Reverse Transcription-PCR in biopsy specimens from leprosy cases

Leprosy Mailing List – October 16th, 2009

Ref.: Reverse Transcription-PCR in biopsy specimens from leprosy cases.

From: Haroen M. S., Jakarta , Indonesia

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Dear Dr Noto,

I am a resident of dermatology in Indonesian University , Jakarta , Indonesia . I am doing a research about leprosy. I would be very grateful if you can give me some information/expanations.

1. RNA M. leprae from skin biopsies.

Biopsy specimens from 30 newly diagnosed, untreated, clinically MB patients were put in RNAlater® in 4⁰C for 1 night then -20⁰C until examined (in 3,5 months) with Reverse Transcription-PCR. All patients were positive for acid fast bacilli (all bacterial index were positive).

RNA extracted using RNeasy® Mini kit & RNeasy® fibrous tissue kit from QIAGEN®

RT-PCR was done using random primer ( to make cDNA)

First PCR was done with primer: LPF & LPR, nested PCR with primer: LP1 & LP2

The result: RNA M. leprae was detected only 17 (56,7%) specimens, the other 13 specimens were negative

What could cause this happened?

From Kurabachew M, et al [Reverse transcription-PCR detection of Mycobacterium lepraein clinical specimens. J Clin Microbiol 1998;36(5):1352-6] said RNA M. leprae was detected in 82% of skin biopsy specimens from untreated leprosy patients (96% from MB patients). Is it because I used different primer that might cause the positive result from my research is very low (56,7%)? Or is there any other explanation that can cause this?

2. RNA M. leprae from skin swabs.

Swab using cotton buds, that had been dipped into phosphate buffer saline, were swab on surface of intact skin lesions from 30 newly diagnosed, untreated, clinically MB patients. The cotton buds were put in RNAlater® in 4⁰C for 1 night then -20⁰C until examined (in 3,5 months) with Reverse Transcription-PCR. All patients were positive for acid fast bacilli (all bacterial index were positive).

RNA extracted using RNeasy® Mini kit & RNeasy® fibrous tissue kit from QIAGEN®

RT-PCR was done using random primer ( to make cDNA).

First PCR was done with primer: LPF & LPR, nested PCR with primer: LP1 & LP2

The result:

a. RNA M. leprae was detected 2 (6,7%) specimens, the other 28 specimens were negative.

Is this mean that M. leprae was viable on skin surface?

b. From 1 patient, RNA M. leprae from skin swab & skin biopsy was positive.

Is there any possibility that M. leprae from skin structure can come out to the surface of the skin lesion?

Is there any possibility that M. leprae can live outside the body (enviroment: water, soil, etc)?

So what is the meaning of M. leprae is an obligate intracellular?

c. But from 1 specimen that positive RNA from skin swab, the RNA biopsy was negative.

Is that mean that M. leprae that viable on the skin surface came from environment? Or is there any other explanation regarding this result?

I did not find other journal that ever did RT-PCR from swab of leprosy skin lesions. I did this research because I want to know the possibility intact leprosy skin lesions as a source of leprosy infection.

One of my colleague did PCR DNA from swab of intact leprosy skin lesions and found 82% positive and from Job CK, Jayakumar J, Kearney M, Gillis TP. [Transmission of Leprosy: A Study of Skin and Nasal Secretions of Household Contacts of Leprosy Patients Using PCR. Am J Trop Med Hyg 2008;78(3):518-21] found 80% of the patients had M. leprae DNA in skin washings.

Please, help me.

Mona

Dr. Mona Safira Haroen (Resident)

Departement Dermatology and Venereology

Faculty Medicine of University of Indonesia

Jl. Diponegoro no 71

Jakarta , Indonesia