The need for molecular tools for the differentiation of isolates of Mycobacterium leprae, the organism that causes leprosy, is urgent in view of the continuing high levels of new case detection, despite years of aggressive chemotherapy and the consequent reduction in the prevalence of leprosy. The slow onset of leprosy and the reliance on physical examination for detection of disease have restricted the epidemiological tracking necessary to understand and control transmission. Two genetic loci in several isolates of M. leprae have previously been demonstrated to contain variable-number tandem repeats (VNTRs). On the basis of these reports and the availability of the full genome sequence, multiple-locus VNTR analysis for strain typing has been undertaken. A panel of 11 short tandem repeat (STR) loci with repeat units of 1, 2, 3, 6, 12, 18, 21, and 27 bp from four clinical isolates of M. leprae propagated in armadillo hosts were screened by PCR. Fragment length polymorphisms were detected at 9 of the 11 loci by agarose gel electrophoresis. Sequencing of representative DNA products confirmed the presence of VNTRs between isolates. The application of nine new polymorphic STRs in conjunction with automated methods for electrophoresis and size determination allows greater discrimination between isolates of M. leprae and enhances the potential of this technique to track the transmission of leprosy.The World Health Organization and its partners created the Global Alliance for Leprosy Elimination in 1999 with the aim of achieving the elimination of leprosy by the end of the year 2005, a goal originally set for the year 2000 (http://www.who .int/inf-pr-1999/en/pr99-70.html). Leprosy is effectively controlled by a multidrug therapy (MDT) regimen composed of dapsone, rifampin, and clofazimine. However, continuing large numbers of new cases are being detected in areas of the world where the disease is highly endemic, despite the application of the MDT program since 1982 (36). In order to comprehend this rising incidence of leprosy, it is necessary to identify the natural reservoir of Mycobacterium leprae, the route of infection, and the mode of its transmission. It is commonly believed that the human is the host and reservoir of M. leprae and that successful MDT will eliminate leprosy. However, other modes of transmission involving nonhuman reservoirs, such as soil (6, 15), water (20), vegetation (16), animals (including armadillos and sooty mangabeys) (22, 33), and arthropods (fleas, ticks, mosquitoes, and flies), have been suggested, as reviewed by Blake et al. (3). The route of infection is also unknown, although the entry and the exit of the bacteria via the nasal passages have been proposed (9). Methods that specifically detect M. leprae DNA in nasal swabs are being developed with the aim of early detection in populations at the community and village levels and for the monitoring of leprosy transmission (13,23). Typing methods for distinguishing cases of relapse from new infections are also required. Molecular typing wi...

Mycobacterium leprae is the noncultivable pathogen of leprosy. Since the genome sequence of an isolate of M. leprae has become available, multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) has been explored as a tool for strain typing and identification of chains of transmission of leprosy. In order to discover VNTRs and develop methods transferable to clinical samples, MLVA was applied to a global collection of M. leprae isolates derived from leprosy patients and propagated in armadillo hosts. PCR amplification, agarose gel electrophoresis, and sequencing methods were applied to DNA extracts from these infected armadillo tissues (n ‫؍‬ 21). We identified polymorphisms in 15 out of 25 short-tandem-repeat (STR) loci previously selected by in silico analyses of the M. leprae genome. We then developed multiplex PCR for amplification of these 15 loci in four separate PCRs suitable for fluorescent fragment length analysis and demonstrated STR profiles highly concordant with those from the sequencing methods. Subsequently, we extended this method to DNA extracts from human clinical specimens, such as skin biopsy specimens (n ‫؍‬ 30). With these techniques, mapping of multiple loci and differentiation of genotypes have been possible using total DNA extracts from limited amounts of clinical samples at a reduced cost and with less time. These practical methods are therefore available and applicable to answer focused epidemiological questions and to allow monitoring of the transmission of M. leprae in different countries where leprosy is endemic.The causative pathogen of leprosy is Mycobacterium leprae. A continued incidence, defying global campaigns to eliminate leprosy even after years of rigorous case finding and the availability of multidrug therapy regimens (28,29,30,31), is attributed to subclinical human and environmental reservoirs of the pathogen (1,8,13). In recent years, molecular strain-typing methodologies have complemented conventional infectious disease epidemiology. With the publication in 2001 of the complete genome sequence of an isolate from Tamil Nadu, India, called the TN strain (4), selection of potential polymorphic genomic markers for strain typing was feasible. The first genetic markers that showed polymorphism were short tandem repeats (STRs) in the M. leprae genome. One was a 6-bp intragenic sequence in the rpoT gene, and the second, a trinucleotide (TTC) repeat element upstream of a pseudogene (17, 23). These sequences exhibit variable numbers of tandem repeats (VNTRs) when sequenced in different isolates. Based on these observations, we short-listed 44 loci (including the rpoT and TTC loci) by in silico analyses of the M. leprae genome and accomplished the screening of 11 STR loci, of which 9 were polymorphic when tested in a small panel of four human isolates derived from passage through armadillos (6). Five were minisatellites (6-to 50-bp repeat units), and four were microsatellites (1-to 5-bp repeat units). Since then, others have also shown that VNTR loci exist in M. lepra...

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.Leprosy is a devastating human disease caused by infection with Mycobacterium leprae bacilli. The disease predominantly affects the skin, although during infection, significant nerve destruction leads to deformities of the hand, foot, face, and, in some cases, eye (1). The disease is represented by a clinical spectrum. Lepromatous leprosy/borderline lepromatous (LL/ BL) patients represent one pole of the spectrum, demonstrating a high bacterial index (BI) and, as such, are classified as multibacillary (MB). LL/BL patients demonstrate high titers of M. leprae-specific antibodies and an absence of M. leprae-specific cell-mediated immunity. At the opposite pole, tuberculoid tuberculoid/borderline tuberculoid (TT/BT) patients demonstrate very low or absent BI and are designated paucibacillary. These individuals demonstrate significant M. leprae-specific cellmediated immunity and very low or absent titers of M. lepraespecific antibodies.Despite the success of multidrug therapy in reducing the number of registered leprosy cases worldwide, the annual rate of new case detection remains unchanged, at approximately 700,000 cases per year (33), with children representing 15% of new cases (18). This suggests that active transmission of M. leprae is still occurring, but the route and me...

In the virulent state (Bvg ؉ ), Bordetella bronchiseptica expresses adhesins and toxins that mediate adherence to the upper airway epithelium, an essential early step in pathogenesis. In this study, we used a rabbit tracheal epithelial cell binding assay to test how specific host or pathogen factors contribute to ciliary binding.

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.

Molecular diagnostic and epidemiology studies require appreciable amounts of high-quality DNA. Molecular epidemiologic methods have not been routinely applied to the obligate intracellular organism Mycobacterium leprae because of the difficulty of obtaining a genomic DNA template from clinical material. Accordingly, we have developed a method based on isothermic multiple-displacement amplification to allow access to a high-quality DNA template. In the study described in this report, we evaluated the usefulness of this method for error-sensitive, multiple-feature molecular analyses. Using test samples isolated from lepromatous tissue, we also evaluated amplification fidelity, genome coverage, and regional amplification bias. The fidelity of amplified genomic material was unaltered; and while regional differences in global amplification efficiency were seen by using comparative microarray analysis, a high degree of concordance of amplified genomic DNA was observed. This method was also applied directly to archived tissue specimens from leprosy patients for the purpose of molecular typing by using short tandem repeats; the success rate was increased from 25% to 92% without the introduction of errors. This is the first study to demonstrate that serial whole-genome amplification can be coupled with error-sensitive molecular typing methods with low-copy-number sequences from tissues containing an obligate intracellular pathogen.

BackgroundNew tools are required for the diagnosis of pre-symptomatic leprosy towards further reduction of disease burden and its associated reactions. To address this need, two new skin test antigens were developed to assess safety and efficacy in human trials.MethodsA Phase I safety trial was first conducted in a non-endemic region for leprosy (U.S.A.). Healthy non-exposed subjects (n = 10) received three titrated doses (2.5 µg, 1.0 µg and 0.1 µg) of MLSA-LAM (n = 5) or MLCwA (n = 5) and control antigens [Rees MLSA (1.0 µg) and saline]. A randomized double blind Phase II safety and efficacy trial followed in an endemic region for leprosy (Nepal), but involved only the 1.0 µg (high dose) and 0.1 µg (low dose) of each antigen; Tuberculin PPD served as a control antigen. This Phase II safety and efficacy trial consisted of three Stages: Stage A and B studies were an expansion of Phase I involving 10 and 90 subjects respectively, and Stage C was then conducted in two parts (high dose and low dose), each enrolling 80 participants: 20 borderline lepromatous/lepromatous (BL/LL) leprosy patients, 20 borderline tuberculoid/tuberculoid (BT/TT) leprosy patients, 20 household contacts of leprosy patients (HC), and 20 tuberculosis (TB) patients. The primary outcome measure for the skin test was delayed type hypersensitivity induration.FindingsIn the small Phase I safety trial, reactions were primarily against the 2.5 µg dose of both antigens and Rees control antigen, which were then excluded from subsequent studies. In the Phase II, Stage A/B ramped-up safety study, 26% of subjects (13 of 50) showed induration against the high dose of each antigen, and 4% (2 of 50) reacted to the low dose of MLSA-LAM. Phase II, Stage C safety and initial efficacy trial showed that both antigens at the low dose exhibited low sensitivity at 20% and 25% in BT/TT leprosy patients, but high specificity at 100% and 95% compared to TB patients. The high dose of both antigens showed lower specificity (70% and 60%) and sensitivity (10% and 15%). BL/LL leprosy patients were anergic to the leprosy antigens.InterpretationMLSA-LAM and MLCwA at both high (1.0 µg) and low (0.1 µg) doses were found to be safe for use in humans without known exposure to leprosy and in target populations. At a sensitivity rate of 20–25% these antigens are not suitable as a skin test for the detection of the early stages of leprosy infection; however, the degree of specificity is impressive given the presence of cross-reactive antigens in these complex native M. leprae preparations.Trial RegistrationClinicalTrails.gov NCT01920750 (Phase I), NCT00128193 (Phase II)

To study initial Bordetella bronchiseptica-tracheal epithelial cell interactions, we coincubated B. bronchiseptica with rabbit tracheal explant cultures and assayed bacterial adherence and host cell Ca 2؉ signaling. Wild-type B. bronchiseptica (RB50) preferentially adhered to cilia and induced ciliated host cell Ca 2؉ transients within 2 min of coincubation, whereas coincubation with an avirulent strain (RB57) resulted in limited binding and Ca 2؉ signaling. The described cell system allows for assessment of initial B. bronchiseptica-host cell interactions that can contribute to pathogenicity or to host cell defense.The tracheal epithelium creates a physical barrier between respired air and the underlying tissue of the upper respiratory tract. In addition to the barrier, the epithelium generates a "mucociliary escalator" to clear particulate materials, including pathogenic bacteria, from the airway and keep them from reaching the lungs (15). Local environmental changes can influence coordinated ciliary movement through second messenger signaling pathways (12). Such changes elicited by respiratory pathogens through toxins and adhesins can help to overcome mucociliary defense and establish infection (16). Bordetella bronchiseptica is a gram-negative bacterium that colonizes the airways of a variety of animals. Similar to other bordetellae, B. bronchiseptica responds to environmental conditions and switches between virulent and avirulent phases via a two-component signal transduction system termed BvgAS (1, 2, 4, 11). In animal studies the Bvg ϩ phase is necessary to establish infection, whereas mutants locked in a Bvg Ϫ state (e.g., RB57) are unable to establish infection (6, 7). It is accepted that establishment of infection involves factors under BvgAS control that allow for adherence of B. bronchiseptica to cilia in the upper airway (3,5,7,10).To investigate initial Bordetella-host cell interactions, we monitored B. bronchiseptica and explant cultures of rabbit tracheal epithelial cells (RTEC) with video and digital imaging microscopy. RTEC were grown at 37°C in 5% CO 2 on collagen-coated glass coverslips in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum as described previously (8). Cultures were washed extensively with Hanks' balanced salt solution (HBSS) and covered with 250 l of HBSS for microscopic observation (Fig. 1A). Wild-type B. bronchiseptica (RB50) or a Bvg Ϫ avirulent strain (RB57) was grown to log phase under constant shaking at 37°C in supplemented stainer Scholte broth (14). Bacterial cultures were resuspended in HBSS to 10 8 to 10 9 CFU/ml. Binding assays were performed in an open cell chamber by video microscopy on an Olympus IX70 inverted microscope with a ϫ100 phase contrast or a ϫ60 differential interference contrast objective and time lapse video capability. RTEC cultures were washed with HBSS and imaged with a DAGE 300T charge-coupled-device camera (Fig. 1B). To initiate interactions, RTEC cultures were initially exposed to bacteria by replacing the HBSS with four con...