1:800, suitable to reduce background PGL-I IgM reactivity, two BL patients with a BI of <4 showed anti-human polyvalent immunoglobulin G, A, and M reactivity measured with a combination of ML0405, ML2331, and natural disaccharide O-linked human serum albumin (NDOHSA) (synthetic PGL-I) that was markedly higher than IgM reactivity to NDOHSA alone. We suggest that ML0405 and ML2331 may have utility in serological leprosy diagnosis.