[Leish-l] inquiry

Patrick Bastien patrick.bastien at univ-montp1.fr
Sat Jun 4 05:44:40 BRT 2011


Dear K.P.,
I did not know you even worked in Tukey !...

Just to correct one of your last messages: I did not say we have no  
problem in isolating L infantum. I wrote "that all L. infantum do NOT  
have difficulties in growing in vitro: (a) it depends upon the culture  
medium you are using; (b) it seems to depend upon the "taxon" (here  
zymodeme), dermotropic zymodemes being in general (but not always)  
more difficult to grow.". So: we indeed noted that some isolates are  
more difficult to grow; and we do experience failures in isolating L  
infantum.

As regards the medium used for isolating from sandflies, it was the  
simple and classical NNN medium (for 1 liter: NaCl 6 gr , BactoAgar 10  
gr; then blood 12%). One important point is the type of blood used.  
Horse blood works less well than rabbit blood, it seems. Human blood  
does not work. But I believe evrybody knows that.
Best wishes
P. Bastien

Professeur Patrick Bastien
Laboratoire de Parasitologie-Mycologie, Faculté de Médecine
UMR MIVEGEC (CNRS 5290 - IRD 224 - Université Montpellier 1)
National Reference Centre for Leishmania
CHU de Montpellier
39 Avenue Charles Flahault
34295 Montpellier Cedex 5, France

"Chang, Kwang-Poo" <KwangPoo.Chang at rosalindfranklin.edu> a écrit :

> Dr. Bastien,
>
> Could you kindly share with us the magic medium you used ?
>
> Anything is worth a try, although we have tried a variety of   
> different formulations of 3N.
>
> KP
>
> -----Original Message-----
> From: Patrick Bastien [mailto:patrick.bastien at univ-montp1.fr]
> Sent: Wednesday, June 01, 2011 7:39 AM
> To: Chang, Kwang-Poo
> Cc: Sharman; Petr Volf; Hiro Goto; elfadil abass;   
> vishwamohan_katoch at yahoo.co.in; lalit Kant ICMR;   
> leish-l at lineu.icb.usp.br
> Subject: Re: [Leish-l] inquiry
>
> Hi everybody !
> Just to let you know that all L. infantum do NOT have difficulties in
> growing in vitro: (a) it depends upon the culture medium you are
> using; (b) it seems to depend upon the "taxon" (here zymodeme),
> dermotropic zymodemes being in general (but not always) more difficult
> to grow.
> Once they are adapted to in vitro culture, there is no more problems.
> Re: isolation from sandflies, Pr Rioux (from whom I learnt in the
> field) used to soak the fly in successive baths of saline with
> antibiotics, before dissecting it upon a sterilized glass slide using
> alcohol-soaked instruments ! A bit awckward but try it !..
> Best wishes
> P. Bastien
>
> Professeur Patrick Bastien
> Laboratoire de Parasitologie-Mycologie, Faculté de Médecine
> UMR MIVEGEC (CNRS 5290 - IRD 224 - Université Montpellier 1)
> Centre National de Référence des Leishmania
> CHU de Montpellier
> 39 Avenue Charles Flahault
> 34295 Montpellier Cedex 5, France
>
>
>
>
>
> "Chang, Kwang-Poo" <KwangPoo.Chang at rosalindfranklin.edu> a écrit :
>
>> Dr. Sharma's cultures produce promastigotes, which cannot be
>> passaged to build up the population, if I remember correctly.
>>
>> Petr will be great to help Dr. Sharma out with the sandfly studies.
>>
>> KP
>>
>> ________________________________
>>
>> From: Sharman [mailto:nandlals at hotmail.com]
>> Sent: Tue 5/31/2011 10:13 AM
>> To: Petr Volf; Chang, Kwang-Poo; Hiro Goto; elfadil abass;
>> vishwamohan_katoch at yahoo.co.in; lalit Kant ICMR
>> Cc: leish-l at lineu.icb.usp.br
>> Subject: Re: [Leish-l] inquiry
>>
>>
>> Hi Petr,
>> Thanks for enclosing the pdf. In fact the strain which we are
>> dealing with is not very difficult to grow but difficult to
>> maintain, It is rK 39 positive and as I mentioned in the
>> conversation below that even the serous exudate from the ulcer is
>> also positive for rK 39 dipstick. In between because of my
>> relocation to a different institute I was little away from this work
>>  but I intend to resume this work in couple of months. Although in
>> past we tried to isolate the parasite from sand flies but did not
>> succeed. We will surely attempt again with the techniques described
>> by you and others. Contamination is the problem.
>> This particular focus seems to be a complex one.
>> NL Sharma
>>
>> From: Petr Volf <mailto:volf at cesnet.cz>
>> Sent: Tuesday, May 31, 2011 12:36 PM
>> To: Chang, Kwang-Poo <mailto:KwangPoo.Chang at rosalindfranklin.edu>  ;
>>  Sharman <mailto:nandlals at hotmail.com>  ; Hiro Goto
>> <mailto:hgoto at usp.br>  ; elfadil abass
>> <mailto:elfadil_abass at yahoo.com>
>> Cc: leish-l at lineu.icb.usp.br
>> Subject: Re: [Leish-l] inquiry
>>
>> Hi K.P.,
>> in Cukurova region, Turkey, cutaneous L. infantum (now it seems that
>>  we are dealing with L.donovani/L. infantum hybrid) grew very
>> poorely  if we isolated them from patients. Only 1 of 25 isolations
>> was  succesfull. However, the same strain (confirmed by molecular
>> methods) grew repeatedly and very well if we isolated them from
>> sandflies. It might be useful for Dr. Sharma try to get isolates
>> from sand flies. It is very laborious but very useful, in Cukurova
>> we got about dozen of isolates by this method (all identical).
>> Patients are rK39 negative, see attached paper.
>> Best wishes
>> Petr
>>
>> 	----- Original Message -----
>> 	From: Chang, Kwang-Poo <mailto:KwangPoo.Chang at rosalindfranklin.edu>
>> 	To: Sharman <mailto:nandlals at hotmail.com>  ; Hiro Goto
>> <mailto:hgoto at usp.br>  ; elfadil abass
>> <mailto:elfadil_abass at yahoo.com>
>> 	Cc: leish-l at lineu.icb.usp.br
>> 	Sent: Sunday, May 22, 2011 1:39 AM
>> 	Subject: Re: [Leish-l] inquiry
>>
>> 	Dr. NL Sharma has been working on an important CL endemic area
>> along the Satluj River valley to the south of Himalaya in India
>> (Please correct me should I be wrong for anything I said here and
>> below). I had the good fortune of visiting the site several years
>> back courtesy of Dr. Sharma.
>>
>> 	I believe Dr. Sharma's finding is important, since the parasites
>> there are different from the familiar Indian L donovani in Bihar.
>> The parasites are refratory to in vitro cultivation. They do
>> differentiate into promastigotes and may grow a little, but  can't
>> really be subcultured to establish passageable lines. This is very
>> much reminescent of L infantum in the Mediterranean area. I recall
>> Dr. Sharma has also found rK39+ dogs, if I remember correctly. If
>> so,  Satluj river valley is endemic to the infantile CL.
>>
>> 	Analyses of several batches of basically clinical CL samples from
>> Dr. Sharma showed evidence of L infantum, but also L tropica as well
>>  as a mixture of the two in one sample. This is the same picture we
>> have noted for samples from Hatay, Turkey.
>>
>> 	These observations make me wonder a lot about our current knowledge
>>  on the clinico-epidemiology based on data collected previously by
>> analyses of cultured promastigotes from one or few 'representative
>> samples'. Nowaday, technology makes it very doable to work with
>> biological samples for Leish DNAs directly from sand flies, patients
>>  and reservoir animals.
>>
>> 	KP
>>
>>
>> ________________________________
>>
>> 	From: leish-l-bounces at lineu.icb.usp.br on behalf of Sharman
>> 	Sent: Fri 5/20/2011 11:31 PM
>> 	To: Hiro Goto; elfadil abass
>> 	Cc: leish-l at lineu.icb.usp.br
>> 	Subject: Re: [Leish-l] inquiry
>>
>>
>>
>> 	Dear all
>> 	I agree with Hiro gito, We are working on a focus where the CL is
>> 	predominantly caused by L. donovani, and the rK 39 STRIPS GIVE POSITIVE
>> 	RESULTS WITH SERA as well as serous exudate from the lesion. The   
>> results are
>> 	dependent upon species.
>> 	NL Sharma
>>
>> 	--------------------------------------------------
>> 	From: "Hiro Goto" <hgoto at usp.br>
>> 	Sent: Wednesday, May 18, 2011 12:09 AM
>> 	To: "elfadil abass" <elfadil_abass at yahoo.com>
>> 	Cc: <leish-l at lineu.icb.usp.br>
>> 	Subject: Re: [Leish-l] inquiry
>>
>> 	> Dear all,
>> 	> In our oppinion, DAT and rK39 for those samples are not indicated  since
>> 	> these tests are produced for the diagnosis of visceral  leishmaniasis.
>> 	> In case of tegumentary leishmaniasis, it is very appropriate the
>> 	> observation of J.J. Shaw appointing species specificity of antibody
>> 	> response in these cases. Titers of antibodies are in general low  
>>  in  these
>> 	> cases therefore depending on the species, it may result    
>> negative. We have
>> 	> published a review recently in Expert Rev. Anti  Infect. Ther. 8(4),
>> 	> 419?433 (2010), Current diagnosis and treatment of  cutaneous and
>> 	> mucocutaneous leishmaniasis, where we raise this point.
>> 	> Hiro Goto
>> 	>
>> 	> Citando elfadil abass <elfadil_abass at yahoo.com>:
>> 	>
>> 	>> Dear all I would recommend using DAT and rK39 strip test to measure
>> 	>> antibody
>> 	>> responses and to evaluate the diagnostic efficiency for both tests  in
>> 	>> such group
>> 	>> of patients.
>> 	>>
>> 	>>  Elfadil Abass
>> 	>>
>> ________________________________________________________________________________
>> 	>> Institute of Medical Microbiology and Hospital Hygiene
>> 	>> Philipps University Marburg
>> 	>> BMFZ / Hans-Meerwein Straße 2
>> 	>> D-35033 Marburg, Germany
>> 	>>
>> ________________________________________________________________________________
>> 	>>
>> 	>>
>> 	>>
>> 	>>
>> 	>> ________________________________
>> 	>> From: Nuha Nuwayri-Salti <racha at aub.edu.lb>
>> 	>> To: saad saad <saad1426 at gmail.com>; "leish-l at lineu.icb.usp.br"
>> 	>> <leish-l at lineu.icb.usp.br>
>> 	>> Sent: Fri, May 13, 2011 9:39:33 AM
>> 	>> Subject: Re: [Leish-l] inquiry
>> 	>>
>> 	>> Dear Saad first precaution to take is to separate your samples into
>> 	>> several
>> 	>> portions each (at least 5 each being no more than a few hundred(200-400)
>> 	>> microliters). This is a necessary precaution to avoid freezing and
>> 	>> thawing
>> 	>> several times the same sample which will be the case should you do
>> 	>> different
>> 	>> studies at different times which is unavoidable.
>> 	>>
>> 	>> What you can do is correlate the type(ulcerated, abscess, furuncle
>> 	>>  etc.. ) the
>> 	>> locale, the number and age of lesions with the levels of   
>> antibody in  the
>> 	>> sera of
>> 	>> these patgients and also monitor cell mediated immunity with leishmanin
>> 	>> skin
>> 	>> test!
>> 	>> I have just published (in print) an article about having circulating
>> 	>> parasites
>> 	>> in some of these patients with apparently pure cutaneous disease. It is
>> 	>> the
>> 	>> first paper that revealed this fact. It would be interesting to confirm
>> 	>> or
>> 	>> de-confirm this fact repeating what we did.
>> 	>> Best wishes
>> 	>>
>> 	>> Nuha Nuwayri-Salti MD
>> 	>>
>> 	>>
>> 	>>
>> 	>> AOA
>> 	>> Medical Honor Society
>> 	>>
>> 	>>
>> 	>>
>> 	>>
>> 	>> -----Original Message-----
>> 	>> From: leish-l-bounces at lineu.icb.usp.br
>> 	>> [mailto:leish-l-bounces at lineu.icb.usp.br]
>> 	>> On Behalf Of saad saad
>> 	>> Sent: Friday, April 29, 2011 11:23 PM
>> 	>> To: leish-l at lineu.icb.usp.br
>> 	>> Subject: [Leish-l] inquiry
>> 	>>
>> 	>> Dear all
>> 	>> Hi. i am Saad from Saudi Arabia. i have 60 sera from positive case of
>> 	>> CL from south west of the country. I would like to have your
>> 	>> recommendations to start a good research line in CL using these sera.
>> 	>> Thanks in advance for your help
>> 	>> _______________________________________________
>> 	>> Leish-l mailing list
>> 	>> Leish-l at lineu.icb.usp.br
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>> 	>> _______________________________________________
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>> 	>>
>> 	>
>> 	>
>> 	>
>> 	> Profa. Dra. Hiro Goto
>> 	> Laboratório de Soroepidemiologia e Imunobiologia
>> 	> Instituto de Medicina Tropical de São Paulo, USP
>> 	> Av. Dr. Enéas de Carvalho Aguiar, 470, prédio II, quarto andar
>> 	> 05403-000 - São Paulo, SP
>> 	> Tel. +55-11-3061 7023, 3061 7056 ou 3061 7027
>> 	> Fax. +55-11-3061-8270
>> 	>
>> 	> _______________________________________________
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>> 	> Leish-l at lineu.icb.usp.br
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>>
>>
>> ________________________________
>>
>>
>>
>>
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