Rapid Multiplex Loop-Mediated Isothermal Amplification (m-LAMP) Assay for Differential Diagnosis of Leprosy and Post-Kala-Azar Dermal Leishmaniasis.

Leprosy and post-kala-azar dermal leishmaniasis (PKDL) are co-endemic neglected tropical diseases often misdiagnosed because of close resemblance in their clinical manifestations. The test that aids in differential diagnosis of leprosy and PKDL would be useful in endemic areas. Here, we report development of a multiplex loop-mediated isothermal amplification (m-LAMP) assay for differential detection of Mycobacterium leprae and Leishmania donovani using a real-time fluorometer. The m-LAMP assay was rapid with a mean amplification time of 15 minutes, and analytical sensitivity of 1 fg for L. donovani and 100 fg for M. leprae. The distinct mean Tm values for M. leprae and L. donovani allowed differentiation of the two organisms in the m-LAMP assay. Diagnostic sensitivity of the assay was evaluated by using confirmed cases of leprosy (n = 40) and PKDL (n = 40) (tissue and slit aspirate samples). All the leprosy and PKDL samples used in this study were positive by organism-specific QPCR and loop-mediated isothermal amplification assays. The diagnostic sensitivity of the m-LAMP assay was 100% (95% CI: 91.2-100.0%) for detecting PKDL and 95% for leprosy (95% CI: 83.1-99.4%). Our m-LAMP assay was successfully used to detect both M. leprae and L. donovani in a patient coinfected with leprosy and macular PKDL. The m-LAMP assay is rapid, accurate, and applicable for differential diagnosis of leprosy versus PKDL, especially in endemic areas.

Addressing the drug-resistant tuberculosis challenge through implementing a mixed model of care in Uganda.

Worldwide, Drug-resistant Tuberculosis (DR-TB) remains a big problem; the diagnostic capacity has superseded the clinical management capacity thereby causing ethical challenges. In Sub-Saharan Africa, treatment is either inadequate or lacking and some diagnosed patients are on treatment waiting lists. In Uganda, various health system challenges impeded scale-up of DR-TB care in 2012; only three treatment initiation facilities existed, with only 41 of the estimated 1010 RR-TB/MDR-TB cases enrolled on treatment yet 300 were on the waiting list and there was no DR-TB treatment scale-up plan. To scale up care, the National TB and leprosy Program (NTLP) with partners rolled out a DR-TB mixed model of care. In this paper, we share achievements and outcomes resulting from the implementation of this mixed Model of DR-TB care. Routine NTLP DR-TB program data on treatment initiation site, number of patients enrolled, their demographic characteristics, patient category, disease classification (based on disease site and human immunodeficiency virus (HIV) status), on co-trimoxazole preventive therapy (CPT) and antiretroviral therapy (ART) statuses, culture results, smear results and treatment outcomes (6, 12, and 24 months) from 2012 to 2017 RR-TB/MDR-TB cohorts were collected from all the 15 DR-TB treatment initiation sites and descriptive analysis was done using STATA version 14.2. We presented outcomes as the number of patient backlog cleared, DR-TB initiation sites, RR-TB/DR-TB cumulative patients enrolled, percentage of co-infected patients on the six, twelve interim and 24 months treatment outcomes as per the Uganda NTLP 2016 Programmatic Management of drug-resistant Tuberculosis (PMDT) guidelines (NTLP, 2016). Over the period 2013-2015, the RR-TB/MDR-TB Treatment success rate (TSR) was sustained between 70.1% and 74.1%, a performance that is well above the global TSR average rate of 50%. Additionally, the cure rate increased from 48.8% to 66.8% (P = 0.03). The Uganda DR-TB mixed model of care coupled with early application of continuous improvement approaches, enhanced cohort reviews and use of multi-disciplinary teams allowed for rapid DR-TB program expansion, rapid clearance of patient backlog, attainment of high cumulative enrollment and high treatment success rates. Sustainability of these achievements is needed to further reduce the DR-TB burden in the country. We highly recommend this mixed model of care in settings with similar challenges.

Ticks as potential vectors of Mycobacterium leprae: Use of tick cell lines to culture the bacilli and generate transgenic strains.

Leprosy is an infectious disease caused by Mycobacterium leprae and frequently resulting in irreversible deformities and disabilities. Ticks play an important role in infectious disease transmission due to their low host specificity, worldwide distribution, and the biological ability to support transovarial transmission of a wide spectrum of pathogens, including viruses, bacteria and protozoa. To investigate a possible role for ticks as vectors of leprosy, we assessed transovarial transmission of M. leprae in artificially-fed adult female Amblyomma sculptum ticks, and infection and growth of M. leprae in tick cell lines. Our results revealed M. leprae RNA and antigens persisting in the midgut and present in the ovaries of adult female A. sculptum at least 2 days after oral infection, and present in their progeny (eggs and larvae), which demonstrates the occurrence of transovarial transmission of this pathogen. Infected tick larvae were able to inoculate viable bacilli during blood-feeding on a rabbit. Moreover, following inoculation with M. leprae, the Ixodes scapularis embryo-derived tick cell line IDE8 supported a detectable increase in the number of bacilli for at least 20 days, presenting a doubling time of approximately 12 days. As far as we know, this is the first in vitro cellular system able to promote growth of M. leprae. Finally, we successfully transformed a clinical M. leprae isolate by inserting the reporter plasmid pCHERRY3; transformed bacteria infected and grew in IDE8 cells over a 2-month period. Taken together, our data not only support the hypothesis that ticks may have the potential to act as a reservoir and/or vector of leprosy, but also suggest the feasibility of technological development of tick cell lines as a tool for large-scale production of M. leprae bacteria, as well as describing for the first time a method for their transformation.

Use of Approximate Bayesian Computation to Assess and Fit Models of Mycobacterium leprae to Predict Outcomes of the Brazilian Control Program.

Hansen's disease (leprosy) elimination has proven difficult in several countries, including Brazil, and there is a need for a mathematical model that can predict control program efficacy. This study applied the Approximate Bayesian Computation algorithm to fit 6 different proposed models to each of the 5 regions of Brazil, then fitted hierarchical models based on the best-fit regional models to the entire country. The best model proposed for most regions was a simple model. Posterior checks found that the model results were more similar to the observed incidence after fitting than before, and that parameters varied slightly by region. Current control programs were predicted to require additional measures to eliminate Hansen's Disease as a public health problem in Brazil.

Socio-economic and environmental effects influencing the development of leprosy in Bahia, north-eastern Brazil.

OBJECTIVES: To investigate spatial clusters and possible associations between relative risks of leprosy with socio-economic and environmental factors, taking into account diagnosed cases in children under 15 years old. METHODS: An ecological study was conceived using data aggregated by municipality to identify possible spatial clusters of leprosy from 2005 to 2011. Relative risks were calculated accounting for the respective covariate gender. The second stage of the analysis consisted of verifying possible associations between the relative risks of leprosy as a dependent variable, and socio-economic and environmental variables as independent. This was performed using a multivariate regression analysis according to a previously defined conceptual framework. RESULTS: Overall rates have decreased from 0.88/10 000 in 2005 to 0.52 in 2011. Spatial scan statistics identified 4 high-risk and 6 low-risk clusters. In the regression model, after allowing for spatial dependence, relative risks were associated with higher percentage of water bodies, higher Gini index, higher percentage of urban population, larger average number of dwellers by permanent residence and smaller percentage of residents born in Bahia. CONCLUSIONS: Although relative risks of leprosy in Bahia have been decreasing, they remain very high. The association between relative risks of leprosy and water bodies in the proposed geographic scale indicates that hypothesis linking M. leprae and humid environments cannot be discarded. Socio-economic conditions such as inequality, a greater number of dwellers by residence and migration are derived from the urbanisation process carried out in this State. Precarious settlements and poor living conditions in the cities would favour the continuity of leprosy transmission.

The production of social discourse on Hansen' disease and health education materials in Brazil: a skin patch as something harmless or a serious disease?

AIMS: Hansen's disease is endemic in Brazil and government control programmes promote publicity campaigns to increase the detection of new cases through the production and distribution of educative material. OBJECTIVES: This study analyses a set of 276 educational materials produced by governmental and non-governmental organisations that work to control Hansen's disease in Brazil. It describes the content of the materials and the way the issues were approached. DESIGN: It is a qualitative study that adopts the theoretical and methodological framework of the semiology of social discourse. RESULTS: Analysis reveals that the relations between the enunciator and recipient of the materials are asymmetrical as a result of the technical and educational language employed. Biomedical information forms the basis for social representations an practices of Hansen's disease, as opposed to historical collective knowledge of 'leprosy'. The prioritised topics are: signs and symptoms of the disease, treatment stigma, cure and surveillance. CONCLUSIONS: The institutionalisation of public education on Hansen's disease in Brazil was not limited simply to the change of terminology from 'leprosy' to 'Hansen's disease,' but was shaped also by new educational practices. It is recommended that the evaluation and production of new materials be incorporated into the set of activities already carried out in health centres so as to expand the discussion on content, language and the best way to address the disease in the materials.

TNF -308G>A single nucleotide polymorphism is associated with leprosy among Brazilians: a genetic epidemiology assessment, meta-analysis, and functional study.

Leprosy is an infectious disease caused by Mycobacterium leprae. Tumor necrosis factor (TNF) plays a key role in the host response. Some association studies have implicated the single nucleotide polymorphism TNF -308G>A in leprosy susceptibility, but these results are still controversial. We first conducted 4 association studies (2639 individuals) that showed a protective effect of the -308A allele (odds ratio [OR] = 0.77; P = .005). Next, results of a meta-analysis reinforced this association after inclusion of our new data (OR = 0.74; P = .04). Furthermore, a subgroup analysis including only Brazilian studies suggested that the association is specific to this population (OR = 0.63; P = .005). Finally, functional analyses using whole blood cultures showed that patients carrying the -308A allele produced higher TNF levels after lipopolysaccharide (LPS) (6 hours) and M. leprae (3 hours) stimulation. These results reinforce the association between TNF and leprosy and suggest the -308A allele as a marker of disease resistance, especially among Brazilians.

Rapid variable-number tandem-repeat genotyping for Mycobacterium leprae clinical specimens.

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.

Treating leprosy: an Erb-al remedy?

The leprosy pathogen Mycobacterium leprae attacks Schwann cells in the peripheral nervous system, causing them to demyelinate. Recent work by Tapinos et al. shows that a direct mechanism of demyelination induced by M. leprae depends on the binding of the bacterium to the receptor tyrosine kinase ErbB2 on Schwann cells and the resulting activation of the Ras-Raf-MEK-ERK pathway. These findings have relevance for the potential treatment of leprosy and they highlight parallels between the dedifferentiation signal in leprosy and that in nerve injury and cancer.

Assessment of viability by normal mouse foot-pad and bacillary ATP bioluminescence assay in multibacillary cases treated with an MDT regimen using conventional as well as newer drugs like minocycline and ofloxacin.

The therapeutic effect of a drug regimen of conventional drugs as well as newer drugs like ofloxacin and minocycline in smear-positive multibacillary (MB) leprosy cases was assessed by mouse foot-pad and ATP bioluminiscence methods. Biopsies were taken before starting treatment and after one year of treatment. They were processed for viability assessment by normal mouse foot-pad inoculation and bacillary ATP assay techniques. The test regimen was quite effective in its anti-bacterial effect as it was found to result in loss of bacillary viability in all the cases, as assessed by both methods.

Microdensitometric scanning procedure for quantitative assessment of hybridization of rRNA targeting probes in leprosy.

In order to develop an objective criteria of grading of positivity of hybridization signals of gene probes targeting rRNA, a microdensitometric scanning procedure was standardised. Ribosomal RNA was extracted from the bacilli harvested from biopsies of leprosy cases across the spectrum and blotted on nitro-cellulose membranes. M. leprae specific rRNA targeting oligonucleotide probes were end-labelled and hybridization was done by the technique standardised and published earlier. The autoradiographs were developed and microdensitometric scanning was done by altering different parameters. Positivity was graded in 5 grades and compared with visual positivity. Microdensitometric scanning procedure and 5 grade system appear to be useful and reproducible. Signals in paucibacillary specimens were in 2+ to 3+ grading range whereas those in multibacillary specimens varied in grades from 2+ to 5+. This approach appears to have potential usefulness for assessing the bacillary load (possibly viable) in the clinical specimens from leprosy cases.

Comparative assessment of viability of M.leprae by mouse foot-pad and fluorescent staining techniques.

Morphological characteristics have been used as a parameter to assess the viability of M.leprae in leprosy patients. However, with the advent of the mouse foot-pad technique, viability of M.leprae is determined by growing the bacilli in the mouse foot-pad. In recent years, a fluorescent staining technique using fluorescent diacetate-ethidium bromide (FDA-EB) has been used to assess the viability of cultivable mycobacteria as well as M.leprae. The purpose of this study was to compare the viability of M.leprae by both mouse foot-pad and fluorescent staining techniques. M.leprae strains from both untreated and treated patients as well as mouse passaged strains of M.leprae were used for the comparison. Percentage of green-stained bacilli in the inoculum was compared with that of multiplication of M.leprae in the mouse foot-pad. It was observed that there was no correlation between the estimates of viable M.leprae by fluorescent staining and by mouse foot-pad inoculation. FDA-EB staining appears to reflect only trends as absence of green staining cells had overall general correlation with loss of infectivity to mouse foot-pad but, the converse was not found to be true.