For more than 10 years, diagnostic methods for TB have been developed globally, and some new methods have been approved for use in the United States, Europe, and other developed countries. For a country with a high burden of tuberculosis like China, the detection of patients basically by conventional bacteriological testing methods may miss many patients with active tuberculosis, causing the spread of tuberculosis and aggravating the danger to society. I will focus on the need of society for new diagnostic methods for TB, the status of routinely used tests and several promising tests, and explore promising diagnostic methods that may be used.
I. The need for new diagnostic methods for tuberculosis
In the absence of a truly effective tuberculosis vaccine, treatment of bacillary-positive tuberculosis remains the most important component of tuberculosis control programs. Rapid and accurate diagnosis is a necessity, and in both developed and backward countries, rapid and more accurate diagnostic methods of TB than conventional methods are an inevitable trend for controlling active TB.
In many places, the value of sputum smear for finding Mycobacterium avium complex growing in the sputum examination of patients has gradually decreased, and the diagnosis of tuberculosis is only a possible diagnosis, combined with the presence of cough and abnormal chest X-ray images in older people, or if the patient is HIV-infected, the diagnosis of tuberculosis is established. This has led to a decline in the specificity of sputum smears in the diagnosis of TB, even down to 50% in some cases. In clinical experience, a test with 50% sensitivity and 95% specificity is of far greater value than one with the same sensitivity but only 50% specificity. In the former test the reliability of a 10% ambulatory diagnosis was 53%, and conversely in the second test the reliability was only 10%, and the value of excluding the diagnosis was 94% and 50%, respectively.
On the other hand, a test that can quickly distinguish between Mycobacterium tuberculosis and Mycobacterium avium complex, for example, with such a device can avoid unnecessary contact with patients at the beginning of the health department to detect bacteriologically positive patients and can allocate limited resources for use where they are more needed. In the United States and other countries, the widespread use of nucleic acid amplification tests has evolved to a certain extent, although these tests have not yet been validated and the devices have not yet been finally adopted.
The lack of rapid and accurate diagnostic tests for tuberculosis has resulted in significant expenditures on isolating patients and unnecessary empiricist treatment. In addition, such treatment may increase costs by causing drug side effects.
In poor countries, the focus is on diagnosing and treating bacillary-positive TB patients, mainly due to.
First, due to the high cost, culture of mycobacteria is not widely available in many parts of the world and diagnosis relies mainly on clinical presentation, X-ray and sputum smear. Second, sputum positivity indicates a high likelihood of infectiousness and there is significant significance in treating such patients to reduce the source of infection. Because of these conditions, this is of course the only way to go, but sputum-negative patients also need to be treated because, first, especially in poor countries, a huge number of TB patients are bacillus-negative cases, and for the reasons mentioned above these patients are undoubtedly going to be misdiagnosed, leading to severe disease progression (which may cause permanent damage such as loss of lung function).
Second, although sputum-positive individuals are responsible for the majority of TB transmission in the population, recent analysis using restriction endonuclease section length polymorphism (RFLP) studies has demonstrated that sputum-negative TB patients are also infectious, not as non-infectious as previously thought, and that infectivity increases with the number of bacillus-negative cases. Thus, accurate diagnosis by sputum-positive individuals is a valuable expense, both for individuals and groups with TB.
In both resource-poor and affluent countries, clinical features (mainly patient symptoms and x-ray presentation) can increase the rate of TB diagnosis by 50% or more, and these can help to target suspicious patients and perform routine diagnostic tests, but it is clear that clinical features alone cannot provide a reliable basis for TB diagnosis.
Second, new diagnostic tests for tuberculosis
1. Diagnostic tests for active tuberculosis
Rapid culture systems for Mycobacterium bifidum: Notably, for example, BACTEC, MGIT, MB/BacT, Septi-check and ESP, when combining these liquid series of media with DNA probes for rapid mycobacterial identification, most sputum smear-positive specimens can have positive results in 2 weeks or less, and smear-negative specimens only take less than 3 weeks. Since this method is the only one that is widely used and can be used for drug sensitivity testing, its value is irreplaceable.
Nucleic acid amplification tests: There are 2 widely used nucleic acid amplification techniques in the United States, MTD (Genprobe) and AmPlicor (Roche), and MTD is primarily used in cases of sputum-negative but highly clinically suspected tuberculosis. It provides a constant temperature environment for nucleic acid amplification, while the AmPlicor device applies PCR technology to multiply unique target nucleic acids that can be used to identify Mycobacterium tuberculosis in clinical specimens. Although the target nucleic acids that are multiplied vary, the published papers suggest that the clinical role of this series of experiments is essentially the same. They almost always provide a correct diagnosis in sputum smear-positive cases, and have a 50% confirmation rate in sputum smear-negative but culture-positive cases. Based on its advantages, the US FDA has used it from the beginning to identify sputum positive in clinically suspicious cases.
Based on the rapid diagnosis of tuberculosis with the NAA test, the American Thoracic Society recently asked: Where is the best use of NAA? The successive papers published on NAA tests have claimed that they can detect TB almost completely with reference to those cases that are reliably sputum positive. At the same time, the papers also state: this technique should be promoted, and it can be more accurate than the current methods of diagnosing TB.
In a recent experiment, NAA was compared with conventional sputum smear and culture, and their specificity was quite high, but their sensitivity was not quite the same, 50% for smear and 100% for culture, although the sensitivity of NAA was at the level of 80-84%, it was repeatedly demonstrated that it could detect all bacillary-positive cases and diagnose half of the smear-negative but culture-positive cases. Therefore, combining all cases (true positive and negative results), NAA is much more sensitive than smear and only slightly less sensitive than culture. Some new experiments report that NAA is also unique in diagnosing smear-negative tuberculosis. In other words, if someone asks, “Can I detect active TB in my patient with NAA?”
The current answer is probably 92-95% accurate, whereas the smear is only 80% accurate (with the caveat that the patient is primary, and if the patient is re-treated, the NAA will be less accurate). In fact, if the cost of the test is lower, NAA can be used instead of sputum smear to diagnose primary TB patients. Of course, its specificity must be equal to or higher than that of the antacid smear method (AFB) now applied in many areas. At present, because of its high cost ($50-$100 per test), NAA is relatively economical only in large central laboratories where large numbers of specimens can be tested quickly and easily at the same time. There are also reports suggesting that NAA could be used in poor countries when its price point is adjusted to a certain level.
The U.S. Centers for Disease Control and Prevention (CDC) recommends NAA for the diagnosis of active tuberculosis and recommends that an AFB smear and NAA be performed at the first sputum collection.
①If both are positive, the diagnosis of TB is basically confirmed.
②If the AFB smear is positive and the NAA is negative, they recommend using an inhibitor to suppress the possible breakdown of Mycobacterium tuberculosis in the specimen and then repeating the test.
③If the AFB smear is negative for NAA, the CDC recommends sending the specimen more often, and if the AFB smear is found to be positive, the patient’s diagnosis is confirmed.
④When both AFB smear and NAA are negative, an additional NAA should be done, and if it is still negative, the diagnosis of TB can be ruled out. It is also suggested that clinical workers must not ignore the relevant clinical basis, because the final diagnosis still depends on the efficacy and culture results.
It follows that the rational use of NAA for rapid diagnosis of TB should be guided by the following principles: NAA can be used to further confirm the diagnosis in those with a positive AFB smear. If the AFB smear is negative and the clinical diagnosis is highly suspicious, a sputum specimen should be taken strictly for NAA testing, which should be either deeply coughed sputum or sputum stimulated by induction of coughing. Although a rapid diagnosis cannot be made with this method in more than 50% of those with negative AFB smears but positive cultures, a fairly high rate of rapid diagnosis is evident in those cases with negative AFB smears. The feasibility of this method has been confirmed by the FDA in the MTD-2 trial, and NAA cannot be used in cases where the AFB smear is negative and the diagnosis of tuberculosis is not clinically adequate.
In the past, the role of sputum smear examination was mainly to determine whether the patient was infectious and to analyze efficacy, but there is no information on whether NAA has this ability. Although it has been claimed that a negative NAA on several different specimens can largely exclude tuberculosis, there is still no evidence on how many times a negative NAA should be done to completely exclude the diagnosis of tuberculosis. In addition, the diagnostic value of NAA for tuberculosis is inconclusive.
For example, if the cerebrospinal fluid (CSF) specimen is positive for NAA, it can be presumed that the patient has tuberculous meningitis, but for those cases that basically meet the clinical diagnostic criteria, even a negative NAA cannot exclude the diagnosis, so at present, the NAA series of experiments still cannot replace the status of specimen culture.
2, rapid determination of drug resistance
In many parts of the world, multidrug-resistant tuberculosis has become a serious problem, so rapid drug-susceptibility experiments have great significance for the treatment and control of tuberculosis. In most cases, finding rifampicin resistance is sufficient to indicate the need to switch to a second-line drug against TB. This is done by testing the actual population for drug resistance (observing the survival status of the organism in the rifampicin environment). One such method, called “nematic analysis”, which determines resistant organisms by detecting changes in the rpoB gene associated with drug resistance, has generated considerable commercial profits in Europe.
This method is particularly popular in places such as Russia, where there is a high number of multi-resistant TB patients, and in places where primary drug resistance is a serious problem. However, it has not been used clinically so far because of its high cost (at least the equivalent of one NAA test per test).
Analysis of luciferase gene report: In this experiment, a specimen (e.g. sputum) is placed in the culture medium and mixed with Mycobacterium tuberculosis phage containing luciferase after noon, and if there are surviving Mycobacterium tuberculosis in the sputum, they will be swallowed into the phage, at which time the luciferase gene starts to work and becomes free luciferin emitting visible light. Then, these specimens containing Mycobacterium tuberculosis are placed in a medium containing anti-TB drugs and are ready for drug sensitivity testing.
Although the test was initially impressive, its clinical progress has been slow. Researchers have struggled to find a technically simple and economical method that can be used in economically disadvantaged countries. The current version of this test is the BronxBox, which uses artificial fluorescent plate film to display and record the results of the test, and is able to detect live Mycobacterium tuberculosis within 2 days.
Molecular signaling: A method to detect Mycobacterium tuberculosis using molecular signaling technology was published with molecules that glow during specific chemical reactions that occur when primers with unique DNA are rolled back to their target fragments in a PCR reaction. In this way, accurate and rapid diagnosis can be made with high accuracy and specificity, and it can be used not only for the diagnosis of tuberculosis, but also for the rapid determination of antibiotic resistance, but the molecular signal requires expensive equipment, so it is not widely available. However, in the future, PCR for detecting those infectious diseases is being developed and improved by clinical laboratories, and molecular signals will be more and more widely used.
3.Detection of latent tuberculosis
Recent research guidelines for detecting latent TB infection have focused on the selection of test targets, with only those patients who will likely be positive being considered, and another method for more accurately determining latent infection (with no cross-reactivity with either BCG or NTM) will greatly assist this strategic goal.
Studies of interferon production by peripheral blood mononuclear cells: As a response to stimulation by M. tuberculosis, these CD4+ and CD8+ T lymphocytes produce gamma-interferon, which has anti-inflammatory effects. Recently, attempts have been made to use these immune responses to diagnose M. tuberculosis infection. Isolated peripheral blood mononuclear cells from patients with established tuberculosis are used to synthesize PPD, and the γ-interferon stimulated by these cells can be simply detected by ELISA. A few studies have reported that this method can accurately detect latent TB infection without the need for patient follow-up to ask for test results compared to the traditional skin test, but it is more technically complex and expensive.
The diagnosis of latent TB infection has been limited by the lack of a gold standard. Initially, the test using γ-interferon produced by tuberculin stimulation was performed on people who were likely to have latent TB infection, and the sensitivity of this method was 90% and the specificity was 98% when analyzed with skin test results. It has also been shown that this method can distinguish between those who are truly infected and those who are BCG vaccinated, and has also demonstrated some ability to distinguish between Mycobacterium tuberculosis and nontuberculous mycobacterial infections.
The specificity of IFN can also be increased by stimulating cells with early “6-antigen (ESAT-6)”, which is a product of mycobacteria, including BCG, and can be used to differentiate between Mycobacterium tuberculosis and other mycobacteria.
However, the feasibility and operability of IFN for the diagnosis of latent tuberculosis is still a question, as it needs to be compared with TST and TFN in areas with high incidence of tuberculosis to see which test is more accurate in predicting the progression of active disease.
III. Conclusion
Although many new techniques for the diagnosis of tuberculosis have been developed and are superior to the AFB smear method in terms of sensitivity, specificity, and statistical accuracy, only one nucleic acid amplification assay (NAA) has actually gained traction. Although these new techniques represent a tremendous improvement in accuracy over the AFB smear method and are more time efficient than Mycobacterium culture, the higher investment and equipment requirements greatly limit the clinical application, and rapid drug sensitivity testing is also feasible for clinical use in the laboratory. However, it must be noted again that none of these new techniques can yet accurately assess a patient’s infection and detect the effects of treatment as can the two AFB smear methods currently in use.
The development of new testing techniques for tuberculosis faces enormous challenges and is difficult to scale up even in relatively wealthy countries, let alone poor ones, because of the high costs. Therefore, in the future, new techniques for diagnosing latent infection and active TB must not only be clinically useful but also cost-effective.