Rapid Diagnosis of Tuberculosis

Transcription

1 Rapid Diagnosis of Tuberculosis Ed DESMOND Richmond, US I. Rapid Diagnosis Methods I would like to discuss rapid diagnosis methods of tuberculosis. The traditional method is acid fast microscopy. However, the method we are all excited about is nucleic acid amplification. The culture method, of course, is not rapid, since mycobacteria grows extremely slowly. 1. Acid Fast Microscopy To measure the sensitivity of the Acid Fast Microscopy method, we conducted a study, published in We examined the sensitivity of the acid-fast smear in patients that had a positive culture. We examined both the direct smear and the concentrated smear, which was obtained following centrifugation. The study included two patient populations. The first consisted of middle-class Americans with health insurance, which means that they have enjoyed high access to medical care. The second group included people from developing countries. We observed that for both populations, the concentrated smear was more sensitive that the direct smear, and for patients from developing countries, its results were rather satisfactory. We have thus concluded that concentrating specimen by centrifugation improves the sensitivity of acid-fast microscopy. We have also observed that the direct smear was more sensitive for patients from developing countries than for the middle class Americans. This interesting finding might indicate that less wealthy people may wait longer before they visit a doctor. By that time, they may be sicker, and cough up higher numbers of TB bacilli. In such as case, even a less sensitive method, such as acid-fast smear, may still offer positive results. In our laboratory, we try to assure that acid-fast microscopy results are available and accurate. However, there are a few problems with the implementation of this method. The first is that sometimes microscopists do not devote sufficient time to reading the slides, resulting in false negative results. I believe it is necessary to examine the results for at least 10 minutes, before it is possible to determine that they are negative. Another problem is that microscopy laboratories might run out of staining reagents. The solution we propose is that regional laboratories produce staining reagents from raw ingredients, and provide them at no charge to local laboratories. A third problem is that sometimes microscopists, who are either inexperienced or poorly trained, will see small deposits of stain on a slide, and mistake these for acid-fast bacilli. Besides more efficient training, the solution to this problem could include panel testing. That is, a reference laboratory sends out slides with known presence of absence of mycobacteria, and asks the participating laboratories to stain them and send them back the results. Blinded slide rechecking is another useful approach for dealing with this problem.

2 2. Nucleic Acid Amplification The method we are probably all excited about is nucleic acid amplification. Several devices that use this method could be used to detect the presence of Mycobacterium tuberculosis. They include commercial products, such as the Gen-Probe MTD, or the Roche Ampllicor. A second development is the LAMP assay, which does not require a thermocycler, and could thus be used in laboratories that do not possess one. Nucleic acid amplification tests could be used for several purposes. in some US laboratories, they are used for all smear-positive specimens. However, for some patient populations, almost all of the smear-positive patients have TB, so it might be wasteful to run the test on all of them. The method could also be used for smear-negative specimens. For example, the Gen-Probe MTD is sufficiently sensitive to detect M. tuberculosis, even in smear-negative samples. However, some of these samples are obtained from patients with a low probability of having TB. Since there is a possibility of obtaining false positive results with this method, using it for smear-negative specimens in patient populations with a low prevalence of TB might lead to poor positive predictive values. In some cases, nucleic acid amplification testing could also replace the culture. However, at the moment, it is not as sensitive as the culture test, and provides no information about drug resistance. 3. A New Generation of Diagnosis Method Newer generations of techniques are currently under development. For example, a nucleic acid amplification test that detects drug resistance, as well as the presence of M. tuberculosis. Another technique is the line probe assays. These assays are already commercially available in Europe, but have not obtained FDA clearance yet. A third development is the molecular beacons assay, which is currently under development by Cepheid. Although it is not a commercial product yet, we nevertheless use it in our laboratory as a home brew test, for which we developed the method ourselves. Finally some automated methods are under development. They involve amplification, followed by an microarray analysis of the PCR product. II. Drug Resistance Testing In our laboratory, we conduct Rifampicin and Isoniazid resistance tests. We try to identify Rifampicin resistance by looking for mutations in the rpob core region, since over 95% of Rifampicin resistant M. tuberculosis strains have mutations in this region. To identify INH resistance, we look for mutations in katg and inha genes. In our patient population, these two genes can enable us to detect 83% of the INH resistant strains. 1. Line Probe Assays Several commercial line probe assays are available for the detection of M. tuberculosis and mutations associated with drug resistance. One example is the Genotype MTBDR from Hain Lifescience, which detects the presence of the TB complex, as well as mutations associated with Rifampicin and INH resistance. I believe they have not obtained US FDA clearance yet.

3 A second line probe assay is INNO-LiPA Rif TB, which was developed by Innogenetics. It detects the presence of TB complex, as well as and mutations associated with Rifampicin resistance. It does not detect mutations associated with INH resistance. However, 91% of the mutations associated with Rifampicin are also INH-resistant. 2. MTBDR Evaluations The best evaluation of Hain MTBDR was conducted by the NY State laboratory, using smearpositive sputum. After using the Hain MTBDR, they obtained valid results for 91% of the specimens. For the remaining 9%, the assay controls did not work properly. With respect to the detection of INH resistance, MTBDR looks merely for KatG mutations, so mutations in inha and other genes are not detected. In the study, MTBDR was able to detect 57% (50/88) of INH-resistant specimens, and 86% (48/56) of strains with high-level INH resistance. The performance of Hain MTBDR was better in detecting Rifampicin resistance. 96% (25/26) of Rifampicin resistant strains were detected through the detection of rpob mutations. It is noteworthy that the MTBDR managed to detected three strains with rpob mutations, which the culture-based method classified as susceptible. These strains were resistant at a lower Rifampicin concentration of 0.5 µg/ml. Thus, although a change has occurred in their Rifampicin susceptibility, the culture-based laboratory test classified them as susceptible. 3. INNO-LiPA Rif TB Evaluation A review article was published in 2005 about INNO-LiPA Rif TB, the line probe assay produced by Innogenetics. The review examined its detection of Rifampicin resistance in tuberculosis, and included four studies in which the LiPA probe assay was applied directly to clinical specimens. Its sensitivity for detecting Rifampicin resistance ranged from 80% to 100%, and was over 90% three of the four studies. In al four, the LiPA recorded a 100% specificity. That is, when a mutation was detected by in the rpob, it was indeed associated with drug resistance in culture-based testing. III. Molecular Beacons In our laboratory, we have been using the molecular beacon testing method for the detection of INH and Rifampicin resistant mutations. A molecular beacon is a DNA probe that has a hairpin shape. When the beacon is in its closed hairpin shape, it is not fluorescent, because a reporter dye located at one end of the probe is close to a quencher that prevents fluorescence. However, if the probe binds to its complementary target, it forms a double stranded DNA, and the reporter fluorescent dye is separated from the quencher, resulting in fluorescence. The method is used in real-time PCR. The probe target is amplified, and hybridization is detected as fluorescence. Our molecular beacons hybridize merely with the wild-type sequence. If the wild-type sequence is present, the molecular beacon opens up, the fluorescent dye is separated from the quencher, and fluorescence is obtained. On the other hand, if a mutant sequence is present, the probe part of the molecular beacon will not hybridize. The fluorescent dye would thus remain close to the quencher, and there would be no fluorescence; the beacon would remain off. The three molecular beacons we use detect mutations in the three most common sites associated with Rifampicin resistance. The method is rather sensitive, as it enables us to

4 detect 98% of Rifampicin resistance using three beacons. The advantage of the instrument output is that we can examine at what PCR cycle we begin to observe the fluorescence developing. This provides us with an idea of how much DNA was originally present in the specimen. The results are more reliable if there is a higher quantity of DNA. Thus, the molecular beacon enables us to evaluate the sample quality by examining the assay results. 1. Advantages Using molecular beacons with real-time PCR offers several advantages. First, the amplification and detection of the amplified product occur inside a sealed tube or microtiter plate. Thus, there is no risk that any of the numerous pieces of amplified DNA will contaminate the laboratory, causing false positive reactions in the future. Second, detection of the amplified product takes place during the amplification, thereby saving time and effort. Consequently, the results can be obtained in approximately two hours. Finally, real-time PCR results allow us to evaluate the quantity and quality of DNA in the sample. 2. Efficacy At the end of the first three years of experience, using both cultures and specimen sediments, we have examined the results of the molecular beacons testing. a. INH Resistance For Isoniazid drug resistance, there were merely nine cases out of 250 specimens, in which the result was not valid. In 50 out of the 56 cultures that displayed Isoniazid resistance, a mutation has been detected in either the KatG or inha genes. Thus, the sensitivity of the test is approximately 50/56. We also obtained a high predictive value. Thus, the detection of a mutation was a strong indication that the culture was indeed resistant. Today, the volume of our work is significantly higher than it was during the first three years, and we conduct about 500 tests per year. b. Rifampicin Resistance The same type of analysis was conducted with Rifampicin. In 39 out of 44 cases in which an rpob the mutation was detected, the culture was Rifampicin resistant. I was actually rather surprised by the fact that there were cases in which we detected a mutation, and yet the culture was Rifampicin susceptible. In addition, 39 out of the 40 cases in which the culture was resistant to Rifampicin, a mutation in the rpob was detected. INH and Rifampicin results are obtained very quickly in our laboratory, using the molecular beacon methods. TB control personnel and physicians who treat TB patients are extremely satisfied with the accuracy of the testing as a preliminary result. Of course, we always follow-up these tests with a culture testing. However, the results of the molecular beacon testing are used to guide therapy, until the culture-based results become available. 3. Success Stories In some cases, molecular beacons can be particularly useful. In one example, a laboratory received a mixed culture of M. tuberculosis and Mycobacterium chelonae, and was therefore

5 unable to conduct drug susceptibility testing. By using molecular beacon testing, we were able to discover that the TB was a multi-drug resistance strain. A second example involved Hmong refugee immigrants, who have spent many years in a refugee camp in Thailand. Many of these immigrants suffer from MDR-TB. Thus, when a patient from this group is acid-fast smear-positive, we are often asked to perform a molecular beacon testing. A fast detection of MDR-TB enables us to provide them with the proper treatment immediately. A third high profile case we worked on involved a recruit in a boot camp. When the soldier was identified as acid-fast smear positive, it was important to obtain a quick drug susceptibility result, in order to find out whether the other soldiers have been exposed to MDR-TB. 4. Limitations Unfortunately, molecular beacon tests do have some limitations. First, a limited number of genes and sites are targeted, so some mutations are not detected, especially for INH resistance. Second, emerging resistance in mixed populations may not be detected. Thus, if there is a mixture of susceptible and resistant strains in the specimen, the molecular beacon might make a mistake. However, the overall agreement between the molecular beacon and the culture-based test results is over 95%, so this problem is rare. Third, sometimes we detect silent mutations, which do not confer resistance. While this phenomenon is rare, it leads to a wrong interpretation. Fourth, some mutations may not confer resistance. For example, mutations in codon 533 of rpob can cause a slight increase in the MIC. However, the MIC would remain lower than the critical concentration level used in susceptibility testing of Rifampicin. Another limitation is that in our laboratory, the molecular beacon method detects merely INH and Rifampicin drug resistance. Thus, phenotypic drug susceptibility testing is still required for other drugs, as well as for the confirmation of the molecular beacon results. In fact, we try to conduct culture-based drug susceptibility testing on all the specimens in which molecular beacons testing were conducted. 5. Usage There are several situations in which a molecular beacon testing is typically performed. First, we use it merely for acid-fast smear-positive specimens, since we observed that there is not enough DNA in the sample of acid-fast smear-negative specimens. Second, it is useful if some of the specimen sediment is available for sending to the reference laboratory. Third, molecular beacon testing is particularly useful if drug-resistant TB is suspected, or when TB suspects have wide contacts. For example, when a healthcare worker or a school teacher is diagnosed with acid-fast smear positive tuberculosis. There are several cases in which we would suspect drug resistance, and perform a molecular beacon testing. First, when the patient has a history of previous treatment. Second, when the patient was born in a country with increased drug resistance. Third, when a patient does not respond well to treatment. Fourth, when a patient is known to have been exposed to a person with MDR-TB. Fifth, molecular beacon testing is also useful for cases that are smear positive, but culture negative. Finally, it is useful when the MTB cultures are mixed with other bacteria

6 IV. Automated Systems Under Development Additional systems for the detection of M. tuberculosis drug resistance are currently under development. They include Autogenomics, Akonni and Cepheid systems. The advantage of all three is that they offer the possibility of multiple assays, using a single instrument. Besides TB detection, they might also be used for detecting respiratory viruses or pathogens that are significant in hospitals. These automated systems may also permit less skilled workers to perform the assay. We thus hope that at least one of them will gain clearance by the FDA in the future, and become widely used. a. Autogenomics The Autogenomics system, called INFINITI, is a single-tube multiplex PCR. The PCR product is analysed using a membrane DNA array. It looks for rpob mutations at five different loci, and at KatG 315. For Pyrazinamide, it looks for pnca 169-G. Of course, it would identify Mycobacterium bovis, but not be able to detect most Pyrazinamide resistance. However, detecting M. bovis could still be useful, since it has a different epidemiology. b. Akonni TruArray A second method is the Akonni TruArray. It is a multiplex PCR, and the PCR products are analysed using a DNA microarray. It also utilises microfluidics. The product is still under development, but I believe it will eventually examine KatG, InhA, and rpob mutations. However, it does have a potential to look for other mutations in the future. c. TruDiagnosis Instrument The TruDiagnosis is also developed by Akonni. It is apparently very easy to use, with a touch screen, a camera and a laser, and its results are presented is an extremely simple manner. Although its producers claim that it could be available for less than USD , I suspect they may charge a high price for the disposable supplies required to operate it. d. Cepheid GeneXpert Another interesting system is the Cepheid GeneXpert, which uses molecular beacons. It combines a sample preparation that uses microfluidics with real-time PCR. It is easy to use, and it detects M. tuberculosis. However, at the moment it predicts merely Rifampicin resistance.

7 V. Recommendations for Rapid Testing in China In conclusion, I would like to offer some ideas for rapid testing in China. I am sure that acid fast microscopy will continue to be performed for all specimens. However, I believe that the nucleic acid amplification system, which detects merely the presence of M. tuberculosis, will be less useful in China than the new testing methods, which also detect mutations associated with drug resistance. Finally, I believe it is important to encourage and promote culture and drug susceptibility testing for all patients, when available resources permit it. VI. Question and Answer Session Brigitte GICQUEL One of the last devices you described processes the sputum, but detects merely Rifampicin susceptibility. However, if MDR-TB or XDR-TB are suspected, it would be necessary to conduct susceptibility tests for additional drugs. Would the sputum be available for other tests? Ed DESMOND I agree that the molecular beacon testing is not as potentially powerful as the DNA array, which offers a practically unlimited capacity of including probes to detect resistance to other classes of drugs, including aminoglycoside and fluoroquinolones. Qian GAO Do you have any information on the impact of early diagnosis on the performance of the TB control programme? Ed DESMOND The California TB control programme has examined the impact of rapid diagnosis. Since there are many MDR-TB patients in California, they have compared patients who have undergone molecular beacon testing with patients who have not. They have discovered that when a molecular beacon testing was used, the patients were put on an effective MDR-TB treatment regimen approximately three weeks sooner. With respect to the cure rate, we do not have data available.