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                  <P>As a country with the second largest population of tuberculosis patients in the world, China has gathered considerable experience of tuberculosis prevention and treatment. But the result of TB control in China seems still barely satisfactory. In order to get more exact understanding about the TB control in China, we went to the Beijing Research Institute for TB Control and local tuberculosis hospital and CDC in Fujian province in China to interview the relevant doctors or researchers. From consulting these front-line practitioners of TB control, we got a comprehensive understanding and inspiration about applying the power of synthetic biology to the diagnosis of TB in clinical practice.  
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                <h3 class="classic-title" id="Doctor LI" ><span>Interview with Doctor LI Bo</span></h3>
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                  <p><b>Q: What are the major methods in current tuberculosis diagnosis and the diagnostic criteria?</b></p>
                   <p><b>A:</b>
                  Early and accurate diagnosis is the key to the treatment of the patients with tuberculosis. For suspected cases, diagnosing active tuberculosis based merely on signs and symptoms is difficult. There are several diagnostic methods currently, including microbiological detection, biochemical detection, immunological detection, nucleic acid detection and imaging detection. Clinical sample culture is a typical part of the initial evaluation and the main definitive diagnosis of TB, called “the golden standard”. People with culture-positive result can be confirmed as TB patients. If the result is negative, combination with other inspection methods should be considered. According to the diagnostic criteria of TB in <span style="color:#00afd1"><a href="https://www.google.com.hk/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&cad=rja&uact=8&ved=0CB0QFjAAahUKEwie16XMrtzHAhXHLYgKHWQbAkA&url=http%3A%2F%2Fwww.beinet.net.cn%2Fpolicy%2Fwbj%2Fwsj%2F201306%2FP020130614537615694411.doc&usg=AFQjCNHi27gvqEzV6WFpl8Mesza9fR6FZw&sig2=PMz-IveBwDmiDZwCzCi82g">Beijing City tuberculosis control work specification &#40;2013 Edition&#41;</a></span>,people with one of the following symptom should be confirmed as the TB patients.
                    <br>
                    1&#41;&nbsp;Chest radiographic examination results are consistent with active pulmonary lesion &amp; Patients have typical TB clinical symptoms such as cough, expectoration and hemoptysis; 
                    <br>
                    2&#41;&nbsp;Chest radiographic examination results are consistent with active pulmonary lesion &amp; The PPD test is strongly positive;
                    <br>
                    3&#41;&nbsp;Chest radiographic examination results are consistent with active pulmonary lesion &amp; Anti-tuberculosis antibody examination is positive;
                    <br>
                    4&#41;&nbsp;Chest radiographic examination results are consistent with active pulmonary lesion &amp; extra pulmonary histopathologic examination verify the tuberculous lesion;
                    <br>
                    5&#41;&nbsp;After diagnostic treatment or follow-up observation, other pulmonary disease such as lung cancer can be ruled out.
                    <br>
                    However, for the culture process being time-consuming, the treatment is often begun before cultures are confirmed.
                    <br> 
                    
                  </p>
                  
                  <p><b>Q: What are the Advantages and Disadvantages of these mentioned methods?</b></p>
                  <p><b>A: </b> 
                   Sputum culture is not sensitive. The positive rate as low as 20% means multiple cultures must be made at the same time. In some situations, cultures need to be performed in several hospitals for further accurately diagnosis. 
                    <br>
                    Besides, difficult culture process for the slowing-growth TB can take 3-8 weeks for sputum cultures. If the drug resistance test is needed, it will take another 3-4 weeks. The long time waiting for the results not only delays the timely treatment, but also takes a high risk releasing a moving infection source to the public. 
                    <br>
                    The specificity of Serologic test is not so good. Positive results appear on the nontuberculous mycobacteria infection cases and healthy people who have been infected but not develop TB disease.
                    <br>
                    Chest X-ray usually works as a supplementary means, since that tuberculosis lesion often appear at the apex and the inferior lobe of the lungs. Such features are quite similar to some other pulmonary disease and it’s easy to confusing. So chest X-ray usually works as a supplementary means.
                    <br>
                    
                  </p>

                  <p><b>Q: What about the nucleic acid test? Is it widely used in MTB diagnosis?</b></p>
                  <p><b>A:</b>
                    So far as I know, the results we get from traditional PCR are susceptible to the environmental change, leading to the condition that the false positive rate is elevated. Meanwhile, the activity of the Taq DNA Polymerase is vulnerable, leading to the elevated false negative rate. Thus it puts forward high demands on the laboratory condition and the level of expertise. Because of the high false positive and false negative rate, it is not easy for clinician to give a reasonable explanation of the PCR results in many cases. Thus, the traditional PCR is out of the market. Before treatment, the PCR results can refer to the MTB infection to some extent. But it can’t be used to distinguish whether the MTB has already been killed or the MTB is still active when the treatment is being conducted.
                    <br>
                    Based on the traditional PCR, some new methods have been put forward. Xpert MTB/RIF can detect M. tuberculosis as well as rifampicin resistance-conferring mutations directly from sputum, in an assay providing results within two hours. It was endorsed by WHO for detection of TB and rifampicin resistance, and made a strong impact on the traditional tools for its accuracy, sensitivity and high detecting speed. PCR-based GenoType MTBDRplus 96 assay (HAINs Lifesciences, Germany) spends about 6-8 hours detecting whether the MTB is rifampin or isoniazid resistant. 
                    <br>
                    Compared to the traditional detection tools, these nucleic acid tests are much more expensive. The traditional PCR is 150 yuan per times. While Xpert is 650 yuan/times and HAINs is 525 yuan/times. In comparison, the smear acid-fast stain is only 15 yuan/times and the sample culture is 60 yuan/times.
                    <br>
                    </p>
                    <img class="col-md-12" src="https://static.igem.org/mediawiki/2015/0/0b/Peking-Practices-Consultation-Li_Bo3.jpeg"style="margin:10px">
                    <p class="text-center"><small>Figure 1. Discussion with Doctor Li in Beijing Research Institute for Tuberculosis Control.</small></p>
                  <p><b>Q: What’s the relationship between diagnosis and prognosis?</b></p>
                  <p><b>A:</b> 
                    If the diagnosis and treatment don’t conduct early, the MTB will spread widely and forms cavities in pulmonary, leading to serious complications and unrepaired cicatrices. Even all the MTB have been killed, the lesion can’t heal and the function of pulmonary can’t recover to normal after treatment. That is to say, the earlier we discover the infection and take proper measures, the better the prognosis will be. This puts forward a high demand for the MTB detecting tool. We need a more sensitive and specific toolkit to detect MTB for discovering the potential patients even before the incipient symptoms appear.
                    <br>
                  </p>
                   
                  <p><b>Q: Then is it feasible to make the asymptomatic carriers receive treatment if the government does a general TB screening on the whole population? </b></p>
                  <p><b>A: </b>
                   If we do like that, it must take the manpower and material resources it consumes into consideration, unless we have a very simple and convenient detecting method with reliable results that can be applied for TB screening. As I mentioned before, PCR requires high laboratory conditions and is expensive. Though the existing PPD screening test for college freshmen is simple, low specificity is still a big problem. Nontuberculosis mycobacteria existing in soil and other natural conditions can also make the body sensitive, leading to a positive result of the PPD test. If PPD test is widely used for TB screening, the therapy cost will be too high to be afforded by the government because there will be a large number of false positives.
                    <br>
                   Thus, the precondition to do such a population TB screening is to develop a simple and convenient detecting method with high specificity.
                  <br>
                
                  </p>

                  <p><b>Q: How to conduct TB screening currently? Is it a good idea to use the PCR as one of the early screening methods?</b></p>
                  <p><b>A: </b>
                  The symptom-testing is being advocating for early screening these days, which means discovery and recognition of suspicious symptoms still depend on the people themselves. At the same time, the screening using chest X-ray and PPD test to the special population such as underclassmen or people with TB contact history also helps finding early cases. Nowadays, national-wide health education of school and community is helping the public to learn more about the incipient symptom of TB.
                    <br>
                  However, reliable testing tools that can be used for wide screening are still desired urgently by our health workers.
                  <br>
                  
                  </p>
                  
                  <p><b>Q: If we develop a new nucleic acid detection method or toolkit, which features are necessary for its popularization and employment?</b></p>
                  <p  id="Director LAO"><b>A: </b>
                  From the clinical point of view, the most important thing is that the sensitivity and specificity being really high. For the sensitivity, the results can be got even the sputum sample contains a few bacteria; for the specificity, the method should have the ability to distinguish MTB from NTB or other bacteria with a close relationship. What’s more, this tool is better to be time-saving and affordable for the common people.
                  </p>
                   
                
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                <h3 class="classic-title" style="margin-top:50px"><span>2)  Interview with Director Wang Sumin</span></h3>
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                   <p><b>Q: Could you please introduce current methods in diagnosis of TB?</b></p>
                   <p><b>A: </b>With the development of molecular biology, various detecting methods have greatly improved our understanding of the biology of the mycobacteria and provide powerful tools to combat the diseases caused by these pathogens. For tuberculosis, different methods have different roles in diagnosis of TB.
                    <br>
                    PCR-based test has been applied to detection of TB since 1990s, allowing detection of small numbers of disease organisms. However, it shows vulnerability to contamination of samples, and the rate of false-positive is high. With the emergence of advanced techniques, things got better since 2000. 
                    <br>
                    The Real-time Quantitative Fluorescence PCR is one of the most powerful and sensitive techniques available. In 2005, German scientists utilize linear PCR to detect MTB and rifampicin resistance at the same time. After that, the Xpert MTB/RIF developed by Cepheid Company was endorsed by WHO for use, and declared as a major milestone for global TB diagnosis for its accuracy, sensitivity and high detecting speed (two hours per sample). It should be mentioned that the melting curve method developed by Xiamen University has gone through the 6th approval of SFDA, which can detect resistance of rifampicin, isoniazid and ethambutol simultaneously. LAMP (Loop-mediated isothermal amplification) also made the headlines recently.
                    <br></p>

                    <p><b>Q: We noticed that the applications of these advanced detecting tools you mentioned are restricted in clinical practice, why does it happen?</b></p>
                    <p><b>A: </b>Getting research into practice is extraordinarily convoluted and difficult. What has been learned in the research setting often is not implemented into daily clinical practices, even for national institutes of TB control.
                      <br>
                      One of the main reasons is the high cost of diagnosis. Expense on reagents and maintenance of facilities raises the price to an extremely high level – about ten times higher than traditional detection methods, not to mention the manpower and other resources which have been put into. Expense of TB examination using new methods is not covered by health assurance, which made it more difficult to be accepted by ordinary people. 
                      <br>
                      Another important reason is the preconceived notions. Most of the doctors in China were born in 1970s, and receive traditional medical education. They tend to believe stained bacteria under the microscope and colonies on the culture medium, instead of numbers or curves on an electronic screen.
                    <br></p>

                    <p><b>Q: What’s your perspective on the future of nucleic acid detection?</b></p>
                    <p><b>A: </b>It makes me thrilled. I believe that this tool will unlock a new era of pathogen detection in no more than five years. There will be one day that we are able to diagnose TB with nucleic acid detection even if all other test results are negative. Compared with nucleic acid detection, current diagnostic methods are far from perfect, considering their sensitivity, specificity and time-cost. As to high cost of nucleic acid detection and conventional mind I have mentioned, I believe that time will change everything.<br></p>

                    <p><b>Q: What the necessary features do you expect in our detecting device?</b></p>
                    <p><b>A: </b>I think these features should be included:
                      1) Able to lyse bacteria and release nucleic acid.
                      <br>
                      2) Able to amplify the nucleic acid fragment.
                      <br>
                      3) Able to show the result intuitively.
                      <br>
                      4) Disposable.
                      <br>
                      5) Low Cost (lower than 100 yuan for an examination)
                      <br>
                      6) High specificity and sensitivity.
                    <br></p>


                    <p><b>Q: Let us suppose that a patient wants to know whether he has recovered from TB after treatment, however, both the living and dead bacterium will lead to a positive result. What’s your opinion on this case? Do you think it will cause misdiagnosis?</b></p>
                    <p  id="Director DING"><b>A: </b>I don’t think it really matters. Note that as a chronic disease, TB has a slow progression and long treatment time. Let’s go into some more details. Patients usually receive more than six months of treatments, 80%-85% of whom will have been recovered in the fourth month. Regarding that it is almost impossible for anyone to expel dead bacteria for two months, which means that, if a patient still has a positive result after six months of treatment, he is not recovered and needs more treatment.</p>

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                   <h3 class="classic-title" style="margin-top:50px"><span>3)  Interview with Director DING Beichuan</span></h3>
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                     <p><b>Q: From the prospects of laboratory workers in the field of TB diagnosis, do you know how the current TB diagnosis methods apply in the clinical practice?</b></p>
                     <p><b>A: </b>At present in Beijing, all the institutes of TB control are able to diagnose tuberculosis through almost all of the current methods. To be specific, sputum smear and sputum bacteria culture were used 110 000 and 80 000 times last year respectively. It is a striking contrast that molecular detection methods are totally used 5 000 times only. Therefore, classic TB diagnosis methods including sputum smear and bacteria culture play a dominating role in the clinical practice, which becomes an obstacle for promoting these molecular detecting methods.
                      <br>
                      Situation can be worse in the other provinces of China with little funds investment. In most institutes for TB control in the county level, there are no molecular detecting platforms, not to mention the applications in clinical practice. It is an obvious fact that the proportion of molecular detecting methods is much lower than that in Beijing.<br></p>
                                         <p><b>Q: What do you think of molecular detecting methods based on nucleic acid in the future?</b></p>
                    <p><b>A: </b>I think the poor applications of nucleic acid detection methods can soon be changed as the TB diagnostic criteria will be amended with the results of nucleic acid assays being included. At the appointed time, its applications must have a great improvement. After all, nucleic acid detection has its excellent superiorities over the classic diagnostic methods. In conclusion, this kind of method has huge potential to be the first choice for patients suspected of tuberculosis. <br></p>
                    <p><b>Q: If we develop a new nucleic acid detection method or toolkit, which features are necessary for its popularization and employment?</b></p>
                    <p><b>A: </b>According to the national stipulation, it must be sensitive and specific reaching a floor level of 85%. As lab operators, our requirements for detecting time may be different from the doctors and the patients. We just hope that our operational process is as simple as possible, without regard to a short detecting time. For instance, a method with a long waiting time is better than one which requires a short but complex operational time for the laboratory workers. However, from the point of TB patients, a quick detection method is obviously better.
                    <br></p>
                    <img class="col-md-12" src="https://static.igem.org/mediawiki/2015/9/91/Peking-Practices-Consultation-Interview-Ding_Weichuan2.jpg">
                    <p class="text center"><small>Figure 2. A visit to the Central Lab of Beijing Research Institute for Tuberculosis Control. The director was showing the experiment of drug-resistance testing.</small></p>
                    <img class="col-md-12" src="https://static.igem.org/mediawiki/2015/2/25/Peking-Practices-Consultation-Interview-Ding_Weichuan3.jpg">
                    <p class="text center"><small>Figure 3. HP group with Director Ding in the Central Lab</small></p>
                                      </div>

                   <h3 class="classic-title" style="margin-top:50px"><span>4)  Visit to primary medical units</span></h3>
                   <div style="padding-bottom:20px"> 
                    <p><i>TB mainly occurs in the middle-sized and small cities and rural area in China. In order to have a comprehensive understanding of the current situation, our team went to Zhenghe, FuJian Provence to conduct some interview and collect some data. The county hospital and Zhenghe research institute for tuberculosis control were our destination.</i></p>
                    <p><b>Destination 1: Zhenghe County Hosiptal.</b></p>
                     <p>We were luckily to have the opportunity to interview the director of infectious disease department, let’s have a look at the impressive interview.</p>
                     <img class="col-md-12" src="https://static.igem.org/mediawiki/2015/e/ea/Peking-Practices-Consultation-Jiceng1.jpg">
                     <p><b>1. What are the current diagnostic methods in Zhenghe county? What about the confirmation criteria? </b></p>
                    <p><b>Director: </b>The mainstream diagnostic methods in prefecture-level hospitals are sputum smear, sputum culture, and imaging examination, of which bacteria culture is the golden standard for TB confirmation. Also, the clinical symptom is a vital reference for the diagnosis. Unfortunately, the nucleic acid detection tools haven’t been recommended and used.</p>
                    <p><b>2. What’s your opinion of the current examination’s shortcomings?</b></p>
                    <p><b>Director: </b>For the bacteria culture, the main deficiency is high time cost and low positive rate (no more than 20%). It needs to emphasize that the bacteriology diagnosis of MDR-TB, which is more and more common, needs almost twice the time. For the imaging examination, its application is too limited which means it can only be used for the pulmonary tuberculosis, not extra pulmonary tuberculosis. Meanwhile, the result of imaging examination can’t confirm the TB, because of the several diseases having similar imaging result.</p>
                    <img class="col-md-12" src="https://static.igem.org/mediawiki/2015/9/93/Peking-Practices-Consultation-Jiceng1-1.jpg">
                    <p class="text center"><small>Figure 4. A visit to the Infectious Disease Department in Zhenghe County Hospital</small></p>
                    <p><b>3. Is there any problem of TB control in Zhenghe County?</b><p>
                    <p><b>Director: </b>On the one hand, the base installation of TB control and expenditure are insufficient, leading to the large gulf between the primary medicine units and metropolis medicine units. We also need more professional and technical practitioners to provide professional service and promote the development of primary medicine care. By the way, the communication between institute of TB control and hospitals is supposed to be more thorough to stop passing the buck to each other. </p>
                    <p><b>4. Have you ever heard of the nucleic acid test and would you like to use it?</b></p>
                    <p><b>Director: </b>There are no such methods or tools (nucleic acid detection) in either hospital or institute of TB control in Zhenghe County and we know little about that. If there is a new tool with high specificity, high sensitivity and low cost, it will surely make a great contribution to our hospital.</p>

                   
                    <p><b>Destination 2: Zhenghe research institute for tuberculosis control.</b></p>
                     <p>We were warmly welcomed by the director of epidemic prevention station, the conversation with him give us lots of inspiration.</p>
                     <img class="col-md-12" src="https://static.igem.org/mediawiki/2015/d/de/Peking-Practices-Consultation-Jiceng2.jpg">
                     <p><b>1. What is the current situation of tuberculosis in Zhenghe County, and what are the demographic characteristics of TB patients?  </b></p>
                    <p><b>Director: </b>At present, Zhenghe County has a population of over 200 000 and there are 170 000 people permanent living here. Among the whole population, about 80 people are tuberculosis patients. TB was a serious disease during the 1990s. It was controlled a few years ago with an ascending trend in the incidence rate. The prevalence in male was higher than in female, and gradually increases by age, with most of the patients between 18 to 60 years old, which accords with the national statistics results. However, with large number of peasant-workers going out for jobs, the morbidity in rural areas is consistent with that in cities, which is different from the current situation of the whole nation. </p>
                    <p><b>2. What are the tuberculosis diagnosis procedures and the diagnostic criteria in Zhenghe County?</b></p>
                    <p><b>Director: </b> In the current situation, the primary diagnostic methods are PPD test, sputum smear, bacteria culture and chest X-ray, along with the patients’ clinical signs. In addition, we would sometimes use erythrocyte sedimentation rate measurement and reactive protein items. But the most frequently used one is sputum smear. Due to the high price and complicated operations, we usually turn over the sample to the municipal institution to get the finally result.</p>
                    <p><b>3. What are the current control measures for TB in the county epidemic prevention station?</b><p>
                    <p><b>Director: </b>All the newborn babies will receive BCG for the early prevention. But the vaccine can only function effectively on pulmonary tuberculosis for 3-4 years. For the suspected cases, we will provide a initial examination freely, but the following examination will be self-paying if the TB is confirmed. Unlike the examination, most of the drugs are free, of which the first-line anti-tuberculosis drugs are all free and the second-line drugs are paid 70% by government.</p>
                    <p><b>4. Is there any problem of TB control in Zhenghe County?</b></p>
                    <p><b>Director: </b>The biggest challenge we are facing is the insufficient fund. It’s difficult for us to make ends meet because we never fully funded by the government while the policy make us foot the bills by ourselves. The insufficient fund leads to the shortage of intelligence and professions, which further results in a vicious circle. Withal, many patients can’t accept the self-paying treatment and examination of which the price is a little bit expensive. From my point of view, the backward regional economic and the distrust for medical Institutions lead to the terrible condition. </p>
                    <img class="col-md-12" src="https://static.igem.org/mediawiki/2015/5/54/Peking-Practices-Consultation-Jiceng2-1.jpg">
                    <p class="text center"><small>Figure 5. Our team member with Director Lv of Epidemic Prevention Station in front of Zhenghe Research Institute for Tuberculosis Control</small></p>
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