[Leish-l] inquiry

Wed Jun 1 19:41:40 BRT 2011

Dear Maowia,

Gald to hear from you with your work-in-progress. May I ask you some questions and share with you some info from my very limited experience with Sudanese L donovani ? Please correct my statements, if inccorrect.

1.	"difficult to maintain" means that the promastigotes emerge after incubation of lesion aspirates/punctures in appropriate media and replicate during successive passages, but are low in cell density and easily lost during repetitive passages for continuous cultivation ?

2.	"difficult to grow" means that the promastigotes never emerge or emerge after incubation of lesion aspirates/punctures in appropriate media, but subsequently fail to replicate or replicate to a limited extent on subculture ?

3.	What is the rate of success in your attempts to isolate parasites in culture from clinical samples in each endemic site over the years ? 

4.	The Sudanese strain used by many in the USA was probably brought over decades ago by Donald Hyneman and established in Syrian Golden hamsters by Leslie Stauber and his students. In the 70's, I had the good fortune of working with Dennis Dwyer on his S1 or S2 strains of this origin. As widely reported in the literature, this is an excellent model of visceral leishmaniasis, producing typical clinical outcome consistently, including invariable death and splenic LD loads of up to 1010 per heavily infected spleen. I recall I had difficult time to differentiate these splenic amastigotes into promastigotes. I did see some motile promastigotes. While a small number remains viable, many died and appeared as ghosts during the subsequent incubation. This has led me to suspect selection of cultivable clones. In some, but not all of these cultures, promastigotes eventually became 'adapted' to the culture conditions and grew, producing consistently up to ~40 X 106 cells/ml in 4-5 days in successive passages (starting from ~106/ml = ~ 4-5 generation or replication cycles). The stationary phase promastigotes of these cultures no longer produce good infection of hamsters, irrespective of the passage numbers (Isolation of 'metacyclics" to produce good infection has since been reported in the literature, if I remember correctly). These promastigotes are more similar to the Mediterranean L infantum than to the Indian L donovani of the Bihar region in the sequence of their single-copy protein-coding genes, as probably already shown by many based on sequencing other genetic sites. 

KP

________________________________

From: Maowia Mukhtar [mailto:mmukhtar at tropmedicine.org] 
Sent: Tuesday, May 31, 2011 2:30 PM
To: 'Sharman'; '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

Dear Colleagues,

We have very interesting experience with Sudanese L.donovani. This group of parasite although are genetically homogenous they still cause diverse clinical forms in Sudan ranging from VL, PKDL, ML to CL. We have isolated  L. donovani MON82 from classic cutaneous ulcers and typed them using both isoenzyme and molecular typing techniques. Please check our manuscript (Elamin et al. 2008. Trans R Soc Trop Med Hyg. 2008 Jan;102(1):54-7.). We have analyzed several virulence genes comparing the isolates from the diverse clinical forms and they turned to be are highly homologous. Immunologically they induce different immune responses (TH1/TH2). We know the vector of L.donovani VL isolates which is P. orientalis, but we still don't know the vector of cutaneous L. donovani parasite. We believe the vector plays an important role in the pathogenesis and the outcome of L. donovani infection together with the host immune response and may be some of the parasite virulence factors. We will soon published our data on parasite typing and host immune responses of the diverse clinical forms. 

Best regards

Maowia

Professor Maowia M. Mukhtar

Institute of Endemic Diseases

University of Khartoum

P.O. Box 11463, khartoum, Sudan

Mobile; +249912234268

Fax: +249183779712

From: leish-l-bounces at lineu.icb.usp.br [mailto:leish-l-bounces at lineu.icb.usp.br] On Behalf Of Sharman
Sent: 31 May 2011 18:13
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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	>> Leish-l mailing list
	>> Leish-l at lineu.icb.usp.br
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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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