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I am a post-graduate student from the Butantan Institute. I will study the Mayaro virus and I would like to know more about this virus. I would also like to learn from someone who has already studied this subject.
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What rapid tests do you recommend to detect Mayaro virus?
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Egypt has the highest prevalence of HCV. What is the major risk factor or history behind it?
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we had a case of right parotid swelling, misdiagnosed as malignancy.Excision of lymph node revealed the diagnosis of Kimura's.
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Access.
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I am trying to calculate GMT for plaque neutralisation in chikungunya/ dengue plaques. Presently I have PRNT-50 plasma dilutions
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PRNT stands for plaque-reduction neutralization titers. We use a plaque assay to determine the level of infectious virus in a solution using 10-fold serial dilutions of the volume. We pick a culture dish with a moderate # of plaques which can be counted, then take the reciprocal to get a plaque titer.
PRNT measures how much you can dilute your antibodies and still reduce the plaque # by 50%.
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Hi guys. Im growing MCF7 cells treated with 1ng/ml TGFb for two weeks. But once after cell passaging, their phenotype changed to a flat, round and large shape which is different from their normal cell shape (See the attahced file). Im wondering what conditions might cause the consequence? Thanks for your help!
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Hi, did you solve the problem? could you please share some experience?
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The laboratory blood picture in typhoid fever is generally characterized by leucopenia with neutropenia, and relative lymphocytosis. However, one study found true lymphopenia in typhoid fever following lab investigations. What could be the reason behind lymphopenia found in this study? Does true lymphopenia occur in typhoid fever?Why?
Could this lymphopenia occur due to haemophagocytosis (not HLP) or hemophagocytic histiocytosis?
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Thanks for all the tips! This was extremely helpful
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infectious diseases speacialist
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Thanks for all the tips! This was extremely helpful
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Detection of new infections from relapses in vivax malaria elimination programme is a big challenge, as a routine diagnostic method that can distinguish these cases from there. Please help us out if you have experience in this field.
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Thanks for all the tips! This was extremely helpful
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Aphthous Ulcer is common during stressful condition. As reactivation of  HSV is always associated with decreased immunity due to any cause. Decreased immunity is most common during prolong stress and so we can imply aphthous ulcer to reactivation of HSV. Recurrence of stress is common and  so recurrence of  aphthous ulcer also common. This recurrence can be prevented by levamisole for many years.
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Hi. Mostly aphthous ulcers, can be a result of stress, certain vitamin deficiency, hormonal fluctuations and even unintended mouth injuries.
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I know that vancomycin is effective only against gram positive bugs but it seems that there is a bacterium that vancomycin covers.
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It's well-known that most Gram-negative bacteria are intrinsically resistant to vancomycin because their outer membranes are impermeable to large glycopeptide molecules (with the exception of some non-gonococcal Neisseria species).
Regards
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I found a lymph node-like granule under the thymus. Is it a lymph node of MLN?
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Here is a diagram of murine lymph nodes. Also an article that explains a method of locating them for the first time. Good luck!
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Does average years of life loss (AYLL) can be used to monitor the progress of a particular disease over time? If so, does it require to be age-adjusted? What does an increasing AYLL over time suggest? 
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For trends analysis purpose, it is recommended to use the age-standardize YLL rate instead of YLL rate. This is important because the age structure of the population changes over time, so it requires to adjust for age as a confounder factor.
I hope this helps.
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For my experiment we infect B6 mice with Plasmodium berghei parasites (IP injection) and after 6 days we measure the percentage of infection = the amount of red blood cells that are infected with our parasites. Last time we did this by making a Giemsa stain of a blood smear from the infected mice. Because this is so much work we were looking for a faster method to determine parasitemia. This week I tried labeling with sybr green. So I took a drop of blood from the mice, added sybr green and CD45 (to stain lymphocytes) and then measured the sample by flow cytometry. The first experiment was fine, I got about the same percentages of infection on the FACS as by counting on the microscope. However, today the results were really bad and I don't now the reason. Does any one already has done this? Or does anyone knows another method to stain DNA of parasites to determine the percentage of infection by flow cytometry? Hoescht is unusable because we don't have an UV laser.
 
I hope someone can help ;-)
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Hi,
Can you test the acridine orange for coloration of DNA ?
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What is the best serotype of AAV for infection/transduction of mouse or human T cells?
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... while none of the rAAV serotypes appeared to infect CD4+T lymphocytes as determined by fluorescence microscopy, flow cytometry and luciferase assay...
 
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Schistosome vaccine development
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not to my knowledge
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How do you coat an antibody on a nitrocellulose membrane in order to develop immunology parasite antigens diagnostic strips?
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We have one HIV positive patient who got failed on first line ART regimen with TDF/ TFC/ EFV, then we need to switch to AZT,3TC,lop/rit (in line with WHO HIV guideline). The patient is also having HBV co-infection, hence we want to keep TDF in that regimen such as TDF,3TC,AZT,lop/rit.
But in our country stock, we do not have a separate tablet of AZT nor TDF . Then the patient will need to take( TDF+3TC) + ( AZT+3TC) + lop/rit. We are concerned about high dose ( 600mg) of 3TC. Are there any study related with 3TC high dose toxicity? Please advice. Thanks
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I understand you wanted to use TDF/XTC or TAF/XTC  in your patient given the activity against both HIV and HBV. The fact is that you can use either of those meds on a fixed-dose combination or individual drug combinations. If you cannot use TDF alone, use entecavir in addition to a fully suppressive antiretroviral regimen but do not use duplicate therapy with 3TC.
Now, my real worry about your question is if you studied the failure? The failure in this case would likely be explained by resistance to EFV but confirmation is needed. What about liver, renal or bone toxicity in your patient?
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I was recently working on the inflammatory factor expression variances between WT and knockout mice by inducing LPS. But I didn't get a convincible result by detecting several IFs. I was wondering if anyone knows the accurate relationship between the inflammatory response and the dose of LPS as well as the injection time. I will deeply appreciate your reply.
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Priya we use Working Conc 1µg/ml in our lab to induce inflammation in cell culture
(Keep LPS Stock Conc 1mg/ml@4degree)
Hope this helps
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As you know, there are many contributing factors in developing Hepatocellular cancer in hepatitis C patients which some of them are immune mediated factors. TLR4 is one of the immune mediated factors. I'm studying about its role in developing HCC IN HCV patients. I need your experience in this regard or your point of view. 
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Looks like TLR4 inhibitors like Metformin can resolve some of interferon's side effects.
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Infectivity relates to how a pathogen is able to establish infection in a healthy but susceptible host and it positively correlates with virulence. Methods to measure infectivity if specific and sensitive will aid prion research.
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Hello! I ligated a DNA in E. coli DH5 alpha, but I got low absorbance of bacteria, it was 0.066 at A500 nm. How will this affect the outcome of the agar culture? 
Sorry for my english, i'm native spanish. Thanks.
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For how long did you give heat shock to the DH5alpha cells? It is recommended to keep your competent cells always in cold ice after taking then out from the -80 degree centigrade freezer. After adding your recombinant DNA ( ligated to your working plasmid) to the competent cells, incubate for a short while in ice. Then give heat shock by placing the vial in water bath at 42 degree centigrade for 45 seconds only. Immediately put back the vial in ice and keep for 5 minutes. Then add LB broth (without any antibiotic) and keep in 37 degree incubator shaker for 1 hour and then plate on your agar plates containing appropriate antibiotic.
Bacterial population increase i.e. growth is measured by checking OD at 600nm. With experience even by sight you can make rough working estimations.
When you get colonies, just proceed for making glycerol cell stock of those, and also isolate plasmid and send for sequencing to confirm presence of desired insert.
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as you know there are some biomarkers which are introduced for different aspects in Glioblastoma (GBM). As I studied, each one has some deficiencies in diagnosis, prognosis or other aspects of the disease. Do you have any new data in this regard? 
Thank you 
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A biomarker actually very useful to study glioblastoma is TLR3. Little studied, but it has a unique theranostic value.
Because the target of TLR3, which is poly IC, has an amazing tumoricidal activity.
Several similar to poly IC, such as Hiltonol, are currently in clinical trials, and are very promising.
The drawback is that these studies do not usually rely on the research of this biomarker.
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We've seen an up regulation of CD27 on both CD4+ T cell and CD8+ T cell subsets in multiple tissues in the body. However, in reading I'm getting a mixed bag of what this could mean for the cells. How does CD27 expression relate to memory phenotype and antigen recall?
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Generally ish,  naive T cells will express CD27, upregulate upon activation but lose when they are effector. Memory cells will go onto re-express CD27.
Ahlers, Blood, 2010 has a nice graphic associated with phenotype markers and their associations. Other good reading is from J. Borst who has done a lot of work looking at the CD27/CD70 pathway and how it relates to antigen specificity etc.
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I am currently studying on the affinity of artificial antibacterial peptides to plasmid DNA of Pseudomonas aeruginosa. Is it possible to use SYTOX Green stain as part of the gel retardation assay (gel electrophoresis; possibly to replace ethidium bromide in the loading buffer)? Thank you for pondering or answering my question. 
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Henry, I think you can use fluorescent intercalators in your case as you are using a plasmid as your DNA substrate so you will have plenty of signal from the DNA-bound dye.
I presume this is supercoiled DNA? In which case, unless you do tritium labeling, you cannot incorporate a radiolabel easily. The best way to do this is to run the EMSA in the absence of DNA stain and then stain your gel after you have run it. 
Are you trying to measure a binding affinity of your peptides for DNA? Plasmid DNA might not be the best substrate in this case as you will most likely just see a upwards trailing smear on the gel as there will be multiple binding sites for the protein and it will be difficult to calculate a binding affinity from this, unless it has a very specific sequence that it binds to in your plasmid.  Using radiolabeled, and much smaller, linear DNA would be a better way to go if this is what you are interested in. One has to empirically determine the binding site size in order to choose the correct substrate length with which to determine the binding affinity.
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I need to use pantothenate as supplement for culture mc2 6020 and mc2 6030.
the pantothenate powder was diluted in MiliQ(water), then filtered with 0.2 um filter, I often find pan contaminated by other bacteria.
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Dear Lihui,
Sterilization through 0.2 um should do the job, however, you may used glass ware or containers that were not sterilized.
The following is a procedure to get sterile pantothenate aliquot:
Pantothenic acid (FW 238.3 hemicalcium salt): Dissolve 0.405 g pantothenic acid in 100 ml water and filter sterilize to yield 17 mM (1000X) stock. Dispense 2 ml aliquots in sterile cryovials and store at -20°C or below; thawed stock can be kept at 4°C for up to 1 week.
Hoping this will be helpful,
Rafik
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How I can block NKG2A receptor signaling for in vitro experiments?
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You might consider to convert your anti-NKG2A antibody of choice (and the respective isotype control) into Fab/F(ab)2-fragments - there are several commercial kits for that purpose that are easy to use and reliable. By using blocking Fab/F(ab)2-fragments you circumvent the potential problem of Fc-receptor-mediated activation. Good luck!
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I already know the work of Miller and co-authors (2001, 2007) about the lived experiences of pwMS taking interferons and glatiramer acetat.
Do you know of any other studies? 
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Elaine Cameron at the University of Manchester is working on a meta-synthesis of experiences of adherence to DMTs: http://www.crd.york.ac.uk/PROSPERO/display_record.asp?ID=CRD42015025254
Also: Johnson et al. (2006) perhaps? http://ijmsc.org/doi/abs/10.7224/1537-2073-8.1.11?=
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Hi. I am planning to test about macropinocytosis ability of RAW264.7 cells
But, I didn't know Which bacteria of shigella and samonella would be more invaded to RAW 264.7 cells. 
If you would do this experiment, Which bacteria would you use for this test?
Please let me know your brilliant thinking. 
Thanx.
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Hi Dong Hyuk Joshua Seo,
I used salmonella typhimurium to check the macrophage phagocytic activity. It worked well for me. 
Best,
Biswaranjan Pradhan
Ph.D Student, SBS
NISER Bhubaneswar
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Is there any dataset/benchmark for ICD-10?
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Have you seen their web page? 
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Please, someone using the inverse algorithm with rapid tests for serological diagnosis of syphilis could clarify me why it is considered positive (and require treatment) a case that is positive by a rapid treponemal test, non-reactive by VDRL or RPR, and positive by TPPA or TPHA; if it is marker of previous syphilis treated? Only in the case of a patient with late manifestations of syphilis could be interpreted these results as a positive case, but not in an asymptomatic patient.
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 Classically, the diagnosis
of syphilis is based on a combination of clinical history,
symptom presentation (if any), and serologic test results,
including non-treponemal and treponemal tests. Nontreponemal
tests (e.g., RPR, VDRL) are typically used
for screening, and measure a non-specific antibody to
treponemal infection. A reactive test can indicate recent
infection, but could be caused by other conditions unrelated
to syphilis. Treponemal tests (e.g., TPPA, TPHA, FTA-ABS,
EIA) detect antibody to syphilis and thus can confirm
exposure to treponemal disease. However, these tests cannot
distinguish venereal infection (syphilis) from non-venereal
treponemal diseases (e.g., yaws, pinta, bejel). Furthermore,
treponemal antibody persists for life, and thus treponemal
tests cannot distinguish between recent, active infection and
previously treated or old, non-contagious infection.
I agree with Dr. Ricardo
2015-cha-guidance-syphilis-testing-lac.pdf
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I used limiting dilution assay for the quantification of parasite burden in the spleen of vaccinated mice post challenged with Leishmania parasite. I need some useful information to use the ELIDA software for data analysis because this has been widely used for analysis in several studies.
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Hi Leila!
good day!
I think the best bet for you to acquire the software is by communicating with the authors of this study:
Comparison of Parasite Burden Using Real-Time Polymerase Chain Reaction Assay and Limiting Dilution Assay in Leishmania major Infected Mouse
Somayeh GHOTLOO,1 Mostafa HAJI MOLLAHOSEINI,1,2 Ali NAJAFI,3 and Farshid YEGANEH1,2,*
Author information ► Article notes ► Copyright and License information ►
this is the link of this paper:
If I cannot access the information thru internet what I usually do is I just contact thru email the authors.
I wish you all the best in your research!
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Serratia marcescens is a broad host-range pathogen and is capable of opportunistic infections of humans. As a member of the Enterobacteriaceae, it is related to Escherichia and Shigella, Salmonella and Yersinia. It is implicated in a wide range of serious infections including pneumonia, lower respiratory tract infection, urinary tract infection, bloodstream infection, wound infection and meningitis. The organism has also been described as an important cause of ocular infection with high incidence in contact lens-related keratitis.
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We may not be able to determine the geographic origin of the whole "species" Seratia marcesens but if there are specifc pathogenic variants of the species which have recently begun causing a disease in humans or plants, it could very well be possible to trace where the most recent source of this spread was.  This may cost many millions of dollars or much less, depending on many aspects of the "outbreak" and the natural range of the organism.   Some recent examples of this type of tracing are found by studying for example how the source of the enteropahtogenic E. coli (STEC serotype 0104H4-2011) which killed people in Europe in May-June 2011, was traced. 
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Hello! Has anyone experience in storing field collected insects (mosquitoes in tropical climates) for insect and included parasites RNA/DNA extraction (using TRIzol/TRIreagent method) and qPCR analysis? No cold chain involved and as cheap as possible. Reviewing the literature I thought about comparing the effect of RNAlater, RNAfix (homemade RNAlater), methylated ethanol or FTA cards. I was wondering whether there are other methods that more cost-effective as possible? Many thanks! 
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Hi Corrado,
Here is a publication for the preservation of insects to be used in spectroscopy. This publication resulted from experience on field trips in Nigeria and Cameroon for insects that would be analyzed in Bamako, Mali. And Dr Floyd Dowell's experience in Tanzania. It is good you are not involving cryo-preservation at any point in the tropics.
I may suggest SILICA GEL for a short period. I had serious degradation of materials preserved in ethanol on one hand and on the other hand, methanol fixes molecules and may not yield very good results during DNA/RNA extraction.
I hope this helps
good luck
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Dear colleagues, could anyone provide information regarding risks of malaria during 1-month field trip in Northern Laos at June - July? We plan to stay at the campground in forested river valleys.
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Lariam uses 1 tablet a week so you need to take 4 tablets.
Better to use. I used Lariam I didn't see side effects.
Zoya
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Many peoples in our district East Godavari and in Agency area having no awareness on Aids and HIV. So that we are planning to aware the children and people from their School level. Itself so we are selecting some Z.P School and A.P.S.W. R Schools and we go for awareness programs and camps to educate the children. And we are also planning to make an HIV Anti- group. We select some children and conduct some competitions, seminars and workshops and aware them against HIV and Aids.
We are requesting you for the fund needed for our plan and requesting for the addresses of any other organizations who are supporting us.
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Bill & Melinda Gates Foundation
Commonwealth Foundation
Elton John AIDS Foundation
European Union
Global Fund to Fight AIDS, Tuberculosis and Malaria
Hewlett Foundation
International HIV/AIDS Alliance
Government of the Netherlands
The Abbott Fund
US President’s Emergency Plan for AIDS Relief (PEPFAR)
UK Department for International Development (DFID)
UNAIDS
United States Agency for International Development (USAID)
United Nations Development Programme
World Health Organization
NACO, 9th Floor, Chanderlok Bldg., 36 Janpath,
New Delhi-110001 Tel: 23325337
Rajiv Gandhi Foundation
Rajendra Prasad Road
New Delhi - 110 001, India
Tel.: +91 11 2375 5117
Fax: +91 11 2375 5119
The Foundation supports HIV training programs for health professionals working with NGOs to treat the underprivileged throughout India. It also supports education, prevention and direct services programs for PLWHA as well as capacity-building grants for CBOs working in this area.
Vasavya Mahila Mandali
Benz Circle, Vijayawada - 520 010
Andhra Pradesh, India
Tel.: +91 86 6247 0966
Fax: +91 86 6247 3056
Vasavya is an intermediary for international and national
funders and governments. It supports home and community-based HIV/AIDS care and prevention programs.
MAMTA MAMTA
Health
Institute for Mother and Child
B-5, Greater Kailash Enclave-II
New Delhi, India 110048
Tel.: +91 11 2922 0210
Fax: +91 11 2922 0575
MAMTA is an intermediary for international and national funders and governments. It supports HIV/AIDS programs focusing on direct care, advocacy, and training throughout the country.
Palmyrah Workers Development Society (PWDS PWDS PWDS )
Crystal Street, Marthandam – 629165
Kanyakumari District
Tamil Nadu, India
Tel.: +91 46 5127 0241
Fax: +91 46 5127 0138
PWDS is an intermediary for international and national funders and governments. It supports CBOs and NGOs working with AIDS orphans as well as organizations providing community-based care and support programs for PLWHA.
LEPRA
Society
Post Box No. 1518
West Marredpally, Secunderabad
Andhra Pradesh, India
Tel.: +91 040 2780 2139
Fax: +91 040 2780 1391
LEPRA is an intermediary for international and national funders and governments. It supports a wide range of HIV/AIDS projects from direct care to advocacy to education and vocational programs throughout the country.
TEST Foundation
4, Sathalvar Street
Mugappair West
Chennai, India 600037
Tel.: +91 044 2624 4100
Fax: +91 044-2625 0315
TEST is both an operating foundation, running its own programs including home-based care and a hospice for AIDS patients, and a grantmaking
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I inoculum 5X10^3 pfu H1N1 influenza virus to mice through intranasal injection, but the result shows that no matter after 48 hrs, 72 hrs or 96 hrs, the virus titer of mice lung tissue still remains in 5x10^3 pfu or even less(From plaques assay results). Are there any possible reasons that cause this problem?
Direct virus  titer test through MDCK cells did not show this problem, only in mice lung tissue. Thanks.
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Yes, it sounds like the problem is with the titre method from the mouse lung as the animals are clearly getting sick. If you're avoiding freeze thawing after homogenization then the other factor that we've found affects titre a lot is the method of homogenization - we used to use hand-held "mixer" devices (with or without glass beads) and would get low titres, whereas the current Qiagen Tissue Lyser II machine gives us 100-fold higher titres from the same experimental set up. Whether this is because it's faster or simply macerates the tissue better (or both) I don't know.
Bottom line - if the animals are losing substantial amounts of weight, your system is basically working.
Paul
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Endotoxin can possibly be neutralized in vivo by diverse substances. The endotoxin- neutalizing substance complex must be removed from the body before it could cause CD14-TLR4-mediated activation of leukocytes and release of inflammatory cytokines leading to ill-effects of endotoxemia. A single mechanism or it will depend on the type of the complex formed?
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Hi Gaurav,
Thanks for the answer. In fact, I was aware of this system (a sort of hemodialysis) to remove endotoxin by polymyxin.  Rather I was curious to know about the fate of endotoxin trapped by its binder in vivo. If we consider reticuloendothelial system, it is likely to invoke innate immune system in somewhat altered manner (LPS in the endosomal compartment also detected by innate PRRs, until and unless it is degraded by the enzymatic system therein).
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Hi all,
I need to know which dilution of serum is better to measure LPS level of a helminth via LAL assay?
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Dear Sally,
I am not sure I fully understand your question.
If you want do measure LPS level by LAL for a given sample.
You need to measure the dilution your sample you will need to fall in the middle of a standard curve (known quantities of LPS vs. OD read).
What I do not get is that helminth does not have LPS, so it will be always negative.
However if you which do Know how much helminth contamination you have in your sample.
Then I would go with a standard ELISA. On one site you do a standard curve with a knonw amount of helminth
Then the same with your sample in serial dilution and you see where you are in comparaison to the standard.
I hope its help.
Best,
Laurent
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I receive conflicting information whether Field stain can show Schuffner dots in malaria parasites.
Information online on this are limited.
What is your knowledge on this?
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Dear Eric
There is an interesting paper you may be want to read:
Am J Pathol. 1988 Apr; 131(1): 48–52.
PMCID: PMC1880566
Immunoelectron microscopy of Schüffner's dots in Plasmodium vivax-infected human erythrocytes.
P. V. Udagama, C. T. Atkinson, J. S. Peiris, P. H. David, K. N. Mendis, and M. Aikawa
I attache the PDF.
Finally, please if you consider this answer appropriate, please upvote it using the green up arrow click. Thanks.
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I am currently looking for research on any clinical study of the direct correlation between oil extraction and specific diseases in Africa, such as cancer, asthma or other respiratory illnesses in Africa.
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Dear Udoh, the study that has been done at Khartoum Sudan in tanneries may in one way correlate industries, chemicals, waste products and skin complications, hope you find some of the points you are looking for.
38. Kamal El-Din H El-Hassan, M.D, Yousif M El-Kordofani, MD, Bashir, A.H.H, MD. The Prevalence of Occupational Dermatosis among Workers in Khartoum State’s Tanneries. Original Article. AJDV, 2014, 3(2).
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Have been documenting an international series of infectious-like events in which deaths and medical admissions simultaneously rise in what can be best described as a rectangular wave effect. Have written a review attempting to link CMV to these suspected outbreaks. Any thoughts or suggestions would be welcome along with potential research which could be relevant. Many thanks for your valuable time. Kind Regards Rod
P.S. even if CMV is not the direct agent I would be highly surprised if it were not taking opportunistic advantage as it does in HIV/AIDS
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I think that this link may be useful for you.
Pathogenesis of human cytomegalovirus infection and ...
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de G Gerna - ‎2004 - ‎Cité 93 fois - ‎Autres articles
In acquired immunodeficiency syndrome patients with human cytomegalovirus (HCMV), disseminated infection, and end-organ disease, autopsy findings show ...
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Drug resistant enteric fever is a harsh reality; with resistance to fluoroquinolones, cotrimoxazole, ampicillin, amoxycillin on the rise. So what is the place of aminoglycosides in the treatment of enteric fever? Could you please cite recent research done in this area? Thanks in advance !
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Aminoglycosides are ineffective for intracellular organisms (as Salmonella often is) and therefore as pointed out by Easow, do not lead to a clinical cure. Quinolone resistance is over 50% in the Indian sub-continent so Ceftriaxone is the current drug of choice. 
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  • hi .. i want to know about the complete procedure of IgA isolation from sputum in MTB .could anyone give a kind approval .regards.
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There is a nice matrix from CaptureSelect™ IgA Affinity Matrix (https://www.thermofisher.com/order/catalog/product/19428801L) able to bind all kind of IgA. I suggest to solubilize all the proteins ins the sputum first. For that, treat the sputum with N-acetylcysteine to break the mucus. Use 5 ml per gram of sputum. Add 10 volumes of PBS, incubate for 10 minutes. Centrifuge at 5000 g for 10 minutes and use the supernatant as binding material for IgA following the instruction of the manufacturer of the matrix. I hope this helps.
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I have isolated human neutrophils (from blood) in the inactivated condition for parasite infection study. I need to fix the cells for staining protocol. I have used 1% and 4% paraformaldehyde. I am getting more ghost cells during staining protocol. What should I use for fixing cells? Also I need it to grow on coverslip. What is the method used for adhering neutrophils on glass coverslip?
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Hi Riva,
If you want to fix PMNs for ICC then fixed cells with 4 or 3.7% PFA at 4 degree Celsius for 30 minutes. Used zero acceleration and break for centrifugation when cells are live. Give minimum jerk to cells during resuspending pellet. Avoid using pipette for resuspending cells. Whenever you fix cells for microscopy then try to use the cells within 3-4 days after fixation.
This procedure works well with me. Try same.
Best luck!
Durgesh Pitale. 
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 monoclonal antibody  against IL2. TNF. IL1. etc.
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There are no current classical antibodies to neutralize DENV, but there is a new development that shows promising results (antibodies against the E dimer conformation region, please refer to Galvin Screaton 2015), also there has been extensive research showing that the permissive cells in vivo are of stem cell which bear megakaryocyte phenotypes, this could also help understand dengue and develop strategies against it
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The START (“Strategic Timing of AntiRetroviral Treatment”) study was a randomized, controlled clinical trial designed to more clearly define the optimal time for HIV- infected individuals to begin antiretroviral therapy. The study was stopped early (May 2015) and showed that starting antiretroviral therapy early prevents a combination of outcomes that includes AIDS events (such as AIDS-related cancer) and serious non-AIDS events (major cardiovascular, renal and liver disease, non-AIDS cancer, and death not attributable to AIDS).
In India, currently we start ART at a CD4 count <350 and even here grapple with a significant percentage of treatment failures due to poor adherence. If every HIV individual were to be started on ART right at diagnosis or quite early in disease, besides economic difficulty, would we not have a large percentage of drug failures (and resistance) in a few years. Would an upward revision of initiating ART at CD4 of 500 or even greater be a good choice, considering drug resistance and poor adherence issues, despite scientific evidence of early therapy being beneficial in RCT. 
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Dear  Shankar Subramanian
It's possible that early initiation of antiretroviral treatment may expose to increasing viral resistance, and early treatment failures. We will have the answer to this concern when the results of ongoing studies in Zambia and South Africa will be published [1, 2].
It is also important to note that the major route of HIV transmission is sexual, more so in poorest areas. Immediate ART initiation thus favorably impacts not only the individual, but also all their sexual partners, providing tangible public health benefit.
The purpose of an early initiation of antiretroviral therapy is therefore not only improve the quality of life of people living with HIV but also to prevent HIV transmission. Thus, we can probably have a lower prevalence of HIV infection.
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1. Iwuji CC, Orne-Gliemann J, Tanser F, et al. Evaluation of the impact of immediate versus WHO recommendations-guided antiretroviral therapy initiation on HIV incidence: the ANRS 12249 TasP (Treatment as Prevention) trial in Hlabisa sub-district, KwaZulu-Natal, South Africa: study protocol for a cluster randomised controlled trial. Trials 2013;14:230.
2. Hayes R, Ayles H, Beyers N, et al. HPTN 071 (PopART): rationale and design of a cluster-randomised trial of the population impact of an HIV combination prevention intervention including universal testing and treatment - a study protocol for a cluster randomised trial. Trials 2014;15:57
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malaria researchers and parasitologists : How can sex hormones influence the outcome of cerebral malaria pathology in humans and mice?
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Dear Bhanu
I believe that the following review is very good for your it has a section about Plasmodium.
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malaria researchers, microbiologists and neuroscientists, how can I detect blood brain barrier breakdown during cerebral malaria in mice other than Evan's blue injection technique?
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There are two approaches: The first, if you can't Perfuse your animals and the second if your can. If you cannot perfuse your animals (and can only fix them by submersion in formalin). One approach is to stain for different molecular wt proteins that exist in the sera. These proteins will leak across the BBB. For example you can immunocytochemically stain for IgG. ( While there are a lot of paper that use this approach, note: B cells found in the Brain may produce a confound with  this approach, so people also stain for other proteins as well). If you can perfuse, one approach is to follow the Saline perfusion with an HRP solution and then fix en-block with formalin. The you can detect the HRP with a DAB reaction.  This is just an explanation to get you started. You can see there are other alternatives to the Evan's blue.
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When the animal is challenged intranasaly or intratracheally will there be any clinical signs in animal? I challenged calves intranasally with 5ml of inoculum containing 109 cfu/ml but observed no clinical signs. Also no clinical signs were produced when the calves were challenged intratracheally with 109 cfu. So in such case will there be any immune response?
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I agree with dr Atef answer. It is important to exclude the presence of Pasteurella before starting experimental infection experiment. You may observe some respiratory signs if the animals are free of Pasteurella. If they are natural carrier of some strains and you use a laboratory strain  immune response  could be unsignifiant, since natural strains of Pasteurella may inhibit growing of laboratory strains
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I have difficulty to transduce unstimulated T cells using VSVG-lentiviral vector. A lot of papers mention that measles virus glycoprotein pseudotyped lentiviral vector is a good option. However, I cannot find any commercial kit. Any suggestions?  Many thanks.
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 Thanks for above answers. 
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I want to grow Mtb for metabolic studies. In some stress conditions people had used Tyloxapol instead of tween 80. Kindly elaborate the main reason behind it and provide and cite a reference, if possible.
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Pamela, Tween has been shown to degrade and inhibit growth of MTB.
1. Davis BD, Dubos RJ. 1948. The Inhibitory Effect of Lipase on Bacterial Growth in Media Containing Fatty Acid Esters. J Bacteriol 55:11–23.
2. R.J. Dubos, B.D. Davis 1946. Factors affecting the growth of tubercle bacilli in liquid media J. Exp. Med., 83,  409–423
and influence acidic stress response
1. Vandal OH, Pierini LM, Schnappinger D, Nathan CF, Ehrt S. 2008. A membrane protein preserves intrabacterial pH in intraphagosomal Mycobacterium tuberculosis. Nat Med 14:849–854.
Also see
1. Tang YJ, Shui W, Myers S, Feng X, Bertozzi C, Keasling JD. 2009. Central metabolism in Mycobacterium smegmatis during the transition from O2-rich to O2-poor conditions as studied by isotopomer-assisted metabolite analysis. Biotechnology Letters 31:1233–1240.
Hope these help.
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We are looking at LPS levels in serum in certain infections and as controls we use serum from healthy individuals. All healthy individuals were fasting for 12 hours and blood was separated in class II safety cabinets with sterile tips. However, the highest LPS levels were detected in healthy individuals and in most the levels were >1000ng/ml which is more than the upper detection limit of the ELISA. 
Why do we get such high levels in healthy individuals?
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Yes, the level of LPS seems pretty high, especially when you look at this paper http://www.ncbi.nlm.nih.gov/pubmed/10515819 and normal donor have ~6pg/ml of LPS in plasma.
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Re: Naegleria Brain damage.
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See the article below. Review on the subject
Pathogenesis of amoebic encephalitis: Are the amoebae being credited to an ‘inside job’ done by the host immune response?
Abstract
Pathogenic free living amoeba like Naegleria fowleri, Acanthamoeba spp., and Balamuthia mandrillaris are known to cause fatal “amoebic meningoencephalitis” by acquiring different route of entries to the brain. The host immune response to these protist pathogens differs from each another, as evidenced by the postmortem gross and microscopic findings from the brains of the affected patients. Cited with the expression of ‘brain eating amoeba’ when the infection is caused by N. fowleri, this expression is making its way into parasitology journals and books. The impression that it imparts is, as if the brain damage is substantially due to the enzymes and toxins produced by this amoeba.
A detailed review of the literature, analysis of archived specimens and with our experimental assays, here we establish that with N. fowleri, Acanthamoeba and Balamuthia spp., the infections result in an extensive brain damage that in fact is substantially caused by the host immune response rather than the amoeba. Due to the comparatively larger sizes of these pathogens and the prior exposure of the amoebal antigen to the human body, the host immune system launches an amplified response that not only breaches the blood brain barrier (BBB), but also becomes the major cause of brain damage in Amoebic meningoencephalitis. It is our understanding that for N. fowleri the host immune response is dominated by acute inflammatory cytokines and that, in cases of Acanthamoeba and Balamuthia spp., it is the type IV hypersensitivity reaction that fundamentally not only contributes to disruption and leakiness of the blood brain barrier (BBB) but also causes the neuronal damage. The further intensification of brain damage is done by toxins and enzymes secreted by the amoeba, which causes the irreversible brain damage
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I have a question
how many ug of protein from inactivated influenza virus simply to immunize (1 swine) help me please, I don’t know what to do I look everywhere and I do not find the information
With my protocol for purification virus I have only 70ug/ml (total protein SIV), IM NOT SURE if 70ug/ml is sufficient to immune 1 swine with 3 booster
Suggestion please
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The approach to the issue reported by Paul Digard and TimothyMiller is very interesting . It enters comparative immunology . it usually solves the equation with one unknown ( human weight of the animal to determine the dose to be injected ) It remains that when it comes to killed virus vaccine should increase the amount compared the live virus vaccine .
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I'm working with Salmonella enterice serovar Typhimurium and when I orally infect mice, I observe 10% of mice having strong dizziness. This bacteria is known to infect systemic tissues such as mesenteric lymph nodes, peyer's patches, spleen and liver but what about the dizziness phenotype? Is Salmonella able to infect the internal ear? Or is there any other explanation for this?
Many thanks in advance for your help!
Best,
Thibault
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Thank you Amar! This indeed answer my question!!
Best,
Thibault
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Dear friends, Suppose if I am interested in finding relationships among symptoms of meningitis across certain test characteristics, can I put test characteristics (CSF test) in levels. For instance, CSF cell count as level 1, level 2 level 3 and etc. or CSFtotal protein levels as level 1, level 2, level 3 and etc.
Thanks in advance.... 
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Thanks a lot! In case if someone interested I may forward my data set such that you may suggest me changes.
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In several clinical settings such as pancreatitis and trauma, a secondary infection can be frequent and often causes death. I wonder if the body in a hyperactivated status would amplify the detrimental effect of bacterial infection. Is there any literatures on this aspect?
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I agree with you. But I can not follow the third explanation. Do you mean the second challenge damage the immune tissue or organs? Such as, altering the total number of T cells or subgroups. I am reasrching the effect of  influenza vaccination on cognitive function during pregnancy. And i have found A(H1N1) influenza vaccine vaccination contribute to neurogenesis in offspring and pregnant mothers. I think the mechanism links to T cells in the periphery, such as the systemic T cells were recruited to the choroid plexes.
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can any explain some genetic differences responsible for EVR,RVR,SVR and NVR
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There are multiple reasons which can be summarised as under :
[a] Stage of liver cirrhosis - those with more advanced disease responds poorly.
[b] IL 28B typing - those with TT genotype fares poorer compared to CC type.
[c] Adherence to treatment - Those needing temporary drug withdrawal or dose modification due to adverse events have poorer outcome. 80% of total dose and 80% of total duration has been found to be effective. Ribavirin dose is more important.
[d] Some other factors which might have genetic basis are that males, presence of NAFLD and other chronic liver disease and coinfection with other hepatotropic viruses have poorer outcome. 
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I would like to immunize different groups of OT-1 mice with SIINFEKL and some variants we have made to determine if there is a difference in T-cell response. The OT-1 model is attractive because all the CD8 T-cells are specific. They are great in vitro tools or for adoptive transfer, but can you directly immunize an OT-1 mouse? The alternative is to use B6 mice, but we have a lot of OT-1 on hand and I'd get a lot more cells from them. 
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Note that because of the high number of T cells there will be competition for antigen, which will limit their response compared to an adoptive transfer model. Responses wiill also be less robust/comparable between animals (http://www.ncbi.nlm.nih.gov/pubmed/11163194). I  recommend for your assay that you stick with the adoptive transfer model.
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is there any research or evidence that advocate locating highly infectious wards in hospital design...is there any scientific evidence about the preferred location regarding the floors (upper vs lower floors).. i found one research that recommends locating infectious patients in the uppermost floors for the prevention of airborne infection transmission... is there any other researches?
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Dear Dr. Hegazy,
The 2009 WHO guidelines entitled "WHO policy on Tuberculosis infection control in health-care facilities, congregate settings and households"  described all the different components on this topic  (http://www.who.int/tb/publications/2009/infection_control/en/). In addition, you can find specific recommendations related to building design in the following document: "TBCTA. IMPLEMENTING the WHO Policy on TB Infection Control in Health-Care Facilities, Congregate Settings and Households. A framework to plan, implement and scale-up TB infection control activities at country, facility and community level. November 2010. (http://www.tbcta.org)."
All the best,
Dr. Daniel Chemtob (Former WHO Medical Officer in charge at the Geneva WHO/HQ  of TB infection control worlwide)
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I wold like to test some newly synthesized  compounds  as aminopeptidase M1 (PfAM1) inhibitors and antimalarial agent. Can any one evaluate our compounds in scientific collaboration or tell me where can I run the  biological evaluation?
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Thank you prof. Amar Safdar.
I have already read this publication, we do not have the facilities  to run the biological testing, we are looking  for someone who can run the testing for us . 
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A number of definitions are found in publications ranging from 6 days to 14 days. Most frequent definitions mention 7 and 14 days. Some use onset of symptoms as staring point. Others use first detection of virus material as starting point. The end of shedding is usually defined by the last detection of viral material followed by negative sampling results. Usual duration of shedding (about 6 days) should probably be considered when the definition is chosen. Any suggestions?
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I have recently added a Commentary article concerning this question: "Prolonged Viral Shedding of Influenza Virus: Which Definition?" by Macé M Schuurmans and Nicolas J Mueller. It deals with some aspects I consider relevant. Surely there are many more aspects to be considered, depending on the setting. Any suggestions or comments are welcome.
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I've been using the commercial Rotavirus ELISAs to detect murine adapted (EDIM and EC wild-type) strains of Rotavirus A, however their sensitivity for this application is quite low - though obviously they are perfectly fine for detecting human rotavirus A infection as viral shedding is so much greater than mice.
Does anyone know of a good sandwich ELISA antibody pair (viral capture and detection) that I can use to detect the relatively low shedding from adult mice? Ideally the detection antibody would be labelled with biotin or directly with HRP / AP.
Appreciate the help. 
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Please try sending message to ROTACLONE manufacturers. They might be able to help.
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due to accelerating roll-out of ARV , HIV/AIDS services are integrated into PHC in order to increase the access of ARV by people living with HIV. however there is ongoing shortage of material and human resource
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You can, may be draw examples from the Indian model, although not completely integrated in its true sense currently. The National AIDS Control program nevertheless has worked progressively towards integration and now envisages to integrate the AIDS program completely in to the national health mission. Integration as I understand occurs at various levels and for various program components of the AIDS program. If your question is about the prevention and treatment component of HIV/AIDS then the answer would be yes it can be integrated one hundred percent. For example, testing for HIV can occur at any general laboratory, in most cases it would not require specialised training or equipment that is not manageable by a certified general laboratory technician. Now the larger problem I assume could be when we talk of the treatment component. If PLHIV have access to ARV medications provided by the government then it could be challenging, in terms of the capacities of the medical officers to provide standardized care based on the guidelines and also the laboratory support for periodic CD4 testing. Our experience in India is that integration is a slow drawn process especially for a concentrated epidemic such as ours. And, most off it depends on the existing public health infrastructure.
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We are interested in sera samples from literally all soil-transmitted helminths and filarial parasites, with a top priority placed on hookworm infected patients, to use sera in screening in immunoassays for vaccine development. Thank you.
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It is hard to find a regulated repository for serum samples from infected individuals as usually an approval from Ethics Committee is required for any specific purposes. It would be best if you find a collaborator in endemic areas.
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Viruses are using cell machinery for its amplification. After  enough number of replication cycle it leaves cell and attack next cell. Is there any chance of changing pH of that infected cell during amplification process?  I mean whether pH values of cell is increasing at the time of  its genome replication and translation process and pH value decreases when cell is dying because of degrading enzymes. 
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Dying cells might exhibit low pH (acidic) due to the process of apoptosis or necrosis. TLR signal pathway is also relevant. 
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I am conducting a qualitative study on HIV/AIDS medication adherence and compliance and seek contributions on how to gather relevant information.
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From your question, you indicate that adherence to HIV and AIDS medicines in Nigeria is high and there are some factors that have been shown to contribute to that, i believe you want to establish which factors may be adapted into strategies for sustained high adherence.
Since you want to conduct a qualitative research, interview with the patients, health care providers, managers, caregivers and community organization representatives (such as support groups and home based care groups)  may be the way to go. You could use semi-structured questionnaires and focused group discussions and your questions should include some of the factors identified to result in high adherence and seek from the participants what they should be done. In your interviews you should have representation in terms of gender, age and if possible, duration on therapy. 
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I am looking for a researcher working on Babesiosis to collaborate with, in order to detect IgG antibodies on human and bovine serum samples from Colombia. Any suggestion?
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La Unidad de Babesiosis del Centro Nacional de Parasitología del INIFAP en Cuernavaca, México realiza tales diagnósticos y mediciones rutinariamente. Sin embargo juzgo muy difícil poder trabajar muestras de sueros de bovinos de Colombia, por las restricciones zoosanitarias. Indudablemente los colegas de EMBRAPA en Brasil ó del INTA en Argentina, son reconocidos internacionalmente y pueden apoyarlos. Igualmente, me imagino el Dr. Benavides que estuvo en CORPOICA muchos años y ahora se encuentra en la Universidad La Salle, podría ser una ayuda más próxima. Ánimo.
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We work with murine model to study Plasmodium berghei NK65 infection, wich causes lung damage in C57BL/6 mice, but some times I don't see this fenotipe. I would like to use 4-aminobenzoic acid to improve the parasites growth , but I don't know if its work.
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The only experience with this is some work with P. chabaudi. The main effect was that when we used pyrimethamine resistant parasites, they were definitely dependent on addition of 4-aminobenzoic acid (which was added to the drinking water for the mice) for a sustainable infection. With wild type parasites, this effect was not seen. My interpretation of that is that the mutation leading to resistance makes the folate production less efficient, and the addition will boost the folate synthesis enough to overcome this. 
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We have used subcutaneous route for MTB infection in mice. We have observed lung bacilli burden at 30 days after infection. I just want to know any supplementary evidence to support my result.
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Please refer to the following very good study and their Methods
If you have further questions, consider contacting the authors of this study
Proc Natl Acad Sci U S A. 2015 Jan 5. pii: 201416951
To assess the bactericidal and sterilizing activity of the first-line regimen, we infected 223 female BALB/c mice by aerosol with M. tuberculosis strain H37Rv, achieving a mean implantation of 2.62 (SD 0.44) log 10 cfus in the lungs, with the spleens being culture-negative the day after infection.
Our objective was to establish a heavy infection with high cfu counts in the lungs and the spleens; as such, our original protocol was to initiate treatment 6 wk after infection. However, by 4 wk after infection, 12 mice had already died and all remaining mice were sick; therefore, we began treatment at 4 wk postinfection.
An additional 10 mice died during the first 4 d of treatment. To compensate for this loss of mice, we reduced the numbers of mice killed on day 0 (the day of treatment initiation) as well as at some of the relapse assessment time points (detailed in Materials and Methods).
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I need to grow influenza viruses from respiratory samples prior to sequencing (due to low viral load), but i don´t know if I can assume for evaluating data the genetic changes that would happen with adapting these viruses to MDCK cells or embrionated eggs.
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We studied this adaptation through passaging in our previous paper (Ramadhany et al. 2012 ; Yasugi et al., 2012), when we cultured human influenza viruses in embryonated eggs, some mutations occurred in HA binding site even only after 1 time passaging. The population of viruses carrying mutation will become dominant through passaging in egg. To lessen these mutations, culturing human influenza viruses in MDCK cell might be better option.
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Does isolate from a stool sample prove a person is infected with TB?
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Yes, in rare cases mycobacteria can be isolated from stool. But that does not indicate tuberculosis at all.
The diagnosis requires clinical evidence (unspecific abdominal and/or general signs, enlarged abdominal  lymph nodes, positive IGRA or Mendel-Mantoux test) and species diagnosis of the AFB´s by conventional or molecular (PCR) methods.
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Should prednisolone be used in the management of immune reconstitution inflammatory syndrome in patients with HIV/AIDS?
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Personally, I think it is case dependent. First, it has to be determined whether its an unmasking or paradoxical IRIS. Starting steroids in under-controlled infections can be detrimental. 
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in the most viral infection experiment, it is common sense that prior to inoculate virus, cells cultures are washed in order to get rid of serum, in some cases, even after infection, the medium are serum free. as I known, for example, flu A viral infection needs plasminogen and its NA protein cooperate to cleavage flu HA protein or exogenous trypsin to cleavage HA.  but why in the many experiments, serum were not added, because calf or human serum contain plasminogen.
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There's a bunch of reasons.
Yes, most strains of flu (excepting HPAI H5 and H7s and a handful of plasminogen-dependent strains) are largely trypsin (or other exogenous protease) dependent for replication in cell culture. This is to cleave the HA so it becomes fusion competent (not receptor-binding competent - that activity is present in the uncleaved trimer). So if you're growing a stock or performing a low MOI multicycle replication experiment, serum is left out to avoid inactivating the trypsin. If you're doing a single cycle/high MOI infection and you don't care about titreing what comes out, then serum can be added back after the adsorption period.
Serum is left out at the point of infection because it contains inhibitors - decoy receptor molecules and other factors. There's a whole load of ancient literature on this (PMID 18904216 is one of the first) and if anyone's interested they can Pubmed "influenza", "serum" and "inhibitor" and read from the bottom up. The inhibition was virus strain and serum species specific, and although I don't recall the story in detail, I suspect the "francis inhibitor" and "beta inhibitors" etc were a mix of different flavours of sialic acid mucoids as well as other protein-based inhibitors that we might now call collectins/defensins etc.
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Maternal diagnosis of VZV was based on clinical symptoms and signs, positive specific IgM and IgG and positive PCR for VZV on samples from cutaneous lesions.
The newborn, was born at 37+5 weeks and IgG and IgM for VZV were tested two times in different laboratories.
No evidence of immunodeficency in mother and child.
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Hi
dear doctor
Your answer is yes!
1. After 8 wk or more of gestation mother with evidence of immunity against varicella will transfer maternal IgG Ab to their fetus, thus their newborn infants already have positive Anti-varicella Ab that reflect passively-acquired maternal Ab (The IgG of the newborn is solely of maternal origin)
2. On the other hand, maternal Ab titers are affected  by her nutritional and immune
status,
3.In rare circumstances, mother may have low or absent levels of circulating IgG antibody. In these cases, the mother cannot transfer protection to her infant
4. You should check IG profile of mother in addition to serum IgG/IgM level of both infant and her mother simultaneously
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We are trying to optimize dried blood spot specimens to quantitatively measure measles IgG in human samples. At low antibody titers, we are getting a lot of non-specific binding. I believe it is due to residual RBC and hemoglobin in eluates. Does anyone have any ideas on how to get rid of the excess RBC and Hemoglobin in whole blood samples? Does centrifugation help get rid of RBCs? I elute samples in 250ul of elution buffer and cannot dilute the samples any further.
Thanks in advance.
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 A quick high speed spin should help to get rid of the big pieces (RBC membranes, various agglutinated protein complexes).  You can also try modifying the elution buffer.  Since I don't know what is in your buffer here are  a few suggestions that might help
Adjust the Ca and Mg concentration down, maybe even to zero
tween or other gentle detergent that won't interfere with subsequent ELISA
mild reducing agents
quick high speed spin to get rid of the big pieces (RBC, protein complexes)
With all of these you will need to ensure they don't cause more problems than they solve
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I want to dissociate virus bound to antibody onto the Protein G sepharose high performance columns. Can anyone suggest a simple protocol which will enable me to separate them while keeping them intact? I came across some suggestions, like using KSCN or 0.1-0.4M MgCl2. I don't want to use any kit for the purpose. Any help will be appreciated. Thanks in advance.
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Hi! Concerning pH change, you have to be careful not to induce virus uncoating (if you want to obtain intact virions)...
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I am interested in the dynamics of the cellular immune response during Leishmania infection, particularly with regards to immune cell recruitment to the site of inoculation. What is the earliest time-point at which antigen-specific CD4+ T cells are present at the site of inoculation? Could you recommend any papers which have used flow cytometry or imaging methods to investigate this in mouse models?
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