Ziehl-Neelsen or Fite-Faraco staining

Leprosy Mailing List – October 13th, 2011

Ref.:    Ziehl-Neelsen or Fite-Faraco staining

From:  Clapasson A., Genoa, Italy

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Dear Salvatore,

Thank you very much to Dr Barreto for his important message (LML Oct. 2^nd, 2011).  I agree with his conclusions and, I would like adding my comments.

The most important factors, to have a good Ziehl-Neelsen’s or Fite-Faraco’s staining, is the concentration of the primary dye.  The staining technique is simple but, there are some pitfalls that should not be underestimated.  There are two important factors that each laboratory technician should value remember; they are:

1. The concentration of carbol-fuchsin and phenol: About this please see the attached paper: “Comparison of variants of carbol-fuchsin solution in Ziehl-Neelsen for detection of acid-fast bacilli”. Selvakumar N, Sekar MG, Rahman F, Syamsunder A, Duraipandian M, Wares F, Narayanan PR. Int J Tuberc Lung Dis. 2005 Feb;9(2):226-9.

  2. The duration of the discoloration:  False negatives increase with excessive time of decoloration.  In Genoa I decolorize each slide separately with 2,5 ml of solution of hydrochloric acid (95ml ethanol 96° and 1ml hydrochloric acid 37% (fuming)), but if it is difficult the acquisition of “alcohol absolute” it is possible to use an aqueous solution of 23,75% sulphuric acid and 3% alcohol.

Best regards,

Andrea

Choosing the decolourizer and its strength to stain Mycobacterium leprae

Leprosy Mailing List – October 10th, 2011

Ref.: Choosing the decolourizer and its strength to stain Mycobacterium leprae. Does it actually matter?

Lepr Rev. 1998 Jun;69 (2):128-33 (see attachments Page 1  2  3  4  5  6)

From:  R de Soldenhoff, Edinburgh, Scotland, UK

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Dear Dr Noto,
 

It may be of interest to LML readers to review the attached article of an operational research study carried out in Indonesia.  The original in Leprosy Review was published prior to all their articles being in electronic form, so my apologies for the mediocre quality scan of the article.
 The reason for the study was the premise that "tuberculosis diagnosing centres with an established system of quality control, can also be responsible for leprosy microscopy. This is easier if the staining technique for both organisms is identical."
 I would be very pleased if another laboratory could repeat the study, in order to confirm these findings.  In addition, a comparison between the "hot method" (usually used in field laboratories for tuberculosis) and the "cold method" and between methylene blue and malachite green (occasionally used as an alternative decolouriser), would also be of interest.
 With kind regards,
Dr. Richard de Soldenhoff

42 Drummond Place

Edinburgh, EH3 6NR

Scotland, UK

Home telephone: 44.131.556.7979

UK mobile: 44.777.0751578

Email: rdesoldenhoff(at)hotmail.com

Is 0.6% fucsin reagent significantly less effective?

Leprosy Mailing List – October 10th, 2011

Ref.:    Is 0.6% fucsin reagent significantly less effective?

From:  L Gilead, Jerusalem, Israel

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Dear Dr. Noto,

Thank you very much to Dr Barreto for his comments (LML Oct. 2^nd, 2011) concerning aspects of skin smears examination for leprosy and Ziehl-Neelsen (ZN) staining for acid-fast bacilli (AFB's).  Following that message we re-evaluated our lab reagents and found out that the concentration of the Ziehl's fucsin reagent we are using (commercially acquired from MERK is 0.6%.

I would appreciate it if any one of the LML members could let us know if this is significantly less effective and we need to prepare our own reagent in the lab with a 1% concentration, or whether any one has a different commercial supplier that can deliver a 1% reagent?

Thank you very much and a Happy New Hebrew year from Jerusalem.

Leon

Dr. Leon Gilead

Head of The Israeli Hansen's Disease Center

24Strauss St., Jerusalem, Israel

Department of Dermatogoy

Hadasssah University Hospital

Mobile: 972-50-7874309

BI and MI results in BB, BL and LL leprosy

Leprosy Mailing List – October 4^th, 2011

Ref.:    BI and MI results in BB, BL and LL leprosy

From:  L Duncan, Peebles, Scotland, UK

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Thanks Salvatore,

I would like commenting on Dr Verduga and Barreto’s letters dated respectively LML 27^th Sept. and 2^nd Oct. 2011.  About Dr Verduga’s clinical cases (see attachment) I would agree that the last 2 slides (cases 4 and 5) should have been with a bacteriological index of 5+ and with a positive morphological index (MI) as well.  The mid borderline (BB) ones are often more difficult to get as positives but again should have been positive with a positive MI.

Dr Barreto’s letter commenting on the methodology for taking the smears is well made ... as was the second point about the strength of alcohol-acid to decolourise ...

I am sure that you will find in many countries the over-simplification of methodology to diagnose and treat leprosy, together with inadequacy of follow up - so many people with leprosy are told 'Here is your treatment, 'MDT Cure' and you do not need to come back to see us for check-up'' has added to the present apparent resurgence of leprosy in many countries.

The failure to teach about the risk of pregnancy for the woman with leprosy and the need to check her during and after delivery, and to follow up for at least a year after delivery also has to be addressed.

I recently saw one such woman who did all that she was told, was not supervised after delivery and was bedridden with severe reaction and neuritis for a year after delivery.  By the grace of God she was seen unofficially / accidentally (!) by someone who recognised the problem and gave her appropriate treatment and advice for the babe.

Lysbeth Duncan

lists of suitable labs for slit-skin smear examination in leprosy

Leprosy Mailing List – October 3^rd, 2011

Ref.:    It would be advisable to provide, lists of suitable labs for slit-skin smear examination in leprosy.From:  Warren G, Sidney, Australia

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Dear Dr Noto,

I am very pleased to read the comments on slit skin smears made by Dr Barreto (LML Oct. 2^nd 2011).  Many times I have found that smears were reported as negative and when rechecked it was found that the stain was not correctly done.

Yes, M. leprae is difficult to stain and easily over decolourised by the use of the wrong acid-alcohol mixture.  This is particularly a problem when slides are sent for acid fast bacilli (AFB) examination to a good lab that does not do M. leprae as a routine.  So I must confess that if I diagnose leprosy and feel the patients ought to have a positive smear I often also send a suitable slide to an experienced lab to be sure, if it is said to be negative by the first lab.

I have had some very embarrassing experiences with laboratory personnel, whom one would think would be reliable, giving negative diagnosis on a patient who is clinically clearly positive.  It is even more embarrassing when pathologists cannot diagnose from a fairly typical biopsy as happened with one patient with a definite red line (typical of BB) where the anaesthesia started – and when we looked at the stained biopsy a relatively inexperienced pathologist spotted the AFB at once but the Professor at the first institute had not seen them and had stated emphatically it was not leprosy.

It is important that we hone our ability to diagnose so that patients with leprosy are not deprived of MDT and the possibility of recovery without deformity.  I have seen many “primary persistent neuritic leprosy” patients who have early ulnar nerve lesions (clawing of fingers) but NO skin lesions and are dismissed by WHO personnel as “not leprosy” because no anaesthetic skin lesion.  Yet in S E Asia these are common and a biopsy of the nerve will prove AFB etc. present - but many places do not have those facilities.  In fact many clinics in third world countries have no facilities to do slit skin smears and may even have no suitable place to send slides on for examination.  We must hone our ability to diagnose by clinical signs and history.

Would it be advisable to provide, lists of the suitable labs who would check slit skin smears and/or biopsies for those who need it and may be make sure all the labs have full detailed instructions of the correct methods and strengths of stains to get reliable results.

Grace Warren  

Previously adviser for the Leprosy Mission in Asia.

Leprosy in Ecuador and slit-skin smear examination

Leprosy Mailing List – October 2^nd, 2011

Ref.:   Leprosy in Ecuador and slit-skin smear examinationFrom: J A Barreto, Bauru, SP, Brazil

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Dear Drs Noto and Verduga,

Thank you very much to Dr Verduga for sharing with us these very interesting cases (see the attachment to LML Sept. 27^th, 2011).  Here in Brazil, where leprosy is highly endemic we also see similar cases quite commonly.  I would like do some comments, particularly about the skin smear technique.

The bacteriological index (BI) in lepromatous (LL) leprosy is usually 5+-6+, mainly when the smears are collected from nodules.  According to the Ridley & Jopling classification the BI ranges in a logarithmic scale from 0 to 6+, from tuberculoid (TT) to the LL forms of the disease.  Nevertheless, BI in skin smears commonly are 1+ lower than in biopsy bacteriological index (BBI).

I refer to your case number 5; LL leprosy with BI 3+ [you do not give information about the morphological index (MI)].  I have three points to comment about and, they are in relation with the apparently low BI (3+):-

1. How the smear was collected?  Was the dermis scraped after incision of the epidermis?  In Brazil, many physicians do not know that this must be done, becauseM. leprae is an intracellular parasite, and does not "flow in the lymph".

2. What was the concentration of Ziehl's fucsin: 1% or 0.3%?  In Brazil, I found that most laboratory technicians think that this is not important, what is wrong, because the resistance to distaining of the cell wall of M. leprae is weaker than M. tuberculosis, and therefore the use of the lower concentration (0.3%) of Ziehl's fucsin can lead to false negative results in cases of borderline leprosy, mainly when smears are not collected from lesions.

3. What was the concentration of alcohol-acid used to distaining the smear?  As well as the problem I have found above, and for the same reason, many laboratory technicians also do not know that the concentration must be lower, i.e., 1%, or this will lead to false negative results, even in lepromatous cases.

For many years, unfortunately, bacilloscopy, an important tool for the diagnosis, classification and follow up of leprosy was neglected by many leprosy control programmes in the world, and now many health professionals just don`t have knowledge about it.  Many even are afraid to become infected when collecting smears.

I hope that these comments be useful and I will be happy to know what other labororatories/programmes do.

Best regards,

Jaison A. Barreto

Dermatologist and Leprologist

Instituto Lauro de Souza Lima

Bauru, SP

The value of Polymerase Chain Reaction (PCR) in the diagnosis of leprosy

Leprosy Mailing List – April 30^th, 2011 

Ref:     The value of Polymerase Chain Reaction (PCR) in the diagnosis of leprosy
From:  Tom Gillis and David Scollard, Baton Rouge, LA, USA

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Dear Salvatore,

In response to the questions about the usefulness of PCR in the diagnosis of PB leprosy: 

Our experience with PCR for detecting M. leprae DNA in human skin biopsies has not changed appreciably since our publication (Scollard et al., Microbiol and Inf Dis 109, 1998) summarizing PCR results and patient diagnoses at the National Hansen’s Disease Programs in Baton Rouge, LA.  We found and continue to find that biopsies from lepromatous (LL) cases result in approximately 90% positivity rates and biopsies from tuberculoid (TT) cases range from 10-25% positive.  TT and indeterminate cases, where no acid-fast bacilli are seen by microscopy, are almost uniformly negative.  Accordingly, our emphasis remains on testing biopsies where AFB are seen but the diagnosis is not conclusive by clinical and histopathological assessment.  We should add that specificity for our tests as well as most tests in the literature are very close to 100%.

While gene target and primer selection are very important, we find that an often overlooked aspect is sample preparation for DNA.  Many reports characterize specificity and sensitivity using purified M. leprae DNA or bacteria from various biological sources (lepromin, mouse, armadillo, human skin biopsy).  Using defined reagents is an important aspect of assay characterization but final specificity and sensitivity for clinical applications of a given PCR test must be determined using patient materials.  Clinical materials can be unfixed biopsies, biopsies fixed in buffered formalin, embedded in paraffin or fixed in 70% ethanol.  Each of these fixed preparations can alter DNA and subsequent extractions are critical for optimal results.  Unfortunately, a well characterized, commercial test for M. leprae DNA in skin biopsies is not available and so many labs continue to report results using their own definitions of sensitivity and specificity and in most cases the results are not comparable across different clinical applications.

To understand why these technical issues are so important, consider that in some instances PCR may be done on specimens from patients classified as “paucibacillary” (PB) by counting lesions, without any support from skin biopsy or skin smear results.  It is well documented that a small percentage of patients classified as “PB” in this manner are actually mid borderline (BB) or borderline lepromatous (BL).  These would be expected to have a higher likelihood of ‘positive’ PCR results than “true PB” (borderline tuberculoid [BT] or TT) patients, but without a biopsy or skin smears they would be considered “PCR + PB”.  

Our recommendation is to link up with a laboratory that has experience in this area and has published results with clinical materials associated with sound clinical assessment of cases.  This would include experienced clinicians and a pathologist who, together, can classify patients using the Ridley-Jopling system.

Tom Gillis, Ph.D.

Chief, Laboratory Research Branch

National Hansen's Disease Programs

Baton Rouge, LA

e-mail:  Tgillis(at)hrsa.gov

David Scollard, M.D., Ph.D.

Chief, Clinical Branch

National Hansen’s Disease Programs

Baton Rouge, LA.

E-mail:  dscollard(at)hrsa.gov

Web:  www.hrsa.gov/hansens 

Skin biopsy from:	PCR positivity rates:
Lepromatous leprosy (LL)	approximately 90%
Tuberculoid leprosy (TT)	10-25%

Polymerase chain reaction in the diagnosis of paucibacillary leprosy in Colombia

Leprosy Mailing List – April 16^th, 2011 

Ref:     Polymerase chain reaction in the diagnosis of paucibacillary leprosy in Colombia
From: Cadorna-Castro N., Medellín, Colombia

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 Dear Dr. Noto,

 We have few but interesting experiences with the use of polymerase chain reaction (PCR) to help in the diagnosis paucibacillary (PB) leprosy.  However as you comment there is not a definitive concept about this technique.  

With the RLEP PCR we have detected new patients at early stage of disease; we do active search among high risk population: household contacts.  In our experience we could confirm several difficult cases of PB leprosy using PCR (RLEP 1-2 and RLEP 3-4), i.e:

1. Boy, 5 years old with a hipo-pigmented skin lesion on the leg, bacteriological index (BI) negative, both parents with multibacillary (MB) leprosy.  The PCR from skin biopsy was positive.

2. Boy 17 years old; contact of leprosy patient.  One skin lesion on leg.  It was difficult to evaluate sensitivity on the lesion; we concluded that there was no evident loss of sensitivity.  The BI was negative.  PCR from skin biopsy was positive.

We can use PCR as a complementary diagnostic tool for this kind of cases.  Kindly, find in attachment the paper of Donoghue et al:-

“PCR primers that can detect low levels of Mycobacterium leprae DNA”. 

Donoghue HD, Holton J, Spigelman M.

J Med Microbiol. 2001 Feb;50(2):177-182”.

Best wishes,

Nora Cardona-Castro.

MD. MSc.

Instituto Colombiano de Medicina Tropical.

Universidad CES.

What is the value of polymerase chain reaction (PCR) in the diagnosis of leprosy?

Leprosy Mailing List – April 14^th, 2011

Ref:  What is the value of polymerase chain reaction (PCR) in the diagnosis of leprosy?
From: C. Smith, Aberdeen, U.K.

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 Dear Dr Noto,

The role of PCR in the diagnosis of leprosy is an important question and I have heard the question asked many time recently.  Over the past 20 years there has been a growing body of literature on PCR in leprosy – much of it on the development and validation of the methods, and various ad hoc studies have been published.  The possibility of including PCR in the diagnostic criteria for leprosy was recently debated but in the end it was agreed that there was not sufficient evidence – the presence of M. leprae by PCR alone was not sufficient evidence to diagnose leprosy as studies show contacts, and indeed members of the community, can be positive by PCR without having any other evidence of disease or going on to develop the disease.   The same is the case with other infections.  But it is clear that PCR can have value in certain circumstances and there are a number of useful studies recently, if not definitive, certainly important.

The JALMA group (1) in India have evaluated the use of PCR from slit skin smears in children where skin biopsies were not possible – they found 13 PCR positives in 20 smear negative children.  In Brazil (2) a study evaluated PCR results from buccal swabs in large numbers of patients (18% positive) and contacts (7% positive), they also looked at anti PGL1 status.  A further recent study in India (3) used PCR to assess over 400 contacts and followed them for 2 years where 2 PCR positive contacts developed disease. 

With the growing evidence, there is a case for a review of the literature to determine the role of PCR in the diagnosis of leprosy and the interpretation of a positive PCR in people with no other signs of disease.

Best regards,

Cairns Smith

1.   Kamal R, Natrajan M, Katoch K, Katoch VM.  Evaluation of diagnostic role of in situ PCR on slit-skin smears in pediatric leprosy. Indian J Lepr. 2010 Oct-Dec;82(4):195-200.

2.   Martinez TS, Figueira MM, Costa AV, Gonçalves MA, Goulart LR, Goulart IM  Oral mucosa as a source of Mycobacterium leprae infection and transmission, and implications of bacterial DNA detection and the immunological status. Clin Microbiol Infect. 2010 Dec 31. doi: 10.1111/j.1469-0691.2010.03453.x. [Epub ahead of print]

3.   Banerjee S, Sarkar K, Gupta S, Mahapatra PS, Gupta S, Guha S, Bandhopadhayay D,Ghosal C, Paine SK, Dutta RN, Biswas N, Bhattacharya B. Multiplex PCR technique could be an alternative approach for early detection of leprosy among close contacts--a pilot study from India. BMC Infect Dis. 2010 Aug 24;10:252

What is the value of polymerase chain reaction (PCR) in the diagnosis of leprosy?

Leprosy Mailing List –  April 14^th, 2011

Ref.  What is the value of polymerase chain reaction (PCR) in the diagnosis of leprosy?

From: A. Clapasson, Genoa, Italy

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 Dear Salvatore,

Thank you very much to Drs Antoine Mahé and Elsa Rada for introducing the above mentioned topic (LML 9 and 11 April 20011).  I would like to point out the following references:

“PCR primers that can detect low levels of Mycobacterium leprae DNA.  Donoghue HD, Holton J, Spigelman M. J Med Microbiol.  2001 Feb;50(2):177-82”.  In this article you find the answers needed.  In my opinion, the paper explains the best PCR technique for the detection of DNA of M. leprae.

Another interesting article is: “Comparison of two different PCR amplification products (the 18-kDa protein gene vs. RLEP repetitive sequence) in the diagnosis of Mycobacterium leprae.  T.-J. Kang, S.-K. Kim, S.-B. Lee, G.-T.  Chae and J.-P. Kim. Clinical and Experimental Dermatology, 2003: 28(4), 420–424”.

PCR has many critics.  However, never forget that the laboratory technician cannotproduce a useful result if the clinician does not provide the appropriate sample for analysis and the right conditions.  You need a suitable biological sample to have a reliable result.

The diagnosis of leprosy can be a puzzle that is assembled only after putting all the data together from both clinical and laboratory examinations.  Relying on a single test is very risky.

Andrea Clapasson

What is the value of Polymerase Chain Reaction (PCR) in the diagnosis of leprosy?

Leprosy Mailing List – April 13^th, 2011

Ref:     What is the value of Polymerase Chain Reaction (PCR) in the diagnosis of leprosy?
From:  Naafs B., Munnekeburen, The Netherlands

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Dear Dr Rada,

I refer to your message dated 11 April 2011 about the above mentioned subject.  As you expect, there are no reliable convincing reports about the diagnosis of active paucibacillary (PB) leprosy with PCR?

With kind regards

Ben Naafs 

What is the value of Polymerase Chain Reaction (PCR) in the diagnosis of leprosy?

Leprosy Mailing List – April 11^th, 2011

Ref:     What is the value of Polymerase Chain Reaction (PCR) in the diagnosis of leprosy? From:  Elsa Rada, Caracas, Venezuela

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Dear Dr Noto,

Thank you very much to Dr Antoine Mahé for his message (LML April 9^th, 2011).  I would also be very interested in knowing if there is any work reporting convincing data about the diagnose of active paucibacillary (PB) patients with PCR?

Yours sincerely,

Dr Elsa Rada (Ms)

Instituto de Biomedicina

Apartado 4043 1010A

Caracas, Venezuela

What is the value of PCR in the diagnosis of leprosy?

Leprosy Mailing List – April 9^th, 2011

Ref:    What is the value of PCR in the diagnosis of leprosy?

From:  Antoine Mahé, Colmar, France

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Dear Dr Noto,

I repeatedly wonder about the value of PCR (on skin lesions, nasal mucus,...) in order to diagnosis difficult cases of leprosy (i.e., mainly PB cases).  I did not find any convincing data about the use of this kind of test.

Would someone from the Leprosy mailing list give me definitive focus about this topic; e.g., sensitivity and specificity of PCR in various variants of leprosy?

Kind regards,
 

Dr Antoine Mahé
Dermatologie
Hôpital Pasteur
39, avenue de la Liberté
68024 Colmar Cedex
Secrétariat (+33) 03 89 12 44 65 / 41 58

Fax (+33) 03 89 12 47 69

Portable (+33) 06 31 27 68 71

Laboratory diagnostic tools for leprosy

Leprosy Mailing List –  March 15th, 2011

Ref:    Laboratory diagnostic tools for leprosy. 

From: Das P K, Amsterdam, The Netherlands

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Dear Leon Gilead,

Thank you for your message dated LML March 8^th, 2011.

Lepromin reagents are still produced by Dr. Esther at the Institute Lauro Souza Lima, inBauru , Brazil .  Among the laboratory tests still the IgM titre to PGL-1 (high bacilli laden forms of leprosy only) is the golden standard. 

In addition, IgG and IgM to gel purified 29/33 KDa doublet and IgG and IgM titre to gel purified 65KDa antigen will diagnose LL /BL and TT/BT cases respectively (kindly, see the publication J. Clin Microbiol by Das PK et al 1990). 

One can, therefore also perform ELISA using gel purified 29Kd/33Kd doublet and 64-65 KD singlet antigens, in parallel to lepromin test and IgM -PGL-1 by ELISA.  These antigens are prepared easily by first electrophoresing the BCG homoheganates in reducing SDS gel;followed by cutting out the bands of 29/33KD doublet and /^4-65 KD singlet and then eluted with distilled water.  By combining the ELISA measurement of IG/IGM titre with 29/33KD doublet and 64-65KD singlet with the western blotting pattern showing 29/33KD doublet bands and strong band of 64-65 singlet band will confirm lepromatous side (LL/BL) and diagnose BT/TT of the spectrum respectively.  In this latter test, true BB cannot be diagnosed.

Further, serum cytokine and chemokine profiling is advisable for monitoring the patients (see the publications by de Silva E et al, Lyer et al etc.).  Recently IDEAL group strongly advise cell based assay like that of quantiferon for TB also for leprosy using post genomic antigens.

A combination of serum anti-PGL-1 Ab test positive plus lepromin skin test negative will be consistent with diagnosis of leprosy on the lepromatous side of the spectrum.  While a combination of serum anti-PGL-1 Ab test negative plus lepromin skin test positive will be consistent with diagnosis of leprosy at the tuberculoid side of the spectrum.  So far this is the best laboratory diagnostic tool that we have got.  It matches with the clinical diagnosis and is robust enough to diagnose >90% of the patients.

Hope that this information is useful.

Pranab

Table. Laboratory diagnostic tools in leprosy: anti-PGL-1 Ab and lepromin tests
serum anti-PGL-1 Ab test positive	+	lepromin skin test negative	consistent with diagnosis of	leprosy at the lepromatous side of the spectrum
serum anti-PGL-1 Ab test negative	+	lepromin skin test positive	consistent with diagnosis of	leprosy at the tuberculoid side of the spectrum

Clinical indications for PCR and serum anti phenolic glycolipid (PGL) -1 Ab test?

Leprosy Mailing List –  March 13^th, 2011

Ref:   Clinical indications for PCR and serum anti phenolic glycolipid (PGL) -1 Ab test?

From:  Burdick A E., Miami , Florida , USA

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Dear Dr. Ishii,

Thank you very much for your message about laboratory tests in leprosy in Japan dated LML March 12^th 2011.  I am interested to learn the clinical situations when you use PCR and the serum anti-PGL-1 Ab test?  At the Miami Hansen's Disease Program, we use slit-smear examination and rarely PCR; never lepromin.

I am presenting the lecture "Leprosy in 2011" at the American Academy of Dermatology Summer 2011 meeting and I would like to include information on the use of antibody testing in special clinical cases.

Regards,

Anne E. Burdick, MD, MPH
Medical Director, University of Miami/Jackson Memorial Hospital Hansen's Disease Program
Professor of Dermatology
Associate Dean for TeleHealth and Clinical Outreach
University of Miami Miller School of Medicine
1150 NW 14th Street , Room 206 (R-350)
Miami , Florida 33136 , USA

Laboratory tests in leprosy in Japan

Leprosy Mailing List –  March 12^th, 2011

Ref:    Laboratory tests in leprosy in Japan

From: Ishii N, Tokyo, Japan

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Dear Dr Noto;

I refer to Dr Gilead’s message dated LML March 8^th, 2011.  In Japan , we do not use Lepromin reagent.  We do not produce it.  We use slit-skin smear examination, polymerase chain reaction (PCR), and sometimes serum anti-PGL-1 Ab test.

We have only few new leprosy patients (2009: 2 foreigners,  2010: 4 foreigners).  Laboratory test is covered at Leprosy Research Center NIID, Tokyo .

Best Regards,

Norihisa ISHII　MD, PhD
Director-General, Leprosy Research Center,
National Institute of Infectious Diseases (NIID)
4-2-1 Aobacho, Higashimurayama,

Tokyo 189-0002 JAPAN

Does any one still use Lepromin for skin testing?

Leprosy Mailing List –  March 8^th, 2011

Ref:    Does any one still use Lepromin for skin testing?  

From: Gilead L., Jerusalem, Israel

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Dear Dr Noto,

I would like to ask the participants of the leprosy mailing list (LML) regarding their use of the Lepromin reagent.  Does any one still use Lepromin for skin testing?  Where can a new batch of Lepromin be obtained nowadays, if at all, and what kind of other skin/blood/lab testing do LML participants use in addition to clinical and skin smear testing in their usual clinical work?

Thank you very much in advance for your collaboration.

Your sincerely,

Leon Gilead, MD PhD

The Israeli Hansen's Disease Center

Reverse Transcription-PCR in biopsy specimens from leprosy cases

Leprosy Mailing List – October 17th, 2009

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

From: Clapasson A., Genoa , Italy

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Dear Dr Haroen,

Thank you very much for your message (LML Oct. 16^th 2009). I read it with interest. Your questions are reported in “black”.

“Is it because I used different primer that might cause the positive result from my research is very low (56,7%)?”

LP1 and LP2 are good primers for first step in research of RLEP and you find these repetitive sequence thirty-nine times. These primers have got interesting characteristics, but if your BAR is viable, its rRNA 16s is amplified more than thirty-nine times. Kurabachew uses primers for rRNA 16s.

“Or is there any other explanation that can cause this?”

Yes there is, are you sure about your extraction? This step is more important than PCR.

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

I ask you: what is the morphological index (MI) in the skin smears? What is the aspect of your BARs in the nasal swab? If there were “solids” we may assume they were viable.

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

The main portals of exit are the upper airways and ulcerated or abraded skin lesions.

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

It may survive with the right conditions of humidity up to 40 days.

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

Interesting question. I can tell you that it is a dogma; but I doubt when I see the big hyper-chromic globes in the nasal smears of untreated LL patients, that this dogma is always true (I suspect it is not!).

“But from 1 specimen that positive RNA from skin swab, the RNA biopsy was negative. Is that means that M. leprae that viable on the skin surface came from environment? Or is there any other explanation regarding this result?”

A very interesting paper is that of Dr Williams. She says that if there are a lot of T cells, these can inhibit your PCR. M. leprae does not live on the skin but into the skin.

My best regards,

Andrea

PS.

I apologize if I was imprecise but, in some points I have expressed my opinion.