Mycobacteria: Part 2

Transcription

1 This is the written version of our Hot Topic video presentation available at: MayoMedicalLaboratories.com/hot-topics Welcome to Mayo Medical Laboratories Hot Topics. These presentations provide short discussion of current topics and may be helpful to you in your practice. 1

2 Our speaker for this program is Dr. Nancy Wengenack, Director of the Mycology and Mycobacteriology Laboratories and Associate Professor of Laboratory Medicine and Pathology in the Division of Clinical Microbiology at Mayo Clinic in Rochester, Minnesota. This is the second of a 3-part series on tuberculosis. In Part 2, Dr. Wengenack compares the strengths and limitations of the various assays available for identification of Mycobacterium tuberculosis complex. Thank you, Cara. In this Hot Topic presentation, I will be discussing methods for the detection and identification of Mycobacterium tuberculosis complex. 2

3 For this program, I wish to acknowledge the receipt of research support from Trek Diagnostics 3

4 The objectives of this Hot Topic are to describe methods available for the identification of Mycobacterium tuberculosis after growth on a culture plate or in broth medium. The specific methods to be discussed are nucleic acid hybridization probes, line probes, DNA sequencing, and MALDI-TOF mass spectrometry. In addition, I will discuss methods available for the direct detection of Mycobacterium tuberculosis complex from specimens without the need to wait for growth in culture. These methods include the Mycobacterium tuberculosis Direct test, line probe assays, laboratorydeveloped PCR assays, and the Cepheid GeneXpert MTB/RIF PCR assay. 4

5 First, I will begin by discussing the identification of Mycobacterium tuberculosis complex after growth of colonies on a culture plate or in broth medium. 5

6 Traditionally mycobacteria including Mycobacterium tuberculosis have been identified after growth in culture using clues such as colonial morphology (in other words, is it rough and buff as discussed in the previous Hot Topic), or growth characteristics (is it a rapidly growing mycobacterium or a slowly growing mycobacterium), or perhaps through the use of selected biochemical tests such as niacin accumulation, nitrate reduction, or pyrazinamidase activity. All of these methods have the potential to be extremely slow and in the past, identification of Mycobacterium tuberculosis complex from culture could require 4 to 8 weeks after the organism grew in culture. In addition, high performance-liquid chromatography (HPLC) or gas-liquid chromatograph (GLC) have been used generally by the CDC or in state public health labs. The chromatographic methods targeted the unique mycolic acids which make up the mycobacterial cell wall and by comparing the mycolic acid pattern of the unknown isolate to those in reference libraries, the mycobacteria could be identified. HPLC and GLC have largely given way to other methods of identification for mycobacteria because of the large numbers of mycobacterial species that have been identified in the past 20 years. Mycolic acid patterns are no longer able to discriminate between the large number of closely related species of mycobacteria. Molecular methods are now the gold standard for the identification of mycobacteria including Mycobacterium tuberculosis complex from culture because they allow for rapid identification and they are able to discriminate between the majority of the 150 species of mycobacteria currently recognized today. 6

7 Nucleic acid hybridization probes are available for the identification of Mycobacterium tuberculosis complex, Mycobacterium avium complex, Mycobacterium gordonae, and Mycobacterium kansasii. There is no PCR or other amplification step that occurs when utilizing these probes so a large amount of target nucleic acid is required. That is why the probes are only useful for mycobacteria growing in culture and they cannot be used for the direct detection of mycobacteria from patient specimens. The probes are FDAapproved products and they are rapid with results available within approximately 2 hours after the growth of the organism in culture, they are highly sensitive and specific, and they are technically simple to utilize. The main limitation associated with the probes is that they are only available for the 4 mycobacterial species or complexes listed on this slide so other tools are needed for the identification of the majority of mycobacteria seen in the clinical laboratory today. 7

8 This slide provides an overview of how the nucleic acid hybridization probes function. The laboratorian obtains an RNA template from the organism to be identified by lysing a small amount of the organism after it is picked from a culture plate using an inoculating loop. A DNA-hybridization probe labeled with a chemiluminescent moiety is added to the solution containing the RNA template. If the hybridization probe sequence is complementary to the RNA target, it will bind tightly and specifically. Then, any unbound probe remaining in the solution is hydrolyzed to remove the potential for nonspecific signal and the DNA-RNA hybrid is detected by measuring a chemiluminescence signal. 8

9 The mycobacterial nucleic acid hybridization probes have excellent sensitivity and specificity as shown in the table on this slide. Sensitivity values range from 93% for Mycobacterium kansasii, up to 100% for Mycobacterium intracellulare. Specificity is also extremely good attaining 99% to 100% for all targeted species. There have been some minor cross-reactions reported with uncommon species of nontuberculous mycobacteria, but this can largely be eliminated by strictly controlling the hybridization time and temperature. 9

10 Another type of probe technology that provided identification of mycobacteria from culture isolates are the line probe assays available from Hain Lifesciences or Innogenetics. These tests have genus- and species-specific probes bound to nitrocellulose membranes in a strip format. DNA from the lysed culture extract is added to the strip and allowed to hybridize with the probe. Nonhybridized nucleic acids are washed off and the strip is read to determine which genus- or speciesspecific probe the unknown isolate was able to hybridize with. Several different strip formats are available. For example, the Hain group offers GenoType Mycobacterium CM and AS strips, which allow for the identification of Mycobacterium tuberculosis complex and 29 species of nontuberculous mycobacteria on 2 strips. The GenoType MTBC strip allows for speciation of the members of the Mycobacterium tuberculosis complex, and the GenoType MTBDR plus strip allows for identification of Mycobacterium tuberculosis complex plus selected drug resistance markers including rpob, katg, and inha. A major limitation of the line probe assays is that they are not cleared by the US FDA and, therefore, the manufacturers will not sell these strips for clinical diagnostic use in the United States. 10

11 So what can a laboratory do if it has an acid fast organism growing in culture, but it does not react with the available nucleic acid hybridization probes? Well, the current gold standard for the identification of mycobacteria is DNA sequencing. The most popular method used is traditional Sanger dideoxy sequencing and there are various targets which have proven to be useful for mycobacteria. These include the 16S ribosomal DNA gene, the beta subunit of the bacterial RNA polymerase or rpob gene, and the heat shock protein hsp65 gene. Other gene targets have also been described in the literature. Using the 16S rdna gene target as an example, the technique utilized broad range primers that amplify the 16S gene or a portion of the 16S gene, usually the first 500-base pairs. Sequence evaluation of a hypervariable region of the 16S target found between the primers allows for the species-specific identification of a wide variety of mycobacterial species when the unknown sequence is interrogated against a known mycobacterial sequence library. The typical workflow used for sequencing of mycobacteria in our laboratory includes selection of the organism to be sequenced from the culture plate. This is done working inside of a biological safety cabinet in a biosafety level 3 laboratory as a precaution in case the organism growing on the culture plate is Mycobacterium tuberculosis. After selection, the organism is lysed and heated to render it nonviable. Then the mycobacterial DNA is amplified using a PCR reaction followed by a template cleaning step. Then a second PCR reaction is done to incorporate the dideoxynucleotides 11

12 required for traditional Sanger sequencing, followed by a second clean-up step to remove unincorporated nucleotides. Cycle sequencing is completed using a capillary electrophoresis instrument and the mycobacterial DNA sequence is available for analysis by the laboratory staff. 11

13 Mycobacterial sequence libraries commonly utilized in our laboratory are the MicroSeq library from Applied Biosystems (now Life Technologies), a custom laboratory-specific mycobacterial library that has been developed over a couple of decades using type strains and well-characterized clinical isolates, and the NCBI GenBank database, which can be queried using a BLAST search. In addition, there are several vended, curated, web-based database tools available for library searching and comparison. These include products from SmartGene and isentio. The turnaround time for sequencing can be as fast as 8 hours after growth of the organism on a culture plate. In our laboratory, we batch isolates into groups of 96 for efficiency's sake. This means that we select colonies to be sequenced from culture plates in the morning, perform PCR amplification in the afternoon, perform the electrophoresis of the cleaned PCR products on the sequencing instrument overnight with analysis and reporting of the sequence results occurring early the next morning so the information is available for the physicians as they do their morning rounds. 12

14 The advantages of DNA sequencing for the identification of mycobacteria are that it allows for the objective identification of a wide variety of mycobacteria species including Mycobacterium tuberculosis. Also, a fairly rapid turnaround time is possible with same day or next day identification to the species level after the organism grows in culture. Limitations of sequencing include the fact that it s fairly labor-intensive and requires highly skilled, trained technologists for performance. In addition, the equipment and reagents costs are not insignificant. Finally, I cannot stress strongly enough how dependent the use of a high-quality library database. Sequencing is generally used for the identification of nontuberculous mycobacteria, but Mycobacterium tuberculosis complex can also be identified by sequence analysis if the specific nucleic acid hybridization probe is not available. 13

15 Another technology which is becoming popular in clinical microbiology laboratories is matrix-assisted laser-desorption time of flight mass spectrometry or MALDI-TOF MS. This technology is changing the way that we identify microbes in the laboratory and it has already been established as an important method in our laboratories for the identification of aerobic bacteria and yeast. We will soon be implementing it for the identification of mycobacteria as well. 14

16 This slide shows the 2 MALDI-TOF MS systems that I am aware of that are commercially available for clinical laboratories to use for the identification of microorganisms. The Bruker BioTyper system is shown on the left, and the biomerieux Vitek MS system is pictured on the right. Both systems are FDA-cleared for the identification of a limited menu of bacteria. Neither system has clearance for the identification of mycobacteria at this time, although clinical trial studies in this area are being planned. Laboratories utilizing this technology must perform a thorough validation and verification study to ensure that mycobacteria are correctly identified. 15

17 This slide depicts the workflow utilized in our laboratory for the identification of mycobacteria using MALDI-TOF mass spectrometry. Working a biosafety level 3 laboratory, the organisms to be identified are selected using a 10-microliter disposable loop. The organism is then added to a small tube containing zirconium beads and 70% ethanol. Exposure to the ethanol for a period of time followed by bead beading renders the organism nonviable and makes it safe to complete the remainder of the processing steps in the biosafety level 2 laboratory. The solution is centrifuged to pellet the proteins and the supernatant is decanted followed by a speed vac step to dry the pellet. Finally, formic acid and acetonitrile are added and the solution is spotted onto the MALDI-TOF plate followed by analysis in the mass spectrometer. 16

18 There are a number of advantages to using MALDI-TOF mass spectrometry for the identification of mycobacteria, including the fact that the work flow for the laboratory is similar regardless of the type of organism you are looking for. Bacteria, yeast, mycobacteria, and molds can all be analyzed on the instrument with only minor changes to the preprocessing steps required to spotting on the plate. In addition, the technology is cost effective and green with very few consumable used. The turnaround time for a result is pretty rapid with only an hour or so required to analyze 24 specimens and the throughput available in a single day per instrument is fairly high. Only a single colony of organism is needed and the instrument itself has small footprint. The chemicals used in the process are extremely low volume and the exposure risk to laboratory personnel is small because organisms are inactivated before they are applied to the plate. Finally, the system is adaptable by the user with the ability to create and expand databases to supplement the manufacturer s database. 17

19 Limitations of MALDI-TOF MS for the identification of mycobacteria include the need to grow the organism in culture first, which is often a rate limiting step. MALDI-TOF requires a pure isolate so mixtures of organisms often give a no identification result. In addition, the phase of growth and the media used can affect the spectra produced. For best performance, the spectral library needs to be composed of spectra produced under comparable conditions to your everyday working practices in the laboratory. The manufacturer s databases need expansion for less common organisms and instrument downtime, whether for scheduled maintenance or due to a breakdown, can be a problem if you are using a single instrument and if it has been the main method of organism identification in your laboratory. Regulatory issues can be a concern for groups of organisms which have not yet received FDA-clearance and, specifically for mycobacteria, the technique may, at times, be a little slower than sequencing because of the need to have more growth for MALDI-TOF MS than for sequencing. 18

20 In this slide, I will talk in very general terms about the costs associated with identifying microorganisms including mycobacteria with MALDI-TOF MS. The instrument itself costs approximately $200,000 dollars, while the steel plates are about $500 each or a total of $5,000 for a set of 10. A significant cost is associated with the service contract and annual maintenance cost, which will be in the neighborhood of $25,000 but which are very important to purchase in our experience. Remember, the good news is that mass spectrometry can be used for the identification of bacteria, mycobacteria, and molds on the same platform; next generation MALDI- TOF instruments will likely be linked with susceptibility platforms as well, which will further increase the utility for the laboratory. 19

21 In the remaining slides, I will discuss the direct identification of Mycobacterium tuberculosis complex without waiting for growth of the organism in culture. 20

22 The Centers for Disease Control and Prevention recommend that nucleic acid amplification testing be performed on at least 1 respiratory specimen, preferably the first specimen, from each patient with suspected pulmonary tuberculosis. The testing is recommended if it would alter case management or if it would alter TB control activities. Nucleic acid amplification testing does not replace the need for performing a mycobacterial culture because culture usually has better sensitivity. 21

23 One nucleic acid amplification test which is FDA-approved for the direct detection of Mycobacterium tuberculosis complex is the Mycobacterium tuberculosis Direct Test or the MTD test from Hologic GenProbe. This test utilizes transcription-mediated amplification of Mycobacterium tuberculosis complex ribosomal RNA directly from respiratory specimens. You may frequently hear this test referred to as the TB Probe assay because it is from the same company the produces the nucleic acid hybridization probes. This is a misnomer, however, because this is actually a nucleic acid amplification test. The MTD test has excellent clinical specificity at 99% to 100% and the clinical sensitivity is also good at 91% to 95% for smear-positive respiratory specimens and 83 to 100% for smear-negative respiratory specimens. 22

24 The limitations of the MTD test include the observation that this is a technically difficult test for laboratory technologists to perform. It is an open PCR system, which requires opening of tubes and transfer of amplified products so the risk for a falsepositive result due to sample cross-contamination or amplicon contamination is high. In addition, potential inhibitors from specimen components is a concern. A negative MTD test does not rule-out Mycobacterium tuberculosis infection and a mycobacterial culture still needs to be performed. In addition, the MTD test detects the presence of nucleic acid, but it doesn t indicate if the organism is still viable. Cross reactions can occur with some rare mycobacteria and the test can be costly. 23

25 Two of the line probe assays described previously from Hain Lifesciences or Innogenetics can also detect Mycobacterium tuberculosis complex directly from respiratory specimens, but as discussed earlier they are not cleared by the US FDA and, therefore, the manufacturers will not sell these strips for clinical diagnostic use in the United States. 24

26 Several laboratories including ours offers laboratory-developed PCR tests for the direct detection of Mycobacterium tuberculosis. These are generally closed PCR systems, which significantly reduce the opportunity for false-positives due to cross contamination. In addition, the tests are reported to have good sensitivity and specificity, although this can be difficult to judge since each test is developed and verified independently. Often these laboratory-developed real-time PCR assays are less expensive that the commercially available test and they can sometimes be utilized on a wider variety of specimen types including smear-negative respiratory specimens and nonrespiratory specimens including formalin-fixed, paraffin-embedded tissue blocks from Pathology. 25

27 The workflow for all of our real-time PCR assays is the same and is depicted in this slide. We begin with the patient specimen or a culture isolate, which undergo a processing step to lyse and inactivate the organism. Next the specimen is extracted to isolate the nucleic acids, which are in turn amplified using real-time PCR. Sequencespecific detection is achieved using fluorescence resonance energy transfer (or FRET) probes. The entire process requires about 4 hours from start to finish and, therefore, the result can be available the same day that the specimen is submitted. 26

28 A direct head-to-head comparison of our laboratory-developed real-time PCR assay with the Hologic GenProbe MTD test demonstrated that the 2 assays show a high degree of concordance with a 99.3% agreement rate for 542 specimens including 52 that were previously found to be positive by the MTD test. The kappa coefficient is 0.96, which indicates almost perfect agreement between the 2 tests. 27

29 Limitations of a laboratory-developed PCR assay for Mycobacterium tuberculosis complex include some of those limitations found for the commercially available tests, namely a lower sensitivity for smear-negative specimens, the fact that a negative result does not rule-out Mycobacterium tuberculosis infection, and a mycobacterial culture is still needed, and the fact that nucleic acid amplification tests detect the presence of amplifiable nucleic acid, but they don t indicate if the organism is still viable. In addition, the cost of an LDT test can also be high. 28

30 The most recent FDA-cleared tests available for the direct detection of Mycobacterium tuberculosis complex directly from respiratory specimens is the Cepheid Xpert MTB/RIF test, which is endorsed by the World Health Organization and which runs on the Cepheid GeneXpert system. This test provides direct detection of Mycobacterium tuberculosis complex and provides information about rifampin resistance. The schematic shown on this slide depicts how simple the Xpert test is to perform. The patient s sputum specimen is inactivated and liquefied by the addition of the sample prep reagent. Once inactivated, 2 ml of the sample is transferred into an MTB/RIF test cartridge and the cartridge is inserted into the GeneXpert instrument. This is all the technologist has to do and the sample is extracted and amplified inside the instrument with no hands-on work required. At the completion of the test, which requires about 2 hours from start to finish, a report is generated which indicates whether Mycobacterium tuberculosis complex is present and whether mutations in the rpob gene have been detected, which correlate with rifampin resistance. 29

31 The have been a large number of studies published on the Xpert MTB/RIF assay performance including a meta-analysis of 18 studies with over 10,000 patients presented in the Journal of Infection in 2012 by Chang et al. Their analysis indicated that the sensitivity of the Xpert assay for smear-positive, pulmonary tuberculosis is approximately 91%, while the sensitivity for smear-negative disease is expectedly lower at approximately 74%. The specificity of the test is excellent at 98%. For extrapulmonary tuberculosis, the sensitivity and specificity of the Xpert assay are lower at 80% and 86%, respectively. Finally, the time to diagnosis for the Xpert assay compared favorably with nonspecific smear microscopy and the Xpert assay, both resulted on the day of specimen submission, while broth culture for tuberculosis required an average of 16 days and solid media cultures required an average of 20 days to complete. 30

32 As mentioned earlier, the Xpert MTB/RIF assay provides information about potential rifampin resistance. The target is the rpob gene, which encodes the beta subunit of the bacterial RNA polymerase. Mutations in an 81-base pair region of the rpob gene are responsible for approximately 96% of rifampin resistance in Mycobacterium tuberculosis. Rifampin resistance also is a predictor of multidrug-resistant tuberculosis since the majority of rifampin-resistant isolates will also be isoniazid-resistant. Some false-rifampin resistance has been reported in the literature by users of the Xpert MTB/RIF assay, so the CDC recommends reporting Xpert-rifampin resistant results as a preliminary result pending confirmation with sequencing. In addition, the positive-predictive value of the assay is lower in low-prevalence settings. Growthbased drug susceptibility testing is also still required. 31

33 Strengths of the Xpert MTB/RIF assay include good sensitivity and specificity for respiratory specimens, a rapid 2-hour turnaround time, the ability to detect Mycobacterium tuberculosis complex and rifampin resistance directly, and the use of a closed PCR system with a low risk of cross-contamination. The GeneXpert platform is multifunctional and can be used for other tests such as Clostridium difficile or HIV viral load testing. The test is extremely simple for operators to perform and no advanced biosafety equipment is needed, making this test extremely attractive in developing countries with a high burden of TB. 32

34 Limitations of the Xpert MTB/RIF assay include the need to still perform a culture, the possibility of false-positive rifampin resistance requiring confirmation by sequencing, and the lower sensitivity and specificity for extrapulmonary specimens. Cost considerations include the need to purchase the instrument and the cartridges, which can be as high as $65 each in the US. The cartridges often have a discounted price of around $10 in countries with high TB burden and in developing countries. The need for continuous electrical power and air conditioning can also make its use a challenge in developing countries. Sample storage is limited to 3 days at room temperature or 7 days under refrigerated conditions. Finally as with the other PCR assays discussed, the test cannot differentiate between live and nonviable Mycobacterium Tuberculosis, so it cannot be used to monitor response to treatment. 33

35 So in summary, I will conclude by reiterating that molecular diagnostics are enabling clinical microbiology laboratories to detect and identify Mycobacterium tuberculosis complex both after growth in culture and directly from specimens with good sensitivity and specificity. In addition, molecular methods allow for the rapid speciation between members of the Mycobacterium tuberculosis complex and for the detection of mutations associated with drug resistance. 34

36 I thank you very much for taking the time to listen to this Hot Topic presentation and I hope you will join me for the last installment in this series when I discuss drug susceptibility testing for Mycobacterium tuberculosis complex. 35

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