Post-exposure prophylaxis (PEP) efficacy of rifampin, rifapentine, moxifloxacin, minocycline, and clarithromycin in a susceptible-subclinical model of leprosy

There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

Abstract

Background

Subclinical infection with Mycobacterium leprae is one potential source of leprosy transmission, and post-exposure prophylaxis (PEP) regimens have been proposed to control this source. Because PEP trials require considerable investment, we applied a sensitive variation of the kinetic mouse footpad (MFP) screening assay to aid in the choice of drugs and regimens for clinical trials.

Methodology/Principal findings

Athymic nude mice were inoculated in the footpad (FP) with 6 x 10 ³ viable M. leprae and treated by gastric gavage with a single dose of Rifampin (SDR), Rifampin + Ofloxacin + Minocycline (SD-ROM), or Rifapentine + Minocycline + Moxifloxacin (SD-PMM) or with the proposed PEP++ regimen of three once-monthly doses of Rifampin + Moxifloxacin (RM), Rifampin + Clarithromycin (RC), Rifapentine + Moxifloxacin (PM), or Rifapentine + Clarithromycin (PC). At various times post-treatment, DNA was purified from the FP, and M. leprae were enumerated by RLEP quantitative PCR. A regression analysis was calculated to determine the expected RLEP value if 99.9% of the bacilli were killed after the administration of each regimen. SDR and SD-ROM induced little growth delay in this highly susceptible murine model of subclinical infection. In contrast, SD-PMM delayed measurable M. leprae growth above the inoculum by 8 months. The four multi-dose regimens delayed bacterial growth for >9months post-treatment cessation.

Conclusions/Significance

The delay in discernable M. leprae growth post-treatment was an excellent indicator of drug efficacy for both early (3–4 months) and late (8–9 months) drug efficacy. Our data indicates that multi-dose PEP may be required to control infection in highly susceptible individuals with subclinical leprosy to prevent disease and decrease transmission.

Author summary

While multi-drug therapy (MDT) has been successful in decreasing the worldwide prevalence of leprosy, the new case detection rate, or incidence, remains consistent. These circumstances indicate that leprosy transmission is still occurring. Subclinical asymptomatic leprosy infections are considered a leading cause of ongoing transmission. One means to control this source is an effective post-exposure prophylaxis (PEP) regimen that would prevent both subsequent progression to clinical leprosy for the individual and transmission of the disease to others. Therefore, in this study, we used a modified kinetic mouse footpad screening assay and sensitive molecular bacterial enumeration in a susceptible-subclinical mouse model to identify effective potential PEP drug regimens for leprosy. Using these methods, we showed that a single dose PEP regimen is not effective in a susceptible host, and multiple intermittent doses of combination therapies are required. This model could provide useful pre-clinical information for the development of PEP regimens for leprosy.

Related collections

Most cited references 33

Record: found
Abstract: found
Article: not found

Effectiveness of single dose rifampicin in preventing leprosy in close contacts of patients with newly diagnosed leprosy: cluster randomised controlled trial.

F Johannes Moet, David Pahan, Linda Oskam … (2008)

To determine the effectiveness of chemoprophylaxis using a single dose of rifampicin to prevent leprosy in close contacts. Single centre, double blind, cluster randomised, placebo controlled trial. Leprosy control programme in two districts of northwest Bangladesh with a population of more than four million. 28,092 close contacts of 1037 patients with newly diagnosed leprosy. 21,711 contacts fulfilled the study requirements. A single dose of rifampicin or placebo given to close contacts in the second month of starting the index patient's treatment, with follow-up for four years. Development of clinical leprosy. 18,869 of the 21,711 contacts (86.9%) were followed-up at four years. Ninety one of 9452 contacts in the placebo group and 59 of 9417 in the rifampicin group had developed leprosy. The overall reduction in incidence of leprosy using a single dose of rifampicin in the first two years was 57% (95% confidence interval 33% to 72%). The groups did not differ between two and four years. The overall number needed to treat (NNT) to prevent a single case of leprosy among contacts was 297 (95% confidence interval 176 to 537). Differences were found between subgroups at two years, both in reduction of incidence and in NNT. A single dose of rifampicin given to contacts of patients with newly diagnosed leprosy is effective at preventing the development of clinical leprosy at two years. The effect was maintained, but no difference was seen between the placebo and rifampicin groups beyond two years. Current Controlled Trials ISRCTN61223447 [controlled-trials.com].

0 comments Cited 88 times – based on 0 reviews      Review now

Bookmark

Record: found
Abstract: found
Article: found

Is Open Access

The Missing Millions: A Threat to the Elimination of Leprosy

William Cairns Smith, Wim H Van Brakel, Tom Gillis … (2015)

Introduction Leprosy is a slow, chronic disease with a long incubation period caused by Mycobacterium leprae. The clinical presentation varies across a wide spectrum from tuberculoid to lepromatous leprosy. The condition is characterized by skin lesions and damage to peripheral nerves leading to physical disability and social problems. The past 50–60 years have witnessed remarkable progress in the fight against leprosy. The introduction of dapsone therapy in the late 1940s was the first effective treatment for leprosy, and this was followed by the move to short course multidrug therapy (MDT) in 1981. The World Health Assembly Resolution in 1991 [1] to “eliminate leprosy as a public health problem” by the year 2000 galvanised extraordinary international support resulting in the fall in the point prevalence of patients registered for treatment of leprosy by over 90% to less than 1 in 10,000 at the global level. The effort was led by the World Health Organization (WHO) and supported by national governments and their health service staff, the Nippon Foundation, Novartis, the International Federation of Anti-Leprosy Organizations (ILEP), local non-governmental organizations (NGOs), and by people affected by leprosy. Since 2000, the focus has moved from prevalence of leprosy to incidence as measured by reported new case detection to sustain the achievements and to reduce the burden of disease, particularly on reduction and prevention of disability associated with leprosy and rehabilitation of those facing the long-term consequences of the disease [2]. Understanding Transmission Despite this remarkable progress, understanding of the pathogenesis of leprosy has remained unclear. Basic knowledge of the transmission of M. leprae, portals of exit and entry, the role of the environment and animal reservoirs, the development of immune responses following infection, and the pathogenesis of M. leprae infection to the disease of leprosy are all limited. A recent expert group, hosted by effect: hope (The Leprosy Mission Canada) and the National School of Tropical Medicine at Baylor College of Medicine in Houston, Texas, United States, reviewed the evidence and recent research on transmission and how to block it. Novel methods in strain typing M. leprae and recent findings in both host genetics and immune responses open the potential for new solutions. However, the very long incubation period, the very low incidence rates in those exposed, and the insidious clinical presentation create real challenges to developing strategies to interrupt transmission [3]. Global Trends in Leprosy Global data on the trends in new case detection in leprosy are collated and published annually by WHO [4]. There are concerns about the quality and completeness of these data [5,6]. These data describe new case trends from detection through the completion of MDT at national, regional, and global levels. Fig 1 plots the number of new leprosy cases by year. The red continuous line represents the observed annual new case detection rate between 1985 and 2012, with extrapolation to 2020 based on the trend after 2005 (red dotted line). The blue continuous line is the predicted new case detection rate based on modeling with the SIMLEP model, applying an intermediate scenario in the presence of an infant BCG vaccination programme [7]. These trends in the last decade show a very striking feature (Fig 1, red line): a dramatic and sudden decline in new case detection of over 60% over a short period of time (2001–2005). Understanding the possible explanations for this dramatic fall is very important. One explanation is that this represents a true fall in the incidence of leprosy following reduction in transmission of M. leprae infection. Disease modeling work [7] has suggested that the long-term underlying trend in leprosy incidence in a good scenario including infant BCG immunization is a slow, gradual decline of around 4.4% per year. A large, sudden fall in transmission seems biologically implausible given the long and variable incubation period in leprosy and the evidence of continuing, significant rates of new cases in children [4]. A second explanation is that there was substantial overdiagnosis of leprosy prior to 2001, which has inflated the previous levels of new case detection. This may be a factor to explain the peak of new case detection between 1996 and 2001, a period of intensified case detection activities [8], such as Leprosy Elimination Campaigns (LEC) and Special Action Projects for the Elimination of Leprosy (SAPEL). However, the new case detection trends between 1985 and 1996 are remarkably stable and sustained overdiagnosis seems unlikely over this period. The third, and most probable, explanation is that the dramatic fall in new case detection is a result of a decline in leprosy activities following the declaration of elimination as a public health problem globally, and in individual countries. This decline includes reduced intensity and coverage of case detection activities, community awareness, and training in the diagnosis and treatment of leprosy often associated with the move from vertical leprosy control activities to integrated approaches. The recent rise in disability in new cases detected and the increasing delay in diagnosis reported by many countries supports this explanation [4]. WHO, along with the Nippon Foundation, called an International Leprosy Summit in 2013 to address what they called “stagnation” in the leprosy control. This resulted in the Bangkok Declaration [9], signed by the health ministers of the major leprosy endemic countries, calling for renewed political commitment to leprosy control. 10.1371/journal.pntd.0003658.g001 Fig 1 Number of new leprosy cases by year. The red continuous line represents the observed annual new case detection rate between 1985 and 2012, with extrapolation to 2020 based on the trend after 2005 (red dotted line). The blue continuous line is the predicted new case detection rate based on modeling with the SIMLEP model, applying an intermediate scenario in the presence of an infant BCG vaccination programme [7]. The Implications of the Decline in New Case Detection for the Elimination of Leprosy Fig 1 shows how the model prediction of the long-term trend in new leprosy case detection based on the observed figures before 2000 compares with the observed new case detection. The resulting difference between the expected and observed numbers of new cases of leprosy between 2000 and 2012 is approximately over 2.6 million. This number will increase to over 4 million by 2020. This analysis implies that there may be a large accumulation of people with leprosy in the community who remain undiagnosed and untreated. This assertion is supported by evidence from recent sample surveys in endemic areas detecting many as yet untreated cases in Bangladesh [10] and in India [11]. This large number of undetected cases represents a major threat to leprosy control and contributes to the increased burden of infection in the community and an increased pressure on transmission. This has major consequences for the road map for NTDs in the London Declaration [12–14], which targets “interruption of transmission” and “global elimination” of leprosy by 2020. Response to This Threat to Leprosy Elimination It is vital that all involved and concerned with leprosy control appreciate this situation and recognise that the London Declaration targets of “global elimination” of leprosy and “interruption of transmission” by 2020 are likely to be unobtainable and that revised targets are needed. Major commitments and resources need to be made available without delay. While local elimination (based on new cases detected in a defined locality) of leprosy through targeted leprosy control activities as recommended by WHO is necessary in the short-term, the complete interruption of transmission at a global level will require new tools based on game-changing discoveries. A significant investment in complementary research efforts, designed to better understand the basic elements of transmission, is necessary for achieving “interruption of transmission.” The development of collaboration with other NTD programmes represents a real opportunity to improve the coverage, quality, and cost-effectiveness of leprosy control with numerous cross-cutting opportunities in drug delivery, surveillance, training, disability prevention, and morbidity management. The commitment called for by health ministers in the Bangkok Declaration is also essential at all levels, internationally, nationally, and locally by national governments and by all agencies that support national programmes, including Governmental and non-governmental agencies, industry, and people affected by leprosy. The global introduction of post-exposure prophylaxis [15–17] is a real opportunity to re-energise leprosy control activities through increased community awareness, capacity building, and active management of contacts. The research opportunity recently launched by the Leprosy Research Initiative leads the way to develop further innovations for leprosy control, but much more support is needed for basic, as well as operational, research to develop strategies to interrupt transmission. For example, recent findings have revealed new insights into zoonotic relationships, genetic markers for host susceptibility and resistance, as well as environmental factors that continue to test our long-held notions of the ecology of M. leprae and leprosy. Understanding these relationships may provide the knowledge to move from management practices to strategies designed to stop transmission. The WHO priority to promote early detection and to monitor this through measuring disability in new case detection is a vital component to evaluate enhanced initiatives designed to reduce transmission. However, addressing the gap between the incidence and case detection of leprosy requires improved strategies for case detection, new tools for early diagnosis, and major efforts to improve community awareness and capacity of health staff to diagnose and manage leprosy and its complications. The challenge is to tackle the research gaps through novel collaborations, to improve operational collaborations with multiple players in all NTDs, and to incorporate new approaches in community engagement that would enhance public health at the community level. The leprosy world, including WHO, national governments, NGOs, the research community, and industry, together with people affected by leprosy, must respond to this situation that, if left unaddressed, could see all the past achievements in leprosy control reversed.

0 comments Cited 71 times – based on 0 reviews      Review now

Bookmark

Record: found
Abstract: found
Article: not found

Enumeration of Mycobacterium leprae Using Real-Time PCR

Richard Truman, P. Kyle Andrews, Naoko Robbins … (2008)

Introduction Because M. leprae can not be grown on synthetic media, the bacilli must be enumerated by direct microscopic counting. Originally developed by Shepard [1] in the 1960's, this technique has survived as the “gold standard” for enumerating M. leprae for almost 50 years. Unfortunately, it is a highly specialized procedure, cumbersome to perform and limited in terms of sensitivity and specificity. Only a few laboratories today have retained the ability to enumerate M. leprae using direct microscopy [2],[3]. Various methods have been described to minimize error in direct microscopic counting of M. leprae, including the use of special slide coatings, staining procedures, and methods to calibrate microscopes [2],[4]. However, these steps add to the complexity of the technique and the inherent insensitivity of the method requires that multiple samples be processed in large group sizes in order to reduce error. In addition, direct microscopy has limited clinical utility. For example, M. leprae cannot be differentiated from other acid-fast bacteria by microscopic examination alone, and clinical assessment of suspect biopsies requires that additional tests also be applied when a mixed infection is suspected [5]–[10]. With the development of nucleic acid-based amplification assays, the identification of difficult to grow microorganisms in tissues, including M. leprae, has become routine [11]–[16]. These assays have enhanced our awareness of clinical disease processes, and in some cases have produced new ways to diagnose and monitor mycobacterial infections. Implementing real-time PCR assays adds another potential advantage of direct or indirect quantitation of target DNA. Therefore, we investigated this approach seeking a more precise and reproducible assay for enumerating M. leprae in tissues based on the M. leprae DNA content of tissue specimens using real-time PCR. The M. leprae chromosome contains a family of dispersed repeats (RLEP) of variable structure and unknown function [17]. Twenty-nine copies of RLEP exist in the chromosome, each containing an invariant 545-bp core flanked in some cases by additional segments ranging from 44 to 100 bps. We identified DNA sequences for TaqMan PCR primers and fluorescent probe from the M. leprae-specific, invariant region of RLEP. We tested the specificity of the assay against a number of microorganisms, including cultivable mycobacteria and evaluated the sensitivity of the assay for detecting M. leprae by comparing it with direct microscopic counting for accuracy in estimating the number of M. leprae under a variety of experimental conditions employing both the mouse foot pad (MFP) model and infected armadillos. Materials and Methods Bacteria M. leprae, strains Thai-53 or NHDP98 were isolated as previously described [18] and maintained in continuous serial passage in nude mice (Hsd∶Athymic Nude-Foxn1nu , Harlan Sprague Dawley Inc., Indianapolis, IN). Briefly, M. leprae were harvested from nude mouse foot pad tissues after infection for approximately 6 months. Following CO2 asphyxiation the hind feet are removed and cleaned with 70% ethanol and Betadine to kill surface contaminants. The skin is removed aseptically and the highly bacilliferous tissue excised, minced and homogenized in 10 ml of Middlebrook 7H12 medium without catalase. Tissue debris is removed by slow speed centrifugation (50×g) for 10 minutes and the bacilli-rich supernatant is pelleted (10 k×g×10 min), resuspended and washed extensively in TE buffer to remove extraneous tissue debris associated with the intact bacilli. The suspension is then enumerated using the method of Shepard et al [1] as described in the MFP Technique below, and viability assessed in axenic culture by the oxidation of 14C-palmitate. Viable M. leprae obtained through serial passage in nude mice were used to infect other mice and armadillos used in this study [19]. Cultivable mycobacteria were grown to late log phase in Middlebrook 7H9 media plus glycerol, Tween 80 and OADC at appropriate temperatures for optimal growth (Table 1). M. lepraemurium was purified from infected mouse spleens and was a gift from I. Brown, Middlesex, England. Genomic DNA was purified from all mycobacteria by enzymatic lysis as described by Belisle and Sonnenberg [20]. Purified genomic DNA from Streptococcus pyogenes, Staphylococcus epidermidis, Clostridium perfringens, Escherichia coli and Corynebacterium glutamicum were purchased from American Type Culture Collection (Manassas, VA). 10.1371/journal.pntd.0000328.t001 Table 1 Specificity of RLEP TaqMan for Mycobacterium leprae detection. Organism 16S rDNA RLEP Organisms 16S rDNA RLEP M. leprae + + M. marinum + − M. avium + − M. phlei + − M. bovis + − M. simiae + − M. bovis BCG + − M. smegmatis + − M. chelonei + − M. tuberculosis + − M. flavescens + − M. ulcerans + − M. gordonae + − C. perfringens + − M. intracellulare + − S. epidermidis + − M. kansasii + − S. pyogenes + − M. lepraemurium + − E. coli + − M. lufu + − Shepard Enumeration and Mouse Foot Pad Technique Growth of M. leprae in the mouse foot pad is determined by direct enumeration of bacilli using the method of Shepard et al [1],[2],[21]. Generally, the bacilli are first inoculated through the planter surface of the foot in 30 ul volumes. A localized infection is established and the bacilli are harvested after a suitable time, often 6 months or more. For enumeration, mice are sacrificed and the plantar surfaces of both hind feet are excised with scalpel and forceps. The tissue is minced with scissors before being transferred to a motorized Potter-Elvehjem tissue grinder where it is homogenized to a fine paste for 1 minute. Trypsin-EDTA (GibcoBRL, Life Technologies, Grand Island, NY) (1 ml) is added and homogenized with the tissue for an additional 30 seconds before the entire preparation is incubated for 15 minutes at 37°C. After incubation the tissue is ground an additional 30 seconds and the entire contents transferred to a glass Mickle homogenizer with 25 glass beads (3 mm), capped, and vibrated for 2 minutes. For bacterial enumeration, 10 ul of the homogenized liquid is added to 10 ul of calf serum containing 2% phenol. The suspension is mixed thoroughly and spread evenly over three, 1 cm2 area circles on a premarked counting slide (Bellco Glass, Inc., Vineland, NJ). After drying in air, slides are fixed in formalin vapor for 3 minutes, using a covered staining dish containing 700 ul of formalin. The fixed slides are then heated on a glass plate over a boiling water bath for 2 minutes. Warmed slides are twice flooded and drained of distilled water containing 0.5% gelatin and 0.5% phenol, and then heated again for 2 minutes between each treatment, and again before being stained. The bacilli are stained using a modified Fite carbol-fuschin for 20 minutes, and decolorized for 30–40 seconds with 5% sulfuric acid in 25% ethanol. Slides are counterstained with crystal violet before a final wash and air drying [1],[4],[21]. Acid-fast bacilli (AFB) are then enumerated by direct examination of 20 oil emersion fields in each of the three, 1 cm2 circles, scanning along the horizontal axis of the stained smear using a calibrated microscope. The average number of bacilli in each of three smears is determined and multiplied by the appropriate calibration factor to yield a mean and standard deviation for the AFB count. Care is taken to enumerate only fully stained and intact bacilli avoiding partially stained organisms or those with atypical morphological shapes. Samples Enumerated Three strains of mice were utilized to assess growth and counting efficiency of M. leprae using real-time PCR. The strains were 1) fully immunocompetent C57BL/6 mice which permit M. leprae growth over an approximate 2-log range of growth from 104 to 106; 2) immune-compromised tumor necrosis factor receptor 1 (TNFR1) knock out (KO) mice (B6.129-Tnfrsf1atm1Mak; The Jackson Laboratory, Bar Harbor, ME). This KO strains exhibits a reduced capacity to control multiplication of M. leprae, although not to the extent seen in nude mice, permitting M. leprae growth over a 3-log range from 104 to 107; and 3) Athymic nude mice which lack T-cells making them unable to control M. leprae infections permitting growth over a 6-log range from 104 to 1010. All studies with animals were previously approved and conducted within the ethical guidelines outlined under the U.S. Public Health Service policy for the care and use of laboratory animals (NHDP IACUC assurance number A3032-01). Conventional Mice Vaccine Trial: C57BL/6 mice (Harlan Inc., Indianapolis, IN) were injected intradermally with 2×107 heat-killed M. leprae (n = 20) or normal saline (n = 14). Thirty days later, each mouse was challenged in each hind foot pad with 5,000 viable, nude mouse-derived M. leprae. Six months later foot pads were harvested from all mice and M. leprae from the infected tissues were prepared for counting as previously described above [1],[21]. The remainder of each bacillary suspension was prepared for TaqMan PCR as described in DNA preparation below. Short-term infection: A fresh suspension of M. leprae was harvested from nude mice as described above and serially diluted in HBSS to contain from 1×107–1×102 bacilli in 30 uL. This volume was inoculated through the plantar surface of both hind foot pads (BHFP) of 5 normal BALB/c mice at each dose level. After 4 hours, three mice in each group were sacrificed under CO2 and both hind foot pads and popliteal lymph nodes were collected for M. leprae enumeration. The remaining two mice in each group were harvested 1 week later and processed in an identical manner. Growth of M. leprae in TNF knock out mice: A total of 24 TNFR1 knock out (KO) mice were inoculated in BHFP through the plantar surface with a 30 uL suspension containing 5000 viable M. leprae. The infections were allowed to progress for 6 months when they were harvested for enumeration as described above. Growth of M. leprae in nude mice: Nude mice were inoculated through the plantar surface of both hind foot pads with a suspension containing 1×107 highly viable M. leprae in 30 uL of HBSS. The infection was allowed to progress for approximately 6 months and foot pads were harvested when they showed moderate enlargement using the procedure described above. Enumerated samples from 28 mouse harvests were collected and compared in this study. Growth of M.leprae in armadillo tissues: Nine-banded armadillos (Dasypus novemcinctus) were inoculated intravenously for large scale propagation of M leprae using 1–4×109 highly viable M. leprae according to the procedure described before [22]. Animals were allowed to progress through their experimentally induced infections for 18–24 months before they were sacrificed and their livers, spleens and lymph nodes harvested for purification of M. leprae [22]. A total of 40 different armadillo liver, spleen and lymph node tissue samples were collected and enumerated in this study. Preparation of DNA Mouse-derived M. leprae M. leprae DNA was obtained from the tissue homogenates used for microscopic enumeration of acid-fast bacilli. A 200 uL aliquot of the homogenate was subjected to 3 freeze- thaw cycles, mixed with 10 uL of proteinase K (10 mg/ml in buffer, pH 7.5), and incubated at 56 C for 2 hrs.. The samples were then mixed again by vortexing and incubated overnight. The proteinase K was inactivated by heating to 95°C for 1 hour and an additional 40 uL of TE was added to bring the volume to 250 uL. Samples were then mixed and diluted 1∶4 prior to testing. Armadillo-derived M. leprae A 1.0 gm sample of highly bacilliferous armadillo liver, spleen or lymph node was homogenized in a motorized Potter-Elvehjem tissue grinder containing 4 mL of 7H12 broth and frozen at −70 C. Upon thawing the homogenate was diluted 1∶100 in DH20 and processed with DNeasy (Qiagen, Inc., Valencia, CA) according to the manufacturers recommendations. Briefly, 10 µL of the 1∶100 tissue homogenate was added to 80 µL of ATL buffer with 20 µL of proteinase K solution. The sample was mixed and incubated for approximately 1 hour at 56° C with occasional mixing. After incubation samples were mixed thoroughly and 200 µL of AL buffer and 200 µL of ethanol were added before mixing again. This mixture was transferred to a spin column and centrifuged at 6000×g for 1 min discarding the flow through volume and transferred to a new catch tube. 500 µL of AW1 buffer was added and centrifuged at 6000×g for 1.0 min. The flow through was again discarded and the column transferred to a new catch tube. 500 µL of AW2 buffer is then added and centrifuged at 20,000×g for 3.0 min discarding the flow through and transferring the column to a sterile 1.5 mL tube. 200 µL of the elution buffer was added and the column is centrifuged at 6000×g for 1 min. The eluate was collected and diluted 1∶4 prior to testing. TaqMan Assays Primers and probe for the RLEP TaqMan PCR were selected from a common region of the RLEP family of dispersed repeats. M. leprae RLEP DNA sequences were acquired from the Sanger Center (www.sanger.ac.uk) and aligned for regions of identity using Omiga 2.0 software (Oxford Molecular Ltd., Madison, WI). RLEP primers and fluorescent probe were chosen using Primer Express software (PE Applied Biosystems, Foster City, CA) based on criteria established for TaqMan PCR reactions. All reagents used in the TaqMan assay were recommended by the manufacturer (PE Applied Biosystems), including AmpErase UNG enzyme and AmpliTaq Gold DNA polymerase. PCR cycling conditions were 40 cycles with 60°C annealing/extension temperature for 60 seconds and 95°C denaturating temperature for 15 seconds. PCR and data analyses were performed on a 7300 RealTime PCR System (Applied Biosystems, Foster City, CA). Results Primers, Sensitivity and Specificity RLEP TaqMan primers and probe were selected by aligning DNA sequences from RLEP 1, 2, 3 and 4. A region of RLEP was selected in which the four families of dispersed repeats were identical and analyzed for optimal TaqMan primers and probe. The sequence selected was 5′-GCAGTATCGTGTTAGTGAACAGTGCAtcgatgatccggccgtcggcgGCACATACGGCAACCTTCTAGCG-3′. Capital letters in bold represent the sequence on which the forward and reverse primers were built. The sequence in lower case italics was selected for building the fluorescent TaqMan probe. When forward and reverse primer sequences were blasted against the M. leprae genome, 19 regions were identified with identical sequences. Another 8 regions were identified with high homology to the primers but amplification would not be likely because of 3-prime mismatches with these primers. Accordingly, amplification of a single M. leprae chromosome with these primers should result in 19 copies of RLEP. Sensitivity of the RLEP TaqMan PCR assay was tested with both purified M. leprae DNA and nude mouse-derived M. leprae. A titration of M. leprae DNA in the TaqMan PCR using RLEP primers/probe gave a lower limit of detection of 10 fg equaling approximately 3 organisms based on the M. leprae chromosome of approximately 3.27 Mb (data not shown). While these conditions measure the sensitivity of the assay under ideal circumstances (no inhibitors), a more realistic assessment of the detection limit was determined using M. leprae harvested from infected mouse tissues. Using nude mouse-derived M. leprae as a source of DNA the RLEP TaqMan PCR was able to detect approximately 300 M. leprae (Fig. 1). 10.1371/journal.pntd.0000328.g001 Figure 1 RLEP TaqMan PCR results from titration of nude mouse-derived M. leprae. Serial 2-fold dilutions of M. leprae were made from 2×106 to 1.56×104/ml. Ten microliters of each dilution were tested in triplicate representing 2×104 to 156 M. leprae in the test sample, respectively. The ordinate is PCR cycle number at threshold and the abscissa is number of M. leprae (log10). Standard deviations did not exceed 0.5% of mean at any dilution. Specificity of the RLEP TaqMan PCR for M. leprae DNA was determined by testing purified genomic DNA from 16 mycobacterial species, 10 of which are associated with human diseases, three gram positive microorganisms often associated with skin infections and E. coli (Table 1). In order to monitor genomic DNA for efficient amplification by PCR, samples were tested for reactivity in a separate PCR designed to detect 16S rDNA [23]. All samples tested for 16S rDNA gave a strong signal based on agarose gel electrophoresis when amplifying 10 pg of genomic DNA for 35 cycles. In contrast, RLEP TaqMan PCR was positive only for M. leprae DNA when samples were tested at the same concentration using 40 cycles. Correlation between Molecular Enumeration with RLEP and Direct Counting After enumeration by direct microscopic counting, we extracted DNA for enumeration by RLEP TaqMan PCR using the highest enumerated sample of each tissue type to establish a standard curve for those tissues. As shown in Figure 2, direct microscopic counts ranged between 4.8×103 and 2.3×1010 bacilli. Estimates based on RLEP TaqMan PCR ranged from 623 organisms in conventional mouse foot pad tissues, to 5.8×1010 bacilli in each gram of armadillo tissue. For Molecular Enumeration, Coefficient of Variation (CV) between individual replicates averaged 14.03% (Mode 0.53%, Median 5.58%). Similar CV data was not available for the direct microscopic counts and no values were excluded based on CV. Enumeration estimates based on RLEP showed good correlation with direct microscopic counting with coefficients (Pearson's r) ranging from 0.78 to 0.89 for individual tissue types examined. Best results were seen with tissue sets that had a broad range of estimated bacillary counts. No significant difference in counting efficiency was seen between the various types (liver, spleen or lymph node) of armadillo tissues examined (data not shown). In combination across all tissues examined, RLEP showed a correlation of 0.98 (Pearson's) with direct microscopic counting. 10.1371/journal.pntd.0000328.g002 Figure 2 Comparison of direct microscopic counting of AFB per standard volume with enumeration of M. leprae by RLEP TaqMan PCR from tissues originating from a variety of host animals. Symbols identify individual samples from sets of conventional, TNFR1 knock-out (KO), and congentially athymic nude mice, as well as from nine-banded armadillos. Pearson's coefficient (r2) is calculated for each tissue set. Enumeration estimates for all tissues combined showed high correlation (r2 = 0.96) between the “gold standard' direct microscopic counting and estimates based on RLEP TaqMan PCR. Application of RLEP TaqMan PCR assay in experimental procedures Fate of M. leprae in short-term infections To better understand the fate of M. leprae after inoculation into the mouse, we used the RLEP TaqMan PCR to enumerate the number of bacilli remaining in the foot pad 4 hours and 1 week after inoculation. The foot pads were injected with equal volumes containing concentrations of bacilli ranging from 1×107 to 1×102 M. leprae per foot pad (Table 2). More M. leprae was retained in the first few hours after inoculation than 1 week later and inocula containing higher concentrations of M. leprae demonstrated better retention (ranging from 2.84% to 20.8%). M. leprae inoculated at doses lower than 1×105/ foot pad did not yield detectable results (data not shown). 10.1371/journal.pntd.0000328.t002 Table 2 The number and percent of bacilli recovered from conventional mouse foot pads within 4 hours and 1 week post inoculation with varying doses of M. leprae as measured by RLEP PCR. 4 hr % of Dose Retained 1 wk % of Dose Retained Dose Bacilli Given Site Bacilli/Foot Pad Recovered Site Bacilli/Foot Pad Recovered 1.00E+07 LF1 2.08E+06 20.80% LF4 1.25E+05 1.25% RF1 1.48E+06 14.80% RF4 2.70E+05 2.70% LF2 1.15E+06 11.50% LF5 3.20E+05 3.20% RF2 8.67E+05 8.67% RF5 7.15E+05 7.15% LF3 5.61E+05 5.61% RT3 1.74E+06 17.40% 1.00E+06 LF1 1.23E+05 12.28% LF4 6.14E+04 6.14% RF1 8.27E+04 8.27% RF4 4.95E+04 4.95% LF2 5.07E+04 5.10% LF5 6.68E+03 0.67% RF2 8.39E+04 8.39% RF5 7.80E+04 7.80% LF3 2.94E+04 2.94% RF3 3.73E+04 3.73% 1.00E+05 LF1 NR NR LF4 2.13E+03 2.10% RF1 NR NR RF4 2.81E+03 2.80% LF2 6.65E+03 6.65% LF5 4.31E+03 4.31% RF2 2.87E+03 2.87% RF5 7.39E+03 7.39% LF3 2.84E+03 2.84% RF3 2.86E+03 2.86% Average (SD) 8.42% (5.57%) 4.21% (2.47%) LF = Left Foot with dose animal number, RF = Right Foot with dose animal number, NR = Not Run. SD = Standard Deviation of average percent bacilli retained in the foot pad. On average, 4 hours after inoculation into the foot pad only 8.42% (+/−5.57%) of any inoculum could still be detected within the foot. After 1 week, the degree of individual variation in the number of bacilli retained within the foot was markedly decreased for all inoculum dose levels; but there appeared to be some continued loss of bacilli from the site. On average, for all the inoculum dose levels considered, only 4.21% (+/−2.47%) of the bacilli originally injected into the mouse foot pad could be detected there after 1 week of incubation. In an attempt to account for bacilli draining from the foot pad we also examined the popliteal lymph node associated with each foot. Within the first 4 hours these lymph nodes were uniformly small and unperturbed. After 1 week they appeared noticeably enlarged, however, M. leprae could not be enumerated in these nodes using RLEP TaqMan at either time period. If the bacilli are retained by these nodes, the amount of amplifiable DNA was below the detectable level of our assay. Estimating Vaccine Efficacy To determine the effect of host resistance towards M. leprae on the efficiency of molecular enumeration, we compared counting results obtained with the two techniques in a standard mouse foot pad (MFP) vaccine study. The seminal work of Shepard, et al [24] and a large number of subsequent studies have demonstrated the suppressive effect of potent vaccines, such as BCG and heat-killed M. leprae, on the growth of M. leprae in the mouse foot pad model. Briefly, prior sensitization with heat-killed M. leprae will result in 1 to 2 logs growth suppression in the mouse foot pad. In our studies mice were vaccinated with heat-killed M. leprae (HKML) or saline, as a sham vaccine, and challenged with 5000 viable M. leprae in their foot pads thirty days following vaccination. M. leprae enumerations were performed 6 months after challenge by RLEP TaqMan PCR and direct microscopic counting. The results of the vaccine trial are shown in Figure 3. 10.1371/journal.pntd.0000328.g003 Figure 3 A comparison of RLEP TaqMan PCR and M. leprae counting results from a vaccine trial using conventional C57/B mice. Bars represent mean plus the standard deviation for each group. ** = probability of statistical significance (p)<0.01, and *** = probability of statistical significance (p)<0.001. Vaccination with HKML resulted in a significant reduction in the growth of M. leprae in the foot pads (p<0.01, Kruskall-Wallace and Dunn's Multiple Comparison's Test) that was detectable by both enumeration techniques. Bacillary counts below 103 are generally not detectable by direct microscopy, but with RLEP enumeration, numerical estimates were derived for 11 samples that otherwise were at or below the threshold of detection for direct microscopic counting. An effective host response that successfully limited growth of the bacilli in the foot pad did not adversely influence the ability to enumerate organisms based on amplification of RLEP DNA amplification. Discussion These results demonstrate that a simple, reproducible test based on genomic DNA can be used to quantify M. leprae in infected tissues. The real time PCR assay yields results similar to those obtained from conventional direct microscopic counting methods, is highly specific, sensitive, and is easily adapted to large scale batch processing of samples. Molecular quantification of M. leprae based on amplification of RLEP TaqMan PCR is a suitable replacement for direct microscopic counting of bacilli. The quantitative sensitivity of RLEP PCR is within the range of other PCR detection assays for M. leprae DNA developed based on a single-copy gene [9]. A major difference between the two assay systems, however, is the time required for analytical testing, and the ability to quantify multiple batch samples at all time points during thermocycling. For example, the 18-kDa traditional PCR with specific probe hybridization, which we developed earlier, requires approximately 48 hours to complete, whereas the RLEP TaqMan PCR can be accomplished with full analysis in as little as 6 hours. Conventional direct microscopic enumeration requires several hours per sample and has no time savings associated with batch processing. The greater sensitivity of the RLEP TaqMan PCR can be especially useful for comparative growth studies in the MFP model and some in vitro techniques. The threshold limit of detection for direct microscopic counting is approximately 1×104 bacilli. Growth results below those levels are not reliable and data baselines in MFP studies are usually plotted as 1×104 or erroneously coded as zero. Since the upper level of growth in the conventional mouse foot pad plateaus at around 1×106 bacilli for BALB/c mice, and perhaps even lower for some other mouse strains, statistical significance in MFP growth results must be drawn from within only a narrow 2 log window. RLEP TaqMan PCR yields reliable quantitative growth results with less variation at a lower detection threshold than direct microscopic counting (about 300 organisms) and the counting efficiency is not influenced by cellular immune processes. The greater sensitivity of RLEP TaqMan PCR can benefit discernment of statistically significant results within more narrow ranges. In addition, since M. leprae is also a notoriously slow growing organism, more sensitive enumeration methods also could lead to shortening MFP trials which now often require 7–12 months to reach completion. Most of our knowledge about the microbiological characteristics of M. leprae is derived from mouse foot pad studies. In the classic Shepard model, mice are typically inoculated in the foot pad with between 5000–10000 bacilli, and the growth of these organisms is assessed after 120–360 days. Even though a large bolus is deposited into the foot, Levy and others observed that the number of bacilli retained in the foot pad 1 week after inoculation was too low to visualize with direct microscopy [25],[26]. The fate of these organisms remains unknown, but our observations that some 90% of the bacilli are lost from the foot within only a few hours after inoculation is in keeping with those original results and confirms a more immediate time for their loss. Foot pad inoculation was originally developed as a means to provide M. leprae a low temperature growth environment. However, the architecture of the foot pad is not ideal for retention of an inoculum or for supporting the growth of obligate intracellular organisms, such as M. leprae. The soft tissue of the foot pad contains few phagocytic cells and consists mainly of dermal and epidermal cells, along with striated muscle. While M. leprae can invade striated muscle cells and other non-professional phagocytes [27], their preferred host cell is the macrophage, and sustained local growth of M. leprae in the foot pad requires a continuous influx of new macrophages to the site. It is notable that popliteal lymph nodes of the mice studied here showed enlargement within one week of foot pad inoculation, even in absence of detectable bacilli in those nodes. The specific mechanisms potentially involved in recruiting macrophages to the foot pad are well beyond the scope of this paper; however, these observations support the notion that there is some systemic stimulation following inoculation of the foot pad and these processes may play an important role in establishing and maintaining that localized infection. Although MFP is the oldest and most widely used method to propagate M. leprae, there is much that remains unknown about the technique. Methods that might enhance the growth environment for M. leprae in the foot pad by priming the host beforehand, or pre-populating the foot pads with receptive macrophages could benefit our ability to better exploit this model. Regardless, evolution of more sensitive methods to detect M. leprae in tissues, such as RLEP TaqMan PCR, can aid that development and help advance this reliable model. Other gene targets also can likely be used for relative quantification of M. leprae. Our results with the RLEP TaqMan PCR are in keeping with those reported earlier for quantification of M. leprae based on genetic sequences in the proline-rich antigen region that used purified DNA as a comparative standard [28]. However, the accuracy of estimates based on comparison to purified DNA standard depends entirely on the efficiency of DNA isolation from different tissues, and the inter-run reproducibility of the extraction method. The use of pre-enumerated standards as employed here (and also available from the NIAID Leprosy Research Support Contract), can help eliminate the inaccuracy inherent in variable recovery of DNA in different runs or conditions, and permits ready comparison of results between individual laboratories. Molecular enumeration of M. leprae using the RLEP TaqMan PCR is a rapid and more accurate method to quantify M. leprae in tissues that can have wide applicability in research. The DNA based technique is more sensitive and reproducible than direct microscopic counting, requires less technical expertise, and can permit ready comparisons of results between laboratories. Utilization of this or other molecular based techniques to enumerate M. leprae will likely aide more careful investigation of growth results in a variety of model systems, and will enhance our ability to propagate this and other difficult to grow microorganisms.

0 comments Cited 54 times – based on 0 reviews      Review now

Bookmark

All references

Author and article information

Contributors

Shannon M. Lenz:

ORCID: http://orcid.org/0000-0001-5705-1819

Role: Data curationRole: Formal analysisRole: Writing – original draftRole: Writing – review & editing

Jaymes H. Collins:

ORCID: http://orcid.org/0000-0001-7509-0109

Role: Data curationRole: Formal analysisRole: Writing – review & editing

Nashone A. Ray: Role: Data curationRole: Writing – review & editing

Deanna A. Hagge:

ORCID: http://orcid.org/0000-0002-3792-2266

Role: Funding acquisitionRole: Writing – review & editing

Ramanuj Lahiri: Role: Formal analysisRole: Funding acquisitionRole: Writing – review & editing

Linda B. Adams:

ORCID: http://orcid.org/0000-0001-6265-3531

Role: ConceptualizationRole: Formal analysisRole: Funding acquisitionRole: Writing – review & editing

Susilene Maria Tonelli Nardi: Role: Editor

Journal

Journal ID (nlm-ta): PLoS Negl Trop Dis

Journal ID (iso-abbrev): PLoS Negl Trop Dis

Journal ID (publisher-id): plos

Journal ID (pmc): plosntds

Title: PLoS Neglected Tropical Diseases

Publisher: Public Library of Science (San Francisco, CA USA )

ISSN (Print): 1935-2727

ISSN (Electronic): 1935-2735

Publication date (Electronic): 16 September 2020

Publication date Collection: September 2020

Volume: 14

Issue: 9

Electronic Location Identifier: e0008583

Affiliations

[1 ] IHRC, Inc. Atlanta, Georgia, United States of America

[2 ] Department of Health and Human Services, Health Resources and Services Administration, Healthcare Systems Bureau, National Hansen’s Disease Programs–Laboratory Research Branch, Baton Rouge, Louisiana, United States of America

[3 ] Mycobacterial Research Laboratories, Anandaban Hospital, The Leprosy Mission Nepal, Kathmandu, Nepal

Adolfo Lutz Institute of Sao Jose do Rio Preto, BRAZIL

Author notes

The authors have declared that no competing interests exist.

* E-mail: slenz@ 123456hrsa.gov (SML); ladams@ 123456hrsa.gov (LBA)

Author information

Shannon M. Lenz http://orcid.org/0000-0001-5705-1819

Jaymes H. Collins http://orcid.org/0000-0001-7509-0109

Deanna A. Hagge http://orcid.org/0000-0002-3792-2266

Linda B. Adams http://orcid.org/0000-0001-6265-3531

Article

Publisher ID: PNTD-D-20-00714

DOI: 10.1371/journal.pntd.0008583

PMC ID: 7494095

PubMed ID: 32936818

SO-VID: 25f971b1-cce9-402c-9dac-9fc60981834f

License:

This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

History

Date received : 28 April 2020

Date accepted : 9 July 2020

Page count

Figures: 3, Tables: 1, Pages: 10

Funding

LBA and DAH received funding from the Leprosy Research Initiative (LRI) and the Turing Foundation under LRI Grant number 703.15.43. LBA and RL received funding from the National Institutes of Health, National Institute of Allergy and Infectious Diseases through an interagency agreement (No. AAI20009) with the Health Resources and Services Administration National Hansen's Disease Program. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Custom metadata

Data Availability All relevant data are within the manuscript.

ScienceOpen disciplines: Infectious disease & Microbiology

Data availability:

ScienceOpen disciplines: Infectious disease & Microbiology

Comments

Comment on this article

scite_

Cited by 5

See all cited by

Most referenced authors 1,468

See all reference authors

Post-exposure prophylaxis (PEP) efficacy of rifampin, rifapentine, moxifloxacin, minocycline, and clarithromycin in a susceptible-subclinical model of leprosy

Read this article at

Abstract

Background

Methodology/Principal findings

Conclusions/Significance

Author summary

Related collections

CHAIN Network Collection

Most cited references 33

Effectiveness of single dose rifampicin in preventing leprosy in close contacts of patients with newly diagnosed leprosy: cluster randomised controlled trial.

The Missing Millions: A Threat to the Elimination of Leprosy

Enumeration of Mycobacterium leprae Using Real-Time PCR

Author and article information

Contributors

Journal

Affiliations

Author notes

Author information

Article

History

Page count

Funding

Categories

Custom metadata

Comments

Comment on this article

Similar content 100

Cited by 5

Most referenced authors 1,468