Tuberculosis - Science topic

Dear Dr. Faiz Ahmed Raza

I am sending you the manual containing the troubleshooting for your ROTINA 380 R (Hettich GmbH ) centrifuge machine.

I hope it helps you to solve your problem.

Best regard for you

Hi! I just completed a protocol to induce autoimmune myocarditis in Balb/c mice and I was told the Pertussis toxin is absolutely required to induce a strong immune reaction in Balb/c. In A/J mice it is not necessary. So my tip for you is to use the toxin as well (500ng per mouse IP).

You can kill the MTB by heating 80°C for 2hrs if solid culture or 80°C for 1hr if liquid culture

.

Until recently urine testing for MTB was unreliable (except for renal TB) even in immunocompromised individuals- such as HIV.

Xpert methodology will change that.

Use the proportion of treatment outcomes and the Wang and Chow (2007) formula for the calculation of the sample size.

Regards

Soumajit Mukherjee Thanks so much.

Yes there's

this list might help you

Dr. Tanya Singh and her colleagues at the National Institute of Immunology (NII) in New Delhi.

Dr. Amit Singh and his team at the Indian Institute of Science in Bangalore

Dr. Pramod K. Jangir and his team at the CSIR-Central Drug Research Institute in Lucknow

Dr. Anil K. Tyagi (ICGEB) in New Delhi

Dr. Manju Y. Krishnan and her team at the Indian Institute of Technology in Madras

Dr. Pradip K. Chakraborty and his team at the Indian Institute of Chemical Biology in Kolkata .

Dear colleague! Unfortunately, they do not work with such software in the Russian Federation. However, for more than 80 years of existence of the tuberculosis service of the USSR and further in the Russian Federation, a list of questions to the population has been developed and standardized, allowing to identify epidemiological chains and risks for the disease and the epidemiological spread of tuberculosis. This makes it possible to identify and promptly sanitize the foci of epidemiological danger. In addition, a system and algorithms have been created to diagnose cases of granulomatous inflammation among the population, which includes not only tuberculosis, but also rheumatoid diseases, infectious pathology of the section of anthropo-zoonotic infections, etc. I am a specialist pathologist in the diagnosis of granulomatous diseases and the scope of my competence is endoscopic and surgical diagnostics based on tissue and cytological material, followed by the identification of the type and properties of the pathogen, the characteristics of tissue reactions, classification of the identified forms of diseases and assessment of their epidemiological danger. If you need help in this area and my expertise to build a diagnostic system, I will be happy to help. If you are interested in the methodology of compiling questions for screening analysis of the possible development of epidemiological situations, as well as building a system of outpatient and inpatient care with the introduction of palliative and recreational zones, then I can arrange for you to consult the necessary specialists.

Sincerely, the Head of the Pathology Department of the Moscow Regional Clinical Tuberculosis Center.

Berezovsky Yuri.

I don’t know your country. I think India heath care system has this kind of Stewardship of Anti-tuberculosis. I have found a literature titled : ''Anti-tuberculosis treatment stewardship in a private tertiary care hospital in South India''... They had a Stewardship team. Correspondence are from Amrita Institute of medical Sciences and Research Centre. You may google it to find their address or number.

Hi,

Usually it happens while in-house development of any real time PCR. Factors such as purity of DNA, initial load in the provided template play a major role in such different results or obtaining lower RFU from clinical isolates when compared to the synthetic controls. This can be adjusted by checking the quality of the DNA by measuring 260/280 ratio and increasing the initial template concentration or template volume while setting up PCR. The increase in concentration need to be done carefully so that the problems such as deficient MgCl2 or dNTP can be avoided. You can do a template (after quantification spectrophotometrically / nanodrop) volume titration to find the optimal concentration which provide better RFU for your mastermix milieu.

I hope these points may shed some light for your progress. All the best

Kalyanaraman

MPT64 is one of the major culture filtrate protein (24 kDa) [6],[7] encoded by the RD2 region genes [8] and has been shown to be a specific antigen that differentiates the M. tuberculosis complex from the mycobacteria other than tuberculosis (MOTT) species. [5],[9] An MPT64-based, simple and rapid immunochromatographic assay was developed by the Standard Diagnostics, Inc. (SD) (Yongin, Korea), known as the SD Bioline TB Ag MPT64 RAPID ® test (SD bioline kit). This lateral flow test has been reported to identify the M. tuberculosis complex from the MOTT using the mouse monoclonal anti-MPT64 antibody.

Utility of MPT64 antigen detection for rapid confirmation of mycobacterium tuberculosis complex Arora J, Kumar G, Verma AK, Bhalla M, Sarin R, Myneedu VP - J Global Infect Dis (jgid.org)

I don't think so, because this test only evaluate degree of inflammation, not the cause of the disease

you can review the personal ( file) of the women .

Factors associated to low case finding of tuberculosis in SEA countries

If I am understanding this properly, it seems your sample input is too low. Can you obtain more cells and prepare more genomic DNA?

If you cannot obtain more DNA, then try adjusting your primer concentrations. Due to low DNA input, your primer concentrations could be too high for this PCR. I also recommend running the DNA products out on a TBE gel. Check to see the amount of primers remaining relative to the PCR product. Also, you probably need to adjust the number of cycles.

Hi Alexander Polster ,

Generally while growing e.coli harboring our plasmid of interest, we incubate the culture for 16 hours. After 12 hours of incubation, you can charge the culture with another dose of selection antibiotic, and finally harvest after 4 more hours. This will keep the bacteria under strict selection pressure to produce the plasmid of interest.

This has helped me.

Best,

Subham.

Suraj Kapoor

Thanks a lot for sharing the paper Sir. Got in touch with Dr Maunank,the corresponding author and gathered further insights. Was really helpful.

Trying to explore more options as well meanwhile.

Also check please the following useful link: https://www.datadictionary.nhs.uk/data_elements/provisional_diagnosis__icd_.html

Also, kindly check:

Pressing the bacteria with the LC50 intermediate generations. Requires level 4 biosecurity

Hevar Neaz,

This published method may be helpful for you

Have a look at this useful RG link.

You cannot combine them in Revman5 but can calculate each and "copy and paste (or screenshot)" by clicking the commands (right above of forest fig, OR, RR, or RD). It is very easy.

Hello,

You can refer to the research article given below. It will be helpful.

Best.

Thank you, this information is a great help.

In addition, the major socioeconomic determinants of TB needs to be addressed.

Tuberculosis is one of the debilitating chronic diseases that exhaust the patient and his immune system, I think here even if the person died due to covid, tuberculosis has increased the susceptibility to infection and the development of severe symptoms of the disease because it exhausted the body's immunity a lot, meaning that tuberculosis became a predisposing factor for that... My sincere gratitude to all.

I assume you are working with mouse hippocampal sections, if this is the case then try to use primary antibody raised in any species other than mouse. Anti-mouse secondary will bind with endogenous mouse IgG and gives false positive signals. As John said, DAB development step can be shorten to couple of minutes, 10 minutes are definitely too much.

Priya Murugan It seems that the plasmid is not commercially available and it is also stored at Addgene. Usually, if you generate a plasmid and publish your work, you should provide other with the plasmid. However, at the moment a lot of people in Europe are on summer vacation, and we are still in the middle of a pandemic. Maybe, you should give Prof. Munro a bit more time and send a nice email again...

As an alternative:

In the original paper

the insert for pHTAB2/Cyp121 was cloned from a cosmid from Stewart Cole from the Pasteur Institute in Paris, France. You might get the cosmid there as well.

If none of this works, Prof. Liu from the University of Texas also generated a plasmid for expression and purification of Cyp121. They did not use mycobacterial DNA as a template, but ordered the sequence.

If even this does not help, you could consider gene synthesis yourself.

Kind regards and good luck

Dear Johannes,

You are going to use the general protocol to isolate RNA. RNA isolation

Starting material: 1* 10^6 📷

Method:

A. miRNeasy mini kit (Qiagen), follow the manufacturer’s instructions.

B. General phenol-chloroform isolation method

1- put your samples in safe lock tubes (larvae sample)

2- Aspirate media and add appropriate amount of QIAzol to each well of cells , then collect your simples in safe lock tupes, and go to step 4.

3- Shake the tubes in the Bullet Blender speed 8 for 3 min

4- leave your samples 3 min on room temperature

5- Add 100 ul chloroform to the tubes and centrifuge them 15min- at 4 degree and 10,8 rcf

6- Remove the aqueous phase to new, fresh 1,5 tube

7- Add 100 ul 2- propanol and shake 15 sec (you will see a cloud like structure when RNA is present). To increase yield, samples can be keep at -20 for a couple of hours or overnight. This step is specially important for zebrafish larvae samples.

8- Centrifuge for 5 min at 4 c degree at maximum speed for pellet formation

9- Discard the solution and wash the pellet with 500ul- 750 ul Ethanol 70%

10-Centrifuge for 10 min at 4ᵒc at maximum speed. Then, a clear white pellet is visible

11-Remove the Ethanol till a small fraction is still covered in Ethanol. Then, Place the tube vertically

12- Let the remaining Ethanol airdry till the pellet no longer white but translucent

13- Add 25 uL RNA free water to the tubes and mix it thoroughly using the pipet.

Note: DNA removal is necessary for certain RNA applications that are sensitive to very small amounts of DNA (e.g., TaqMan RT-PCR analysis with a low-abundant target). Please, Follow the instruction in Appendix E: DNase Digestion of RNA before RNA Cleanup in RNeasy MINi kit.

14- Store your sample at -80ᵒc

Although it has some of these issues that you listed, it is still very reliable in TB research.

With great pleasure!

Tina Mamaladze ,cytopathologist from Tbilisi Georgia

Yes, you can. TB media and super broth both are rich media with high peptone and yeast extract contents. TB media also contains additional carbon source. Add 20mM of sterile glucose in super broth.

Using only LB media should also work.

All the best.

Dear Vipul Dhasade you can go through this article

You could compare difference in proportion adhering (%) before and after intervention. Then to detect a desired difference at 5% alpha significance with power 80% (say) use the standard normal approximation method given in any elementary statistics textbook.

an online calculator is given in the attached ref.

Have a look at this useful RG link.

Hi Roger,

Yes, I have used the same condition as I wanted to check in a given condition whether using rosetta enhances my expression compared to BL21(DE3). As my gene was already codon-optimized, I assumed that rosetta would at least express the protein.

Thank you for the suggestion; I will give it another try.

Robert Boer, Mohanad Kamaleldin Mahmoud Ibrahim Babak Jamshidi Thank you everyone for answering. Robert Boer I'm currently conducting a systematic review on the availability of TB services pre- and post- pandemic. I've included that report in my review, and I need to know the study design for reporting on study characteristics and study quality assessment. May I ask how would I assess the study quality for a surveillance study? This is my first time conducting a systematic review and there are a few things I'm still unsure of, any advice would be highly appreciated.

because it is autoimmune disease?

Hi Gulnaz,

Could you control the contamination of your culture? Please share your experience

Hi Hammad,

What gene(s) have you decided to use to detect TB? Keep in mind that you should run a gold standard (solid or liquid culture) to determine the performance of your qPCR test. Good luck

During follow-up, tuberculosis occurred in contacts of index patients in 120 (13%, ... A simplified risk score including only five variables performed similarly, with ... an in-house MDR/XDR-TB Colour Test thin-layer agar assay, ... tuberculosis within 3 years of the date that the index patient started treatment. Household contacts of patients with tuberculosis (TB) are at great risk of TB infection. ... The incidence of TB disease among the contacts of index cases was 4.4% ... for the individualized prediction of TB transmission among household contacts. ... 5.8 million men, 3.2 million women, and 1 million children (≤15 years) .

Dear Fiazall

it is quite strange:

1 liter in which flask?

It do not growth or it stop to growth at certain OD?

best regards

Manuele

..GI Endoscopy

..Barium studies

..Peritoneal fluid microscopy, Gene Xpert and TB culture

..Peritoneal gross examination and biopsy

..abdominal lymph node biopsy

..look for chest/lungs or other organs involvement

..history of TB contact(may need good inquiry)

..symptoms of fever especially afternoon or evening/night, anorexia, +/-constipation/or diarrhea, weight loss, lethargy,...

These points may help upto great extent in making diagnosis...

If physician is convinced to start anti tuberculous treatment, good response may be noted in 15-45days in majority of patients...important exclusion include drug resistant tuberculosis and other illnesses closely related to TB.

Can consult nearby physician experienced in the diagnosis and management of TB.

@eke692000 u try TB/NTM PCR kits equally use d conventional biochemical test for comparison dis help u 2 know d specificity of dis kits

You should use Atomic analyzer.

Hi,

According to your description, I think it may be fungi or phage contamination during cell culture. It is recommended to strictly sterilize the experimental materials. Or it could be that the target protein is toxic to cells.

in vivo

Thanks

Its really interactive map

Please go through the following RG links and a PDF attachment.

Please go through This PDF

Combined therepay for TB/HIV co-infection is effective, for a newly diagnosed HIV with TB, needs to start ant TB for the first 2 weeks, while assessing, IRIS, then we introduce ART based regimen, however remember to effect of rifampicin to DTG, we need to double the dose of DTG, as far as TLD based regimen is concerned, as this medication lowers the level of rifampisin, therefore, be cautius while administer TB drugs with the ART, know the issue of drug-drug interaction, thank you

Of course, although the key is to chose relevant inputs of data that is uncorrelated. And the decision of what is relevant at the moment is human based

I think it is possible to have several fabricated crises due to global conflicts

The two critical words in the Third Law are 'equal' and 'opposite'. The essence of 'Opposite' may be perceived well in various biological systems.

As pieces of exemplary shreds of evidence, in cancer therapeutics, tumor exerts strong pro-tumor response against applied treatment and imposes therapeutic resistance, one of the major problems seen in preclinical and clinical studies. The same goes for the multidrug resistance in various medically relevant bacteria.

However, measuring/gauging the essence of 'equal' may be a bit difficult task in biological systems.

AFTERALL, BIOLOGY IS A SCIENCE OF EXCEPTION. The rules of physics/chemistry may be applied directly in some cases while in others, they might seem to be bit 'diluted' or rather entwined with greater intricacy... reason: living systems are not isolated systems, which are often considered for the derivation of the rules/laws of physics and chemistry. It is not about the violation of the rules/laws... rather a manifestation of the same in different perspectives- unexplored/unexplained milieu...

The short response is: NOT indicated

The long response is: It can be an alternative to other anti-inflammatory treatments in exacerbator patients in the case of non-response to this treatment (for example: macrolides), always at the lowest dose possible.

The exceptions: 1. The presence of concomitant asthma. In this case inhaled steroids are needed. 2. Use of systemic steroids (like COPD) in severe exacerbations (usually required hospitalization).

Dear Israt,

you posted a very interesting question. Tuberculosis is an impressive sanitary burden since about 5000 years, but alas the Scientific Community treat such a disease as a secondary importance threat, probably because, nowadays, it is mainly diffused in Third World Countries. With this in mind, we can clearly see how Covid-19 pandemic nearly erased other diseases from the current emergencies agenda. In my opinion, you are absolutely right; tuberculosis is a very dangerous pathology, and it could cause a true disaster if we do not remember that Covid-19 is not the only danger. Alas, being the root of such problem a socioeconomic bias, it could be very difficult to bring such dilemma to the international agencies' ears.

Best,

Dave

You can autoclave the Yeast extract, tryptone and pH salt: K2HPO4, KH2PO4 separately. It will no precipitation anymore!

https://www.carlroth.com/medias/Info-Brochure-Phosphates-in-Media-EN.pdf?context=bWFzdGVyfHRlY2huaWNhbERvY3VtZW50c3wyNTc3MTl8YXBwbGljYXRpb24vcGRmfHRlY2huaWNhbERvY3VtZW50cy9oMzEvaDk3Lzg5NzY2Mjk5NTY2MzgucGRmfDZmMGRiOTE1YWUwYmFlZWM5M2QxZWI5ZTRkNjk2MzFmNGFjMmM0ODNiNDEzYzk5OGY0MDRkMjJiNjY5ZmEwM2Q

Thank you Michael. I induce at around 0.8-1 O. D. . Yes I have done the supernatant - pellet check on SDS page and there is a significant amount in the pellet in both untagged and tagged proteins.. I am actually concerned that the total lysate ( soluble and insoluble protein combined) is less for the tagged as compared to the untagged. And I have seen lower yields with decreasing temp. But I haven't tried cooling down first and then adding IPTG. I can try that. The last option you suggested is what I have been doing, at least with the second protein. I wanted to avoid doing this for both proteins . Takes a lot of labor and is time Intensive , since I am the only one prepping proteins. Not allowed to hire undergrads or masters students now coz that would have been helpful. Thank you nonetheless.

Qureshi MA, Afzal W, Khalique AB, Pasha IF, Aebi M. Tuberculosis of the craniovertebral junction. Eur Spine J. 2013;22 Suppl 4(Suppl 4):612-617. doi:10.1007/s00586-012-2497-3

you many find this useful

Despite the fact that emerging and re-emerging diseases pose a serious threat to public health in developing as well as developed nations, we are destroying our ecosystem by damaging the forest. We have published many papers on emerging and re-emerging zoonoses. All our papers are easily available at the Research Gate and Academic.

Jeff Falcone

I suggest you to look at the protocol down below in the link, would certainly help you:

https://www.cytivalifesciences.com/en/us/news-center/how-to-determine-dynamic-binding-capacity-10001

Government of India has launched tuberculosis control program through DOTS. The program is not affected by COVID-19.

Secukinumab may be an option. We have experience in psoriatic arthritis and tuberculosis. Considering the similar IL17 signature of the two diseases, it should be theoretically possible. Don't know of secukinumab data for IBD though.

Secondly, whether you include the countries with zero TB, you should both consider the statistical principle and practical meaning of your results. I'm not sure what kind of correlation you used, so suggest you to refer to the statistical book and see if your data met the use condition. If met, then you could consider whether the results of including and excluding zero TB countries are the same. If not, think about the differences and which one is more feasible.

I had the BCG vaccine when I was a kid and have always had a Negative TB skin test.

Hello dear dr. Yazi .. see attached link please

very attractive ideas.

Remarkably, the repositioning drugs that are currently utilized for treatment of COVID-19 either have pyrazine, quinoline s or adenine moieties. Modification of these structures by incorporation of halogens and hydrophobic parts at the vacant positions of the heterocycles will lead to promising candidates as antiviral agents that might be used to fight against COVID-19.

BMI is the only thing i have for nutritional assessment. My plan is to compare TB treatment outcome among well nourished and undernourished using BMI. It is secondary data.

Whilst NMR may identify some systemic changes in biofluids as a response to the Covid-19 virus, it is unlikely to progress specific mechanistic understanding. Thus a combination of NMR and MS would be a better approach.

From literature around SARs an MERs, it seems that both cytokine response and lipid transport would be important. There are several metabolic profiling laboratories that are investigating the metabolic response using these technologies including Rutgers and the Australian National Phenome Centre

If multiple centres undertake similar research, there would be substantial merit in creating a broader international database

The main problem is that many people could fake not to have the virus just because of being scared of being labelled as “the one with the Coronavirus.

Thanks James Leigh !

Circulating IFNg levels? I don't know about very high. Usually they are significantly increased but I won't considered them very high. The IFNg at the site of infection is much higher.

You have to post the paper, or post the title and authors. How did they define TB patients, LTBI or active, untreated?