[Leish-l] Leish-l Digest, Vol 42, Issue 12

Raj raj at cellabs.com.au
Mon Jun 6 02:05:57 BRT 2011


Hello Professor Arora and Dr Sharma,
Inclusion of Non-essential amino acids (MEM-NEAA -Gibco), glutamine, and 
Beta mercaptoethanol  into the leishmania culture medium may help in 
obtaining a stable Leishmania culture.

Also please try Xanthine (Sigma X3627) which is  supportive for growing 
leishmania at a very low concentration.
If it is difficult to obtain this chemical,  practically it is easier  to 
supplement  human  fresh urine (5 to 10%) (substituting xanthine) into the 
growing medium. This would be useful for growing leishmnia parasites.

Please make sure to finally filter the medium after adding these 
supplements,



Raj
Dr G-Halli Rajasekariah
Cellabs
----- Original Message ----- 
From: <leish-l-request at lineu.icb.usp.br>
To: <leish-l at lineu.icb.usp.br>
Sent: Saturday, June 04, 2011 1:25 AM
Subject: Leish-l Digest, Vol 42, Issue 12


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>   1. Re: inquiry (Dr.Sunil Arora)
>
>
> ----------------------------------------------------------------------
>
> Message: 1
> Date: Thu, 2 Jun 2011 21:55:27 -0700 (PDT)
> From: "Dr.Sunil Arora" <skarora_in at yahoo.com>
> Subject: Re: [Leish-l] inquiry
> To: "Chang, Kwang-Poo" <KwangPoo.Chang at rosalindfranklin.edu>,
> "mmukhtar at tropmedicine.org" <mmukhtar at tropmedicine.org>, Sharman
> <nandlals at hotmail.com>, Petr Volf <volf at cesnet.cz>, Hiro Goto
> <hgoto at usp.br>, elfadil abass <elfadil_abass at yahoo.com>,
> "vishwamohan_katoch at yahoo.co.in" <vishwamohan_katoch at yahoo.co.in>,
> lalit Kant ICMR <lalitkant at icmr.org.in>
> Cc: "leish-l at lineu.icb.usp.br" <leish-l at lineu.icb.usp.br>
> Message-ID: <901936.30699.qm at web33208.mail.mud.yahoo.com>
> Content-Type: text/plain; charset="utf-8"
>
> Dear KP and all
> I have been reading the exchange of emails regarding characterisation of 
> leishmania isolates from Himachal in India by Dr Sharma and KP. Actually I 
> have also been associated with that though very briefly and had a chance 
> to try-n-culture some of isolates?sent by?Dr Sharma. The cultures became 
> positive but we failed to establish good passagable cultures from any of 
> aspirates/biopsy specimen?sent by Dr Sharma, although my lab has been able 
> to establish some good number of primaray cultures from spleen/BM 
> aspirates of Ld strains in Chandigarh,?although almost all the source 
> patients?belonged to Bihar originally for these. So there is definetely 
> some thing very different about the leishmania strains that are 
> circulating in that geographical area of India. we also had a chance to 
> use a specific PCR (that was developed in my lab and can distinguish 
> between an Ld and any cutaneous strain). we found about 40-50% of isolates 
> got Ld specific amplification
> while others didnot show Ld genome. So I do believe, there is a mix of Ld 
> and some?cutaneous strain in that area which KP is also refering too. It 
> needs to be investigated further... I know Dr Sharma is on to such effort 
> soon...
> ?
> regards
> sunil
>
> Dr Sunil K.Arora
> Professor
> In Charge HIV diagnostic and Disease Monitoring Laboratory
> Department of Immunopathology
> PGIMER, Chandigarh-160 012
> Ph.: 0091-172-2755192(Off)
> FAX: 0091-172-2744401, 2745078
> Ph:+91-172-2732863 (Res); 9872866087 (cell)
> email: skarora_in at yahoo.com; skarorain at gmail.com
>
> From: "Chang, Kwang-Poo" <KwangPoo.Chang at rosalindfranklin.edu>
> To: mmukhtar at tropmedicine.org; Sharman <nandlals at hotmail.com>; Petr Volf 
> <volf at cesnet.cz>; Hiro Goto <hgoto at usp.br>; elfadil abass 
> <elfadil_abass at yahoo.com>; vishwamohan_katoch at yahoo.co.in; lalit Kant ICMR 
> <lalitkant at icmr.org.in>
> Cc: leish-l at lineu.icb.usp.br
> Sent: Friday, June 3, 2011 12:03 AM
> Subject: Re: [Leish-l] inquiry
>
>
> Dear Maowia,
> ?
> Thanks again for the info.
> ?
> Are there any record available for the number of successful isolations 
> versus the total number attempted for a given endemic site ?
> ?
> KP
> ?
> From:Maowia Mukhtar [mailto: mmukhtar at tropmedicine.org ]
> Sent: Wednesday, June 01, 2011 10:41 PM
> To: Chang, Kwang-Poo; 'Sharman'; 'Petr Volf'; '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 KP,
> Thanks for your comments and valuable notes. We have a high success rate 
> inc culturing Leishmania from clinical aspirates. We have even succeeded 
> in culturing Leishmania directly from PKDL lesions without using hamsters 
> although the parasite is known to be scarce in these lesions. ?We have 
> noticed that the rate of success of ?cultures varies between endemic 
> regions in Sudan as well as between the clinical forms. Sudanese 
> Leishmania ?donovani is known to be different from the Indian donovani 
> even in their response to antileishmania drugs and in their reaction using 
> different diagnostic serology tests.
> Best regards
> Maowia
> ?
> ?
> ?
> Professor Maowia M. Mukhtar
> Instituteof EndemicDiseases
> Universityof Khartoum
> P.O. Box 11463, khartoum, Sudan
> Mobile; +249912234268
> Fax: +249183779712
> ?
> From:Chang, Kwang-Poo [mailto:KwangPoo.Chang at rosalindfranklin.edu]
> Sent: 02 June 2011 01:42
> To: mmukhtar; Sharman; Petr Volf; Hiro Goto; elfadil abass; 
> vishwamohan_katoch at yahoo.co.in; lalit Kant ICMR
> Cc: leish-l at lineu.icb.usp.br; Chang, Kwang-Poo
> Subject: RE: [Leish-l] inquiry
> ?
> 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 ?
> ?
> 1. ?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 ?
> ?
> 1. What is the rate of success in your attempts to isolate parasites in 
> culture from clinical samples in each endemic site over the years ?
> ?
> 1. 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
> Instituteof EndemicDiseases
> Universityof 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
> Sent:Tuesday, May 31, 2011 12:36 PM
> To:Chang, Kwang-Poo ; Sharman ; Hiro Goto ; elfadil abass
> 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
>>To:Sharman ; Hiro Goto ; elfadil abass
>>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 Stra?e 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
>>>> http://lineu.icb.usp.br/cgi-bin/mailman/listinfo/leish-l
>>>> _______________________________________________
>>>> Leish-l mailing list
>>>> Leish-l at lineu.icb.usp.br
>>>> http://lineu.icb.usp.br/cgi-bin/mailman/listinfo/leish-l
>>>>
>>>
>>>
>>>
>>> Profa. Dra. Hiro Goto
>>> Laborat?rio de Soroepidemiologia e Imunobiologia
>>> Instituto de Medicina Tropical de S?o Paulo , USP
>>> Av. Dr. En?as de Carvalho Aguiar, 470, pr?dio II, quarto andar
>>> 05403-000 - S?o Paulo , SP
>>> Tel. +55-11-3061 7023, 3061 7056 ou 3061 7027
>>> Fax. +55-11-3061-8270
>>>
>>> _______________________________________________
>>> Leish-l mailing list
>>> Leish-l at lineu.icb.usp.br
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>>>
>>_______________________________________________
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