As the second largest country of tuberculosis patients in the world, China has gathered considerable experience of tuberculosis prevention and treatment. But at the same time, we are facing unprecedented challenges. In order to investigate the real situation about the prevention and treatment of TB and the scientific research on TB, we went to some authorities of TB control in Beijing and local tuberculosis hospitals and CDCs. From consulting these front-line practitioner of TB control, we’ve got lots of information and inspiration. Also we introduced and presented the conception and application of synthetic biology to them.
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LI Bo, the associate chief physician in Beijing research institute for tuberculosis control, has fought in the front line of TB control more than 10 years. During the interview, she told us about the transition of TB therapy and prevention in the last 10 years.
Q1. What are the major methods in current tuberculosis diagnosis and the diagnostic criteria?
Doctor Li:
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
1) Chest radiographic examination results are consistent with active pulmonary lesion & Patients have typical TB clinical symptoms such as cough, expectoration and hemoptysis;
2) Chest radiographic examination results are consistent with active pulmonary lesion & The PPD test is strongly positive;
3) Chest radiographic examination results are consistent with active pulmonary lesion & Anti-tuberculosis antibody examination is positive;
4) Chest radiographic examination results are consistent with active pulmonary lesion & extra pulmonary histopathologic examination verify the tuberculous lesion;
5) After diagnostic treatment or follow-up observation, other pulmonary disease such as lung cancer can be ruled out.
However, for the culture process being time-consuming, the treatment is often begun before cultures are confirmed.
Q2. What is the Advantages and Disadvantages of these mentioned methods?
Doctor Li:
Sputum bacterial culture
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.
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.
Serologic test
The specificity 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.
Imaging diagnosis
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.
Q3. What about the nucleic acid test? Is it widely used in MTB diagnosis?
Doctor Li:
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.
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.
Compared to the traditional detecting tools, these nucleic acid tests are much more expensive. The traditional PCR is 150yuan per times. While Xpert is 650yuan/times and HAINs is 525yuan/times. In comparison, the smear acid-fast stain is only 15yuan/times and the sample culture is 60yuan/times.
Q4. What’s the relationship between diagnosis and prognosis?
Doctor Li:
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.
Q5. Then is it feasible to make the asymptomatic carriers receive treatment if the government does a general TB screening on the whole population?
Doctor Li:
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.
Thus, the precondition to do such a population TB screening is to develop a simple and convenient detecting method with high specificity.
Q6. How to conduct TB screening currently? Is it a good idea to use the PCR as one of the early screening methods?
Doctor Li:
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.
However, reliable testing tools that can be used for wide screening are still desired urgently by our health workers.
Q7. If we develop a new nucleic acid detection method or toolkit, which features are necessary for its popularization and employment?
Doctor Li:
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.
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Q1. Could you please introduce the current method in diagnosis of TB?
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.
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.
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.
Q2. We noticed that the applications of these advanced detecting tools you mentioned are restricted in clinical practice, why does it happen?
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.
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.
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.
Q3. What’s your perspective on the future of nucleic acid detection?
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.
Q4. What the necessary features do you expect in our detecting device?
1) Able to lyse bacteria and release nucleic acid.
2) Able to amplify the nucleic acid fragment.
3) Able to show the result intuitively.
4) Disposable.
5) Low Cost (lower than 100 yuan for an examination)
6) High specificity and sensitivity.
Q5. 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?
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.
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Q1. 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?
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.
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.
Q2. What do you think of molecular detecting methods based on nucleic acid in the future?
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.
Q3. If we develop a new nucleic acid detection method or toolkit, which features are necessary for its popularization and employment?
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.
