• View in gallery

    Photomicrograph showing the epidermis of skin of a lepromatous leprosy patient containing several acid-fast bacilli (arrows) in the keratin layer (Fite stain). This figure appears in color at www.ajtmh.org.

  • 1

    Fine P, 2007. Leprosy: what is being “eliminated.” Bull World Health Organ 85 :2–4.

  • 2

    Meima A, Richardus JH, Habbema JD, 2004. Trends in leprosy case detection worldwide since 1985. Lepr Rev 75 :19–33.

  • 3

    Anonymous, 2006. Global leprosy situation, 2006. Wkly Epidemiol Rec 81 :309–316.

  • 4

    Job CK, Chehl S, Hastings RC, 1990. New findings on the mode of entry of M. leprae in nude mice. Int J Lepr 58 :726–729.

  • 5

    Chan PC, Reyes LM, Demerre-Lopez B, Gonzaga EM, De los Santos MFA, Gillis TP, 1997. Leprosy infection and disease in the national capital region. Philippine J Microbiol Infect Dis 26 :159–162.

    • Search Google Scholar
    • Export Citation
  • 6

    Klatser PR, van Beers S, Madjid B, Day B, de Wit MY, 1993. Detection of Mycobacterium leprae nasal carriers in populations for which leprosy is endemic. J Clin Microbiol 31 :2947–2951.

    • Search Google Scholar
    • Export Citation
  • 7

    Smith WC, Smith CM, Cree IA, Jadhav RS, Macdonald M, Edward VK, Oskam L, van Beers S, Klatser P, 2004. An approach to understanding the transmission of Mycobacterium leprae using molecular and immunological methods: results from the MILEP2 study. Int J Lepr Other Mycobact Dis 72 :269–277.

    • Search Google Scholar
    • Export Citation
  • 8

    Chatterjee BR, 1976. Carrier state in leprosy. Lepr India 48 :643–644.

  • 9

    Abraham S, Mozhi NM, Joseph GA, Kurian N, Rao PS, Job CK, 1998. Epidemiological significance of first skin lesion in leprosy. Int J Lepr Other Mycobact Dis 66 :131–139.

    • Search Google Scholar
    • Export Citation
  • 10

    Williams DL, Gillis TP, Booth RJ, Looker D, Watson JD, 1990. The use of a specific DNA probe and polymerase chain reaction for the detection of Mycobacterium leprae. J Infect Dis 162 :193–200.

    • Search Google Scholar
    • Export Citation
  • 11

    Williams DL, Gillis TP, Fiallo P, Job CK, Gelber RH, Hill C, Izumi S, 1992. Detection of Mycobacterium leprae and the potential for monitoring antileprosy drug therapy directly from skin biopsies by PCR. Mol Cell Probes 6 :401–410.

    • Search Google Scholar
    • Export Citation
  • 12

    Pedley JC, 1970. Summary of the results of a search of the skin surface for Mycobacterium leprae. Lepr Rev 41 :167–168.

  • 13

    Hosokawa A, 1999. A clinical and bacteriological examination of Mycobacterium leprae in the epidermis and cutaneous appendages of patients with multibacillary leprosy. J Dermatol 26 :479–488.

    • Search Google Scholar
    • Export Citation
  • 14

    Hatta M, Van Beers SM, Madjid B, Djumadi A, de Wit MY, Klatser PR, 1995. Distribution and persistence of Mycobacterium leprae nasal carriage among a population in which leprosy is endemic in Indonesia. Trans R Soc Trop Med Hyg 89 :381–385.

    • Search Google Scholar
    • Export Citation
  • 15

    Gelber RH, Humphres RC, Fieldsteel AH, 1986. Superiority of the neonatally thymectomized Lewis rat (NTLR) to monitor a clinical trial in lepromatous leprosy of the two regimens of rifampin and dapsone. Int J Lepr Other Mycobact Dis 54 :273–283.

    • Search Google Scholar
    • Export Citation
  • 16

    Fieldsteel AH, Levy L, 1976. Neonatally thymectomized Lewis rats infected with Mycobacterium leprae: response to primary infection, secondary challenge, and large inocula. Infect Immun 14 :736–741.

    • Search Google Scholar
    • Export Citation
  • 17

    Wakade AV, Shetty VP, 2006. Isolation of Mycobacterium leprae from untreated borderline tuberculoid, mid-borderline and indeterminate cases using the mouse foot pad technique—a study of 209 cases. Lepr Rev 77 :366–370.

    • Search Google Scholar
    • Export Citation
  • 18

    Franzblau SG, Chan GP, Garcia-Ignacio BG, Chavez VE, Livelo JB, Jimenez CL, Parrilla ML, Calvo RF, Williams DL, Gillis T, 1994. Clinical trial of fusidic acid for lepromatous leprosy. Antimicrob Agents Chemother 38 :1651–1654.

    • Search Google Scholar
    • Export Citation
  • 19

    Levy L, Shepard CC, Fasal P, 1976. The bactericidal effect of rifampicin on M. leprae in man: a) single doses of 600, 900 and 1200 mg; and b) daily doses of 300 mg. Int J Lepr Other Mycobact Dis 44 :183–187.

    • Search Google Scholar
    • Export Citation
  • 20

    Anonymous, 1997. Action Program for the Elimination of Leprosy: Status Report. Geneva: World Health Organization.

  • 21

    Sehgal VN, Rege VL, Vadiraj SN, 1970. Inoculation leprosy subsequent to small-pox vaccination. Dermatologica 141 :393–396.

  • 22

    Sehgal VN, 1971. Inoculation leprosy appearing after seven years of tattooing. Dermatologica 142 :58–61.

  • 23

    Seghal VN, 1986. Leprosy following mechanical trauma. Lepr Rev 57 :272.

 

 

 

 

Transmission of Leprosy: A Study of Skin and Nasal Secretions of Household Contacts of Leprosy Patients Using PCR

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  • 1 St. Thomas Hospital and Leprosy Centre, Chettupattu, India; Department of Pathobiology School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana; National Hansen’s Disease Programs, Baton Rouge, Louisiana

It is generally held that dissemination of Mycobacterium leprae is from nasal mucosa and not through the skin of infected patients. In this study, we evaluated M. leprae in the unbroken skin and nasal secretions of multibacillary (MB) leprosy patients and their contacts. Specimens were examined by direct microscopy and polymerase chain reaction (PCR) for M. leprae DNA. Results showed that 60% of untreated MB leprosy patients examined histologically had acid-fast bacilli in the keratin layer. By PCR studies it was found that 80% of the patients had M. leprae DNA in skin washings and 60% had M. leprae DNA on swabs obtained from the nasal mucosa. Ninety-three contacts of the untreated MB cases were also tested for exposure to M. leprae by analyzing skin washings and nasal secretions by PCR. PCR analysis showed significant skin (17% positive) and nasal muscosal (4%) exposure in contacts before instituting treatment of the index cases. After 2 months of treating the index cases, all contacts tested were negative for M. leprae DNA. These data suggested that both skin and nasal epithelia of untreated MB leprosy patients contribute to the shedding of M. leprae into the environment and contacts of untreated MB cases are at risk for contact with M. leprae through both the nasal mucosa and exposed surfaces of their skin.

INTRODUCTION

The estimated global prevalence of leprosy has been greatly reduced as a result of the multidrug therapy (MDT) program advocated by the World Health Organization (WHO) and its implementation with the help of governmental and non-governmental organizations. Although > 14 million cases have been cured with MDT since 1985, new case detection rates have decreased only marginally over the same time period.1 Therefore, the global use of MDT seems to have had only minimal, if any, effect on transmission of the disease,2 and an adequate explanation for this situation is lacking.3

It has been shown by experimental studies using nude mice that abraded skin and nasal mucosa are routes of transmission in mice and may be operative in humans.4 In human studies, nasal carriage of Mycobacterium leprae has been shown in 5–8% of household contacts of leprosy patients using polymerase chain reaction (PCR) methodology to detect M. leprae DNA.5,6 However, no invasive lesion has been reported in these contacts nor has the persistence of this condition been shown,7 making the case for infection through the nasal route associative at best. Accordingly, there is no conclusive proof for the nasal mucosal route of infection in humans.

Using conventional techniques for staining acid-fast bacilli (AFB) in skin, Chatterjee8 has shown 5.8% of contacts of leprosy patients carry AFB in their skin. Also, Abraham and others9 showed that, when sites of single leprosy skin lesions from children were superimposed with areas associated with skin abrasions and scars from age- and sex-matched children from the same environment, a statistically significant correlation was observed between the leprosy skin lesions and skin abrasions and scars. The authors concluded that these data supported skin as a potential route of infection for M. leprae.

The argument has been put forward that M. leprae–positive nasal secretions may represent transient contamination from the environment. The same case could be made for M. leprae found on surfaces of the skin. In both cases, however, continual exposure could result in increased risk of infection caused by active invasion by the bacteria or through passive entry, resulting from a breach in the epithelium of the skin or nasal mucosa after physical trauma. Defining the exposure rate for these two sites may help elucidate the intensity of exposure and possible route of entry of M. leprae in humans. Techniques, such as PCR, allow precise monitoring of M. leprae in relatively low concentrations on or within skin or nasal mucosa (nasal secretions). In this study, we tested the hypothesis that contacts of untreated multibacillary (MB) patients are exposed to M. leprae, both on the skin and nasal mucosa, at a rate higher than that observed in contacts of MB cases that have successfully completed WHO-recommended MDT for leprosy. We also compared the exposure of skin and nasal mucosa for M. leprae in contacts of untreated MB patients to establish the degree and duration of exposure to M. leprae after implementation of MDT in the index cases.

MATERIALS AND METHODS

Patients and household contacts.

The St Thomas Hospital and Leprosy Center at Chettupattu, India, manages a leprosy control program covering 450,000 people and an outpatient clinic where patients with leprosy and other dermatologic complaints are treated. From among these patients were recruited newly diagnosed, untreated MB patients (nine lepromatous leprosy [LL] and one borderline leprosy [BL]) with bacterial indices of 3+ or greater, MB patients (six LL, four BL) treated for 1 year, and 101 household contacts of 43 untreated MB patients for the study. MB patients consisted of both LL and BL patients. All contacts studied were carefully examined clinically for signs and symptoms of leprosy. Informed consent was obtained from all human adult participants, and approval of the project was obtained from both the Louisiana State University and St Thomas Hospital institutional review boards.

Sample acquisition.

Skin.

For MB leprosy cases, a defined area (312 cm2) of the skin from the posterior surface of both upper arms (3 cm × 12 cm × 2 = 72 cm2) and the back of both sides of the chest (12 cm × 10 cm × 2 = 240 cm2) was washed as follows. In a 50-mL beaker, 20 mL of sterile saline was taken. With sterile gauze dipped in the saline, the areas of the skin defined above were stroked 20 times, taking care not to spill any of the fluid from the swab. The skin washings from the beaker were transferred to two 10-mL centrifuge tubes and were centrifuged for 25 minutes at 8,000g. From the sediment, direct smears were prepared on slides, fixed, stained for AFB, and examined by light microscopy at ×100 magnification. The remainder of the sediments was fixed in 70% ethyl alcohol for PCR study. For contacts, a similar strategy of skin washings was performed except that the swabs were taken from the posterior aspect of the forearms of the contacts between the elbows and the wrists of both arms and combined into one sample per person.

Nasal secretions.

Secretions were collected from both nostrils of each contact or index case using dry, sterile dacron swabs by gently swabbing of the outer nares. Each swab tip was placed into a 1.5-mL microfuge tube containing 1.0 mL of freshly prepared sputolysin (CalBiochem; EMD Biosciences, San Diego, CA). After the addition of Tween 20 to a final concentration of 0.05%, the tubes were mixed by vortexing and allowed to stand at room temperature for 15 minutes. Fluid was expressed from the swabs, and the particulate fraction was collected by centrifugation at 10,000g for 10 minutes. The supernatant fluid was decanted, and the pellet was resuspended in 100 μL of sterile deionized water. An aliquot was spotted on a slide and stained for bacterial enumeration, and the remainder was extracted for DNA as described below. As a control for false-positive PCR reactions, 100 nasal samples were obtained from student volunteers attending the LSU School of Veterinary Medicine. The samples were processed as described above and run blindly along with the patient and contact samples.

Rehydrated sediments obtained from skin and nasal washings were extracted for DNA after digestion with proteinase K (1 mg/mL) dissolved in 10 mmol/L Tris-HCl, 1 mmol/L EDTA, and 150 mmol/L NaCl, pH 8.0 (TE) buffer. Equal volumes of the proteinase K (PK) digest and phenol/chloroform/isoamyl alcohol (25:24:1) were mixed, and the resultant aqueous phase was removed and mixed with an equal volume of chloroform/isoamyl alcohol (24:1). The resultant aqueous phase was removed, and the DNA was precipitated at −20°C after adding two volumes of chilled absolute ethanol after increasing the final NaCl concentration to 100 mmol/L. The precipitate was washed once with chilled 70% ethanol, dried at room temperature, and resuspended in 30 μL of TE. Ten microliters of the DNA extract (neat or diluted 1:5 in TE) was added to the PCR buffer and primer mix, including AmpliTaq, as per instructions of the manufacturer (Applied Biosystems, Foster City, CA). PCR conditions and primers used to amplify M. leprae DNA were described previously.10,11

Slot blotting with a digoxigenin-labeled probe specific for the M. leprae PCR amplicon was used to monitor the presence of M. leprae in clinical samples. Briefly, after treating PCR products in denaturant (50 mmol/L NaOH, 150 mmol/L NaCl) for 10 minutes, the samples were transferred to a nylon membrane in a slot blot apparatus. The membrane was air dried, heated at 80°C for 2 hours, and hybridized with the digoxigenin probe, and samples containing M. leprae DNA were detected by chemiluminescence using the Lumigen PPD detection system as described by the manufacturer (Boehringer Ingelheim Pharmaceuticals, Ridgefield, CT). Biopsies of the skin were also obtained from the back of the chest in seven patients and the back of the upper arm in six patients to further evaluate the location of bacilli in the skin.

Statistics.

The SAS statistical package (version 9.1.3; SAS Institute, Cary, NC) was used to analyze the data in a series of McNemar tests of agreement and calculated κ coefficients for repeated-measures two-way tables. Comparisons were made both within and between nasal and skin washing treatments across time points. All comparisons were considered significant at P ≤ 0.05.

RESULTS

Histopathology of the skin using the modified Fite stain from the untreated MB leprosy patients showed that 6 of 10 had AFB in the keratin layer of the skin and hair follicles (Figure 1). Direct smears of skin and nasal washings of the untreated MB patients showed that 9 of 10 and 7 of 10 samples, respectively, were positive for AFB (Table 1). PCR results from these same specimens were concordant, except for one specimen in each sampling group, confirming the AFB observed was M. leprae. Skin washings proved to be slightly more reliable than nasal washings for detecting M. leprae DNA in these patients before treatment; however, the comparison did not reach statistical significance. Nasal secretion samples from all 100 non-endemic controls tested negative by PCR. These results indicated that both anatomic sites from untreated MB cases may contribute to the dissemination of M. leprae and, therefore, to transmitting leprosy.

Duration of treatment had little effect on detection of bacilli for the first 3 months in the skin and nasal secretions, whether monitoring AFB or M. leprae DNA by PCR (Table 1). However, follow-up studies of MB patients treated for 1 year with MDT indicated that the majority of patients tested negative for M. leprae both in skin washings and nasal secretions (Table 2). This was true for direct microscopic observation and PCR of skin washings and nasal secretions at both the 1- and 2-year follow-up testing (Table 2). These same patients remained BI positive when analyzed by routine skin-slit scrapings and biopsy after 1 year of MDT (data not shown).

Contacts of the untreated MB cases were also tested for exposure by analyzing skin washings and nasal secretions by PCR. PCR analysis showed 16 of 93 (17%) household contacts had skin exposure to M. leprae before instituting treatment of the index cases (Table 3). The same group of contacts showed 4% positivity by PCR in their nasal secretions. None of the PCR-positive contacts tested positive for M. leprae DNA at both anatomic sites. After 1 month of MDT of index cases, 1 of 93 (1%) contacts tested positive for M. leprae DNA by PCR from skin washings, whereas 6 of 93 (6%) contacts tested positive in nasal secretion specimens. Two of the six PCR-positive (nasal secretions) contacts tested positive in both their pretreatment and 1-month post-treatment testing, but both were negative at their 2-month post-treatment testing.

After 2 months of MDT of index cases, nasal secretion and skin washing specimens from 26 contacts plus the 7 contacts that had tested positive at 1 month post-treatment were examined. PCR results were negative for all 33 contacts tested.

DISCUSSION

In an innovative study of the skin of leprosy patients, Pedley12 concluded that the number of M. leprae discharged from intact skin of lepromatous patients was negligible and, therefore, nasal secretions were the major source of infection. This study used a technique referred to as composite skin contact smears, in which a glass slide was pressed repeatedly against the skin lesion of lepromatous patients and, after staining, examined microscopically for AFB. From 34 slides prepared from 11 patients, only 20 AFB were observed, leading to the conclusion that few bacilli are discharged from the intact skin of a lepromatous patient. More recently, Hosokawa13 reported a histopathology study of the skin of lepromatous leprosy and concluded that, whereas viable bacilli could be excreted from non-ulcerating skin lesions, the possibility seemed to be small. These studies coupled with recent PCR studies6,7,14 monitoring M. leprae in nasal secretions of contacts have reinforced the hypothesis that leprosy transmission is primarily through the nasal route. We undertook this study to compare the potential for M. leprae excretion in nasal secretions and from the skin of MB patients with an alternative procedure for sampling skin. We also compared the exposure of skin and nasal mucosa to M. leprae in contacts of untreated MB patients in an attempt to establish the degree and duration of exposure to M. leprae after implementation of MDT in the index cases.

Sites selected for sampling the skin of MB index cases were chosen with the idea that the upper arms and the back would provide a general measure of skin-associated M. leprae in disseminated leprosy and be less likely to be contaminated with M. leprae from exogenous sources through contact with the external environment. In contrast, household contacts were monitored for exposure to M. leprae by sampling exposed areas of the skin. Accordingly, contacts were sampled by preparing washings obtained from the area of each arm between the elbow and the wrist.

Our results showing AFB and PCR detection of M. leprae DNA in 80% of skin washings and 60% of nasal secretions from untreated MB leprosy cases suggests that both anatomic sites serve as potential sources for transmission of leprosy. These results support earlier findings regarding the presence of M. leprae in nasal secretions of both index cases and their contacts. In addition, our results extend the concept of exposure of contacts through the skin and argue against earlier assertions that the number of M. leprae discharged from intact skin of MB patients is negligible. Although our study tested neither the viability nor the absolute number of M. leprae in the skin washings, it has been documented numerous times that viable organisms can be recovered from skin biopsies and expanded in rodent foot pads routinely.1517 To our knowledge similar viability studies have not been reported using M. leprae harvested from nasal secretions. However, a granulomatous lesion in the nasal mucosa of some leprosy patients is not an uncommon clinical finding and is likely the site of nasal dispersion of viable M. leprae.

Earlier studies have shown that PCR positivity associated with M. leprae from skin biopsies from patients receiving successful anti-leprosy therapy was reduced significantly as early as 2 months after initiating therapy.11,18 Similarly, in our study, successful treatment of MB patients for 1 year showed reductions in PCR reactivity from both sites sampled, suggesting that neither nasal secretions nor skin are likely portals of exit for M. leprae from patients receiving appropriate therapy. Earlier time points, ranging from 1 to 3 months, showed minor reductions in PCR positivity rates from both skin washings and nasal secretions. Because viability of M. leprae from the skin drops precipitously after three pulses of rifampin19 or six doses of MDT,20 it is likely that the positivity recorded by PCR in the specimens, taken during the first few months after initiation of treatment, is caused by residual undegraded DNA from dead M. leprae and not viable M. leprae. It has been shown repeatedly that macrophages in MB leprosy patients are not able to clear M. leprae efficiently from lesions because dead organisms and their fragments persist for a period even after treatment is completed.

The data also showed that M. leprae was present in a small percentage of samples taken from both nasal mucosa (4%) and skin (17%) of contacts of untreated MB leprosy cases. The presence of M. leprae from skin washings, which we define as skin exposure, was approximately four times greater than nasal mucosa exposure. Although the PCR positivity rates of the two anatomic sites were no different when compared statistically, the finding supports a role for skin and nasal exposure in transmission of leprosy. Further supporting the role of skin in transmission are reports by Sehgal and others2123 and Abraham and others,9 documenting cases of leprosy that developed in the skin at sites of trauma.

Treatment of index cases with MDT for 1 month greatly reduced skin exposure in contacts but had little effect on the low level of exposure through the nose as judged by PCR positivity of nasal secretions. This finding could be interpreted to suggest the establishment of a nasal carriage state in these contacts as has been proposed earlier.7 However, only two of the six positive nasal secretion specimens tested positive at two time points and all six contact’s specimens were negative on the third testing, which coincided with 2 months of treatment of the index cases.

Our results add to the basic understanding of how M. leprae is shed from index cases and further defines the risk of exposure to contacts. Both nasal secretions and skin from untreated MB cases of leprosy are capable of shedding M. leprae to the environment, which may be deposited on either or both the nasal and skin epithelia of contacts with the potential for initiating infection. What remains unclear is whether one or both sites are critical in establishing a productive infection. It is likely that under the appropriate circumstances either site is capable of sustaining an initial infection, but definitive evidence of primary infection will require better immunologic tests for monitoring early infection with M. leprae.

Table 1

Acid-fast bacilli and M. leprae DNA from skin and nasal washings from multibacillary leprosy patients before and after treatment

Skin washingsNasal washings
Direct smearPCRDirect smearPCR
Treatment+ve−ve+ve−ve+ve−ve+ve−ve
* Samples taken after 1 month of treatment with MDT.
† Samples taken after 2 months of treatment with MDT.
‡ Samples taken after 3 months of treatment with MDT.
None91827364
MDT 1*73825537
MDT 2†73556428
MDT 3‡64554655
Table 2

Acid-fast bacilli and M. leprae DNA in skin and nasal washes from multibacillary patients treated for 1 year

Skin washingsNasal washings
Direct smearPCRDirect smearPCR
Treatment period+ve−ve+ve−ve+ve−ve+ve−ve
ND, not done.
One year19191919
Two years010NDND01019
Table 3

Acid-fast bacilli and M. leprae DNA in skin and nasal washes from household contacts of untreated multibacillary patients

Skin washings (PCR)Nasal washings (PCR)
Study+ve− ve+ve−ve
* After index case had been treated with MDT for 1 month.
† After index case had been treated with MDT for 2 months.
First1677489
Second*192687
Third†033033
Figure 1.
Figure 1.

Photomicrograph showing the epidermis of skin of a lepromatous leprosy patient containing several acid-fast bacilli (arrows) in the keratin layer (Fite stain). This figure appears in color at www.ajtmh.org.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 78, 3; 10.4269/ajtmh.2008.78.518

*

Address correspondence to Thomas P. Gillis, Laboratory Research Branch, National Hansen’s Disease Programs at LSU School of Veterinary Medicine, Skip Bertman Drive, Baton Rouge, LA 70893. E-mail: tgillis@lsu.edu

Authors’ addresses: Charles K. Job, St. Thomas Hospital and Leprosy Centre, Chettupattu 606801, Tiruvannamalai District, Tamil Nadu, India, Telephone: 0416-221055, Fax: 4181-252366. Joseph Jayakumar, St Thomas Hospital and Leprosy Centre, Chettupattu 606801, Tiruvannamalai District, Tamil Nadu, India, Telephone: 4181-252365, Fax: 4181-252366. Michael Kearney, Department of Pathobiology, LSU School of Veterinary Medicine, Skip Bertman Drive, Baton Rouge, LA 70893, Telephone: 225-578-9667, Fax: 225-578-9665. Thomas P. Gillis, Laboratory Research Branch, National Hansens ’ Disease Programs at LSU School of Veterinary Medicine, Skip Bertman Drive, Baton Rouge, LA 70893, Telephone: 225-578-9836, Fax: 225-578-9856.

Acknowledgments: The authors acknowledge the technical assistance of Naoko Robbins and Cheryl Lewis and David M. Scollard for the photograph used in Figure 1.

Financial support: This study was supported in part by the American Leprosy Missions, Greenville, SC.

REFERENCES

  • 1

    Fine P, 2007. Leprosy: what is being “eliminated.” Bull World Health Organ 85 :2–4.

  • 2

    Meima A, Richardus JH, Habbema JD, 2004. Trends in leprosy case detection worldwide since 1985. Lepr Rev 75 :19–33.

  • 3

    Anonymous, 2006. Global leprosy situation, 2006. Wkly Epidemiol Rec 81 :309–316.

  • 4

    Job CK, Chehl S, Hastings RC, 1990. New findings on the mode of entry of M. leprae in nude mice. Int J Lepr 58 :726–729.

  • 5

    Chan PC, Reyes LM, Demerre-Lopez B, Gonzaga EM, De los Santos MFA, Gillis TP, 1997. Leprosy infection and disease in the national capital region. Philippine J Microbiol Infect Dis 26 :159–162.

    • Search Google Scholar
    • Export Citation
  • 6

    Klatser PR, van Beers S, Madjid B, Day B, de Wit MY, 1993. Detection of Mycobacterium leprae nasal carriers in populations for which leprosy is endemic. J Clin Microbiol 31 :2947–2951.

    • Search Google Scholar
    • Export Citation
  • 7

    Smith WC, Smith CM, Cree IA, Jadhav RS, Macdonald M, Edward VK, Oskam L, van Beers S, Klatser P, 2004. An approach to understanding the transmission of Mycobacterium leprae using molecular and immunological methods: results from the MILEP2 study. Int J Lepr Other Mycobact Dis 72 :269–277.

    • Search Google Scholar
    • Export Citation
  • 8

    Chatterjee BR, 1976. Carrier state in leprosy. Lepr India 48 :643–644.

  • 9

    Abraham S, Mozhi NM, Joseph GA, Kurian N, Rao PS, Job CK, 1998. Epidemiological significance of first skin lesion in leprosy. Int J Lepr Other Mycobact Dis 66 :131–139.

    • Search Google Scholar
    • Export Citation
  • 10

    Williams DL, Gillis TP, Booth RJ, Looker D, Watson JD, 1990. The use of a specific DNA probe and polymerase chain reaction for the detection of Mycobacterium leprae. J Infect Dis 162 :193–200.

    • Search Google Scholar
    • Export Citation
  • 11

    Williams DL, Gillis TP, Fiallo P, Job CK, Gelber RH, Hill C, Izumi S, 1992. Detection of Mycobacterium leprae and the potential for monitoring antileprosy drug therapy directly from skin biopsies by PCR. Mol Cell Probes 6 :401–410.

    • Search Google Scholar
    • Export Citation
  • 12

    Pedley JC, 1970. Summary of the results of a search of the skin surface for Mycobacterium leprae. Lepr Rev 41 :167–168.

  • 13

    Hosokawa A, 1999. A clinical and bacteriological examination of Mycobacterium leprae in the epidermis and cutaneous appendages of patients with multibacillary leprosy. J Dermatol 26 :479–488.

    • Search Google Scholar
    • Export Citation
  • 14

    Hatta M, Van Beers SM, Madjid B, Djumadi A, de Wit MY, Klatser PR, 1995. Distribution and persistence of Mycobacterium leprae nasal carriage among a population in which leprosy is endemic in Indonesia. Trans R Soc Trop Med Hyg 89 :381–385.

    • Search Google Scholar
    • Export Citation
  • 15

    Gelber RH, Humphres RC, Fieldsteel AH, 1986. Superiority of the neonatally thymectomized Lewis rat (NTLR) to monitor a clinical trial in lepromatous leprosy of the two regimens of rifampin and dapsone. Int J Lepr Other Mycobact Dis 54 :273–283.

    • Search Google Scholar
    • Export Citation
  • 16

    Fieldsteel AH, Levy L, 1976. Neonatally thymectomized Lewis rats infected with Mycobacterium leprae: response to primary infection, secondary challenge, and large inocula. Infect Immun 14 :736–741.

    • Search Google Scholar
    • Export Citation
  • 17

    Wakade AV, Shetty VP, 2006. Isolation of Mycobacterium leprae from untreated borderline tuberculoid, mid-borderline and indeterminate cases using the mouse foot pad technique—a study of 209 cases. Lepr Rev 77 :366–370.

    • Search Google Scholar
    • Export Citation
  • 18

    Franzblau SG, Chan GP, Garcia-Ignacio BG, Chavez VE, Livelo JB, Jimenez CL, Parrilla ML, Calvo RF, Williams DL, Gillis T, 1994. Clinical trial of fusidic acid for lepromatous leprosy. Antimicrob Agents Chemother 38 :1651–1654.

    • Search Google Scholar
    • Export Citation
  • 19

    Levy L, Shepard CC, Fasal P, 1976. The bactericidal effect of rifampicin on M. leprae in man: a) single doses of 600, 900 and 1200 mg; and b) daily doses of 300 mg. Int J Lepr Other Mycobact Dis 44 :183–187.

    • Search Google Scholar
    • Export Citation
  • 20

    Anonymous, 1997. Action Program for the Elimination of Leprosy: Status Report. Geneva: World Health Organization.

  • 21

    Sehgal VN, Rege VL, Vadiraj SN, 1970. Inoculation leprosy subsequent to small-pox vaccination. Dermatologica 141 :393–396.

  • 22

    Sehgal VN, 1971. Inoculation leprosy appearing after seven years of tattooing. Dermatologica 142 :58–61.

  • 23

    Seghal VN, 1986. Leprosy following mechanical trauma. Lepr Rev 57 :272.

Author Notes

Reprint requests: Thomas P. Gillis, Laboratory Research Branch, National Hansen’s Disease Programs at LSU School of Veterinary Medicine, Skip Bertman Drive, Baton Rouge, LA 70893.
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