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Newer diagnostic methods in tuberculosis detection

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One-third of the world's population has been infected with Mycobacterium tuberculosis, with new infections occurring in about 1% of the population each year. However 90–95% of infections remain ...

One-third of the world's population has been infected with Mycobacterium tuberculosis, with new infections occurring in about 1% of the population each year. However 90–95% of infections remain asymptomatic. Thus early diagnosis of tuberculosis and drug resistance improves survival and helps to promote contact tracing, implementation of institutional cross-infection procedures, and other public-health actions. There have been many advances and modifications to the methodology for tuberculosis diagnosis some of which are very promising. But these advances have not kept pace with the explosion of tuberculosis or the outbreak of drug resistant tuberculosis. This review describes some of the newer advances in tuberculosis diagnostics and the challenges they face.

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    Newer diagnostic methods in tuberculosis detection Newer diagnostic methods in tuberculosis detection Document Transcript

    •                                                                                                   Ne                                      ewer dia        agnostic de methods etection    s in tuberculosis
    • Review Article Newer diagnostic methods in tuberculosis detection Suneetha Narreddy a,* , Sujit Muthukuru b a Infectious Diseases Consultant, Apollo Hospital, Jubilee Hills, Hyderabad, India b Research Assistant, Apollo Hospital, Jubilee Hills, Hyderabad, India a r t i c l e i n f o Article history: Received 9 April 2014 Accepted 2 May 2014 Available online xxx Keywords: Tuberculosis TB Diagnostics NAAT Xpert a b s t r a c t One-third of the world's population has been infected with Mycobacterium tuberculosis, with new infections occurring in about 1% of the population each year. However 90e95% of infections remain asymptomatic. Thus early diagnosis of tuberculosis and drug resistance improves survival and helps to promote contact tracing, implementation of institutional cross-infection procedures, and other public-health actions. There have been many ad- vances and modifications to the methodology for tuberculosis diagnosis some of which are very promising. But these advances have not kept pace with the explosion of tuberculosis or the outbreak of drug resistant tuberculosis. This review describes some of the newer advances in tuberculosis diagnostics and the challenges they face. Copyright © 2014, Indraprastha Medical Corporation Ltd. All rights reserved. 1. The history Yaksma, Consumption, Romantic disease, White plague, Scrofula, Phthisis, Pott's disease, chaky oncay; all these are different terms used to refer to tuberculosis (TB) throughout history. TB was discovered to have been prevalent as early as 9000 years ago1 but it was not until 1882 that the Tubercle bacilli was first isolated by the German Physician Robert Koch using staining techniques and forced the medical community to accept TB as an infectious disease.2 Shortly after, a method to culture the Tubercle bacilli was developed. Tragically the development of TB diagnostics and their implementation neither kept pace with the advances in medical technology nor the calamitous explosion of TB in the wake of the Human Immunodeficiency Virus (HIV) pandemic nor the outbreak of multi-drug resistant tuberculosis (MDR TB). The older smear microscopy and culture methods underwent slight modifica- tions overtime and are still in use even today but they have low sensitivity and more over drug susceptibility, which is the need of the hour, is not known immediately. 2. The present diagnostic methods Chest X-ray followed by acid-fast staining and microscopy of the sputum sample along with culture are the most widely used diagnostic methods today. Chest X-ray is highly sensitive in the diagnosis of pulmo- nary TB but is of low specificity, especially when co-existing with HIV infection. Radiography is important in the diag- nosis of several forms of extrapulmonary TB such as pleural, vertebral and joint types. * Corresponding author. Tel.: þ91 99660 22225. E-mail address: suneethanarreddy@hotmail.com (S. Narreddy). Available online at www.sciencedirect.com ScienceDirect journal homepage: www.elsevier.com/locate/apme a p o l l o m e d i c i n e x x x ( 2 0 1 4 ) 1 e5 Please cite this article in press as: Narreddy S, Muthukuru S, Newer diagnostic methods in tuberculosis detection, Apollo Medicine (2014), http://dx.doi.org/10.1016/j.apme.2014.05.008 http://dx.doi.org/10.1016/j.apme.2014.05.008 0976-0016/Copyright © 2014, Indraprastha Medical Corporation Ltd. All rights reserved.
    • The major drawback of sputum smear microscopy is that it is laborious and has poor sensitivity, estimated to be ~70%.3 It's sensitivity further drops to around ~35% in some settings with HIV co-infection and highly prevalent TB.4 This leads to increased workload, as more sputum tests are needed per patient leading to diagnostic delay and pa- tient loss to follow-up. The fluorescent AuramineeRhod- amine stain is an additional armor apart from Ziehl Neelsen acid-fast stain for smear microscopy, decreasing the time to review the slide and improving the yield. Extrapulmonary TB needs histopathological examination of the tissue spec- imens; sensitivity and specificity depending on the ease of sample collection; but the facilities and resources needed for such methods are often unavailable in developing countries.5 Culture can be performed using solid media, such as LowensteineJensen (3e8 weeks), or liquid media, such as Dubos' medium or Middlebrook 7H9 Broth etc. (10e14 days), using the commercially available automated systems. Cul- ture was also a prerequisite for phenotypic drug susceptibil- ity testing until recent advances in molecular tests were made. The longer waiting time for culture results makes it difficult for clinicians to prove a diagnosis of TB in cases of diagnostic doubt, especially in populations with low TB incidence, and in the management of suspected drug- resistant TB. Tuberculin skin testing using purified protein derivative (PPD) has poor sensitivity and specificity for active TB and is used mainly to screen high risk population and diagnose Latent TB. 3. Newer diagnostic tests e the need, barriers and the impact they can have With the limitation of the present day tests there is a need for faster, user-friendly, low cost, highly specific and sen- sitive diagnostic tests. New ways of performing “old” tests (e.g. sputum smear microscopy) and completely innovative tools (e.g. new technologies for molecular diagnosis) are under investigation or have already been endorsed by WHO.6 3.1. Optimizing smear The strength of the smear test is its simplicity and low cost. Thus approaches, which increase its sensitivity and reduce the need for multiple visits will be beneficial.7,8 Practices, which combine improved techniques with different ap- proaches, have been endorsed by WHO to optimize yield of microscopy. Some of them include,  Collecting two supervised specimens in one visit (e.g. spot, spot) instead of the age-old three early morning sputum samples.9  Other practice which is not yet endorsed by WHO, but still very promising, is the use of Light Emitting Diode (LED) based microscopy as a replacement for conventional fluo- rescent microscopy.10 3.2. Culture The advances in culture methods are mostly in use already. They employ the use of a liquid medium like Dubos' media, Middlebrook 7H9 Broth, Sula's or Sauton's or Proskauer and Beck's medium over the more traditional LowensteineJenson medium. Not only is it more sensitive, it also reduces the delay of drug sensitivity testing to about 10 days. 3.3. Antigen and antibody based tests Antigen detecting test if developed into a point-of-care test would allow for immediate diagnosis and initiation of treat- ment. Urine samples and Breathalyzer are the most commonly used. A urine specimen would be particularly useful for children, who can have difficulty providing sputum. In patients suspected of extrapulmonary TB, an antigen detection test might prevent the use of more invasive tests. The major and most promising antigens currently under study are Lipoarabinomannan (LAM),11 a major glycolipid component of the cell wall of Mycobacterium tuberculosis; ESAT-6 (early secretory antigen target-6) and CFP-10 (culture filtrate protein-10), both located in the RD1 (region of difference-1) region that is lacking in BCG and in most Atypical Mycobacteria.12 Antibodies to several antigens such as malate synthase, TBF613,14 and cord factor are in use now, especially in the developing countries. The accuracy of these tests has not been found encouraging.15 There are still a large number of commercially available serological tests in use, in developing countries in spite of no International Guideline recommen- dation. WHO issued a policy against these tests in 2011. The Indian government banned antibody-based serological tests in 2012. 3.4. Interferon-g release assays These tests are currently used in many countries as a substi- tute for Tuberculin test to diagnose Latent TB. These are based on T-cell responses to antigens such as ESAT-6 and CFP-10, which are more specific to M. tuberculosis than PPD. Presently two such tests, in use in many countries, are the blood based QuantiFERON-TB Gold In-Tube and T-SPOT.TB. They are found to be more specific than the Tuberculin skin test16,17 in diagnosing Latent TB and have good sensitivity but decreased specificity to active TB.18 WHO has recommended against the use of these tests for active TB diagnosis in high burden countries, as these tests, like Mantoux, cannot separate latent infection from active TB disease. 3.5. Molecular diagnostics These tests use nucleic acid amplification techniques like Polymerase Chain Reaction (PCR) for the diagnosis of TB and drug susceptibility testing. The sensitivity of these tests has been found to be >95% in sputum smear positive samples with specificity around 90e100%,19 but in smear negative/culture positive samples the sensitivities has been found to be reduced. The main advantage of these tests, like GeneXpert, is a p o l l o m e d i c i n e x x x ( 2 0 1 4 ) 1 e52 Please cite this article in press as: Narreddy S, Muthukuru S, Newer diagnostic methods in tuberculosis detection, Apollo Medicine (2014), http://dx.doi.org/10.1016/j.apme.2014.05.008
    • Table 1 e Widely used and WHO endorsed tuberculosis diagnostic tests.29,30 Tests Intended use Advantages Drawbacks Comments Chest X-ray Case detection of pulmonary TB Can diagnose other pulmonary conditions too Low specificity Widely in use Acid-fast smear and microscopy Rapid, point-of-care test Rapid. Low cost. Minimal equipment and training needed Low sensitivity. Cannot detect drug resistance and substantial quality assurance is needed Widely in use Culture on solid media Confirmation of TB and drug susceptibility testing Good sensitivity Takes 8e12 weeks for results to be available Widely in use Culture on liquid media Confirmation of TB and drug susceptibility testing Higher sensitivity than solid media, takes lesser time (10e14 days); gold standard for drug susceptibility testing as of now Time to detection is still slow. Requires quality Lab environment Endorsed by WHO Tuberculin skin test Used to diagnose Latent TB Low cost and easy to administer Low sensitivity and specificity with active TB, immunocompromised host. Not recommended for active TB. Low specificity for Latent TB because of cross-reaction with BCG vaccine and Nontuberculous Mycobacteria Widely in use Interferon release assay Used to diagnose Latent TB More specific than Tuberculin skin test and doesn't cross react with BCG vaccination Cannot differentiate active TB from Latent TB, therefore not recommended for active TB diagnosis. More expensive than Tuberculin skin test Widely in use Serological tests Not recommended NA Highly inaccurate and expensive. Used commercially in some developing countries In 2011 WHO issued a negative policy against their use Nucleic acid amplification test (NAAT) TB case detection High specificity but sensitivity lesser than that of culture Requires moderate training, increased labor. Potential for cross contamination Widely in use Xpert MTB/RIF assay Detect TB and susceptibility to Rifampin Minimal processing time, high accuracy even with some forms of extrapulmonary TB like TB meningitis and lymphadenitis Relatively expensive than other conventional tests WHO approved Hain Genotype MTBDR plus assay Confirm smear positive pulmonary TB and susceptibility to Rifampin and Isoniazid Minimal processing time if done singly but usually done in batches. Highly accurate Poor sensitivity in extrapulmonary and smear negative TB; expensive, requires extensive training and stringent quality control WHO approved apollomedicinexxx(2014)1e53 Pleasecitethisarticleinpressas:NarreddyS,MuthukuruS,Newerdiagnosticmethodsintuberculosisdetection,Apollo Medicine(2014),http://dx.doi.org/10.1016/j.apme.2014.05.008
    • the simplicity of the fully automated machinery and that re- sults can be obtained within a few hours. Currently the most common commercially available NAAT tests are the amplified Mycobacterium Tuberculosis Direct Test (MTD, Gen-Probe) and COBAS® TaqMan® MTB Test (Roche Diagnostics). Variations of the NAAT tests are currently in use to detect drug resistance. Currently these tests are only useful in identifying resistance to Rifampin and Isoniazid and such tests for other anti-TB drugs are much less developed and are currently being studied.20 Xpert MTB/RIF assay is a new test that is revolutionizing the TB world. It detects the Tubercle bacilli and resistance to Rifampin in less than 2 h21 compared to the weeks it takes by culture method. It's accuracy and minimal processing time help in selecting treatment regimens quickly. It is relatively expensive, has a sensitivity of 98% in smear positive samples, 67% in smear negative samples and a specificity of 99%. Xpert also detected 95% of rifampicin-resistant TB cases with specificity of 98%.22 In 2013 Xpert MTB/RIF was endorsed by WHO as the initial diagnostic test for pulmonary TB, MDR pulmonary TB and HIV associated pulmonary TB in both adults and children and as a follow-up test for smear negative pulmonary TB in adults who are not at risk of MDR TB and HIV associated TB. WHO also strongly recommended the use of Xpert MTB/RIF for cerebrospinal fluid samples for rapid diag- nosis of TB meningitis and conditionally recommended it for diagnosis of other extrapulmonary TB samples.23 It's sensi- tivity and specificity being 83% and 94% for lymph nodes, 81% and 98% for cerebrospinal fluid and 46% and 99% for pleural fluid samples.24 Another WHO endorsed molecular test is the Hain Geno- type MTBDR plus assay. It is a rapid test that has been found to be accurate only on smear positive pulmonary samples and has been approved by WHO for that purpose.25 It can also detect resistance to both Rifampin and Isoniazid unlike the Xpert test; sensitivity and specificity being 98.1% and 98.7% for Rifampin and 84.3% and 99.5% for Isoniazid.26 Though these are already a great improvement on the widely used tests they can still be improved and investment is needed.  To develop and implement new and better technologies for DNA extraction and concentration methods.  Analyze the cost benefit aspects. 3.6. Adenosine deaminase Adenosine deaminase testing, which has been in use in some developing countries, has been abandoned now. It has been found to have higher sensitivity than conventional tests when performed on pleural fluid samples.16 3.7. Nose technology It was documented that in ancient Greece, TB could be diag- nosed by strong odors emitted from specimens. This was also favored in Roman times and by traditional Chinese healers. More recently there are reports suggesting that African pouch rats can be trained to identify TB cases by smelling sputum.27 Instrument-based technologies using Electronic Noses28 have been developed and neural patterns were established by exposure to a series of known TB-positive and TB-negative samples and once such a pattern predictable of the disease is established, it is programmed into the device. They have the potential to be highly sensitive, portable and be able to screen samples in a few minutes. The drawbacks being, it maybe unstable over time, vulnerable to interference from water vapor and background odors, and variation may occur be- tween instruments requiring calibration. Some of the widely used tests and those approved by WHO are listed in Table 1.29,30 4. Conclusions The future for TB diagnostics is bright with the tests like Xpert, Hain and liquid cultures in use in many parts of the world and many other advances in their late stages of evaluation. There is the presence of large amount of funding and there is also a huge challenge in translating these technological advances into the public health settings. Initial studies show that these advances are highly accurate in the normal setting but more research is required to assess their performance in TB affected children, MDR TB, HIV co-infection and with different types of extrapulmonary TB.31 An ideal test, as also stated by the STOP TB partnership, would have  ~100% sensitivity and specificity Be affordable  Have a quick turn around time of possibly <2 h  Be fully automatable with internal controls  Have a closed system to minimize contamination  Require minimal specimen processing As of now existing tools like LED microscopy, Xpert, Hain and liquid cultures should be scaled up as they are now more affordable and accessible, even in India. Conflicts of interest All authors have none to declare. r e f e r e n c e s 1. Hershkovitz I, Donoghue HD, Minnikin DE, et al. Detection and molecular characterization of 9,000-year-old Mycobacterium tuberculosis from a Neolithic settlement in the Eastern Mediterranean. PLoS One. 2008;3(10):e3426. 2. Brock Robert Koch 1999:120. 3. Steingart KR, Henry M, Ng V, et al. Fluorescence versus conventional smear microscopy for tuberculosis: a systematic review. Lancet Infect Dis. 2006;6:570e581. 4. Corbett EL, Watt CJ, Walker N, et al. The growing burden of tuberculosis: global trends and interactions with the HIV epidemic. Arch Intern Med. 2003;163:1009e1021. 5. Parsons Linda M, Somosk€ovi Akos, Gutierrez Cristina, et al. Laboratory diagnosis of tuberculosis in resource-poor a p o l l o m e d i c i n e x x x ( 2 0 1 4 ) 1 e54 Please cite this article in press as: Narreddy S, Muthukuru S, Newer diagnostic methods in tuberculosis detection, Apollo Medicine (2014), http://dx.doi.org/10.1016/j.apme.2014.05.008
    • countries: challenges and opportunities. Clin Microbiol Rev. Apr 2011;24(2):314e350. 6. Norbis L, Miotto P, Alagna R, et al. Tuberculosis: lights and shadows in the current diagnostic landscape. New Microbiol. 2013 Apr;36(2):111e120. Epub 2013 Mar 31. 7. Keeler E, Perkins MD, Small P, et al. Reducing the global burden of tuberculosis: the contribution of improved diagnostics. Nature. 2006;444(suppl 1):49e57. 8. Mase S, Ramsay A, Ng V, et al. Yield of serial sputum specimen examinations in the diagnosis of pulmonary tuberculosis: a systematic review. Int J Tuberc Lung Dis. 2007;11:485e495. 9. Hirao S, Yassin MA, Khamofu HG, et al. Same-day smears in the diagnosis of tuberculosis. Trop Med Int Health. 2007;12:1459e1463. 10. Steingart K, Henry M, Ng V, et al. Fluorescence versus conventional sputum smear microscopy for tuberculosis: a systematic review. Lancet Infect Dis. 2006;6:570e581. 11. Hamasur B, Bruchfeld A, Haile M, et al. Rapid diagnosis of tuberculosis by detection of mycobacterial lipoarabinomannan in urine. J Microbiol Methods. 2001;45:41e52. 12. Munk Martin E, Arend Sandra M, Brock Inger, et al. Use of ESAT-6 and CFP-10 antigens for diagnosis of extrapulmonary tuberculosis. J Infect Dis. 2001;183(1):175e176. 13. Weldingh K, Rosenkrands I, Okkels LM, et al. Assessing the serodiagnostic potential of 35 Mycobacterium tuberculosis proteins and identification of four novel serological antigens. J Clin Microbiol. 2005;43:57e65. 14. Wanchu A, Dong Y, Sethi S, et al. Biomarkers for clinical and incipient tuberculosis: performance in a TB-endemic country. PLoS One. 2008;3:e2071e2079. 15. Steingart KR, Henry M, Laal S, et al. Commercial serological antibody detection tests for the diagnosis of pulmonary tuberculosis: a systematic review. PLoS Med. 2007;4(6):e202. 16. Dinnes J, Deeks J, Kunst H, et al. A systematic review of rapid diagnostic tests for the detection of tuberculosis infection. Health Technol Assess. 2007;11(3):1e314. 17. Menzies D, Pai M, Comstock G. Meta-analysis: new tests for the diagnosis of latent tuberculosis infection: areas of uncertainty and recommendations for research. Ann Intern Med. 2007;146(5):340e354. 18. Kang YA, Lee HW, Hwang SS, et al. Usefulness of whole-blood interferon-{gamma} assay and interferon-{gamma} enzyme- linked immunospot assay in the diagnosis of active pulmonary tuberculosis. Chest. 2007;132(3):959e965. http:// dx.doi.org/10.1378/chest.06-2805. 19. Ling DI, Flores LL, Riley LW, Pai M. Commercial nucleic-acid amplification tests for diagnosis of pulmonary tuberculosis in respiratory specimens: meta-analysis and meta-regression. PLoS One. 2008;3(2):e1536. 20. Campbell Patricia J, Morlock Glenn P, Sikes R David, et al. Molecular detection of mutations associated with first- and second-line drug resistance compared with conventional drug susceptibility testing of Mycobacterium tuberculosis. Antimicrob Agents Chemother. May 2011;55(5):2032e2041. 21. http://www.cdc.gov/tb/publications/factsheets/testing/ Xpert_MTB-RIF.htm. 22. Steingart KR, Schiller I, Horne DJ, et al. Xpert® MTB/RIF assay for pulmonary tuberculosis and rifampicin resistance in adults. 23. http://www.stoptb.org/wg/gli/assets/documents/WHO% 20Policy%20Statement%20on%20Xpert%20MTB-RIF%202013% 20pre%20publication%2022102013.pdf. 24. Denkinger CM, Schumacher SG, Boehme CC, et al. Xpert MTB/ RIF assay for the diagnosis of extrapulmonary tuberculosis: a systematic review and meta-analysis. 25. http://www.who.int/tb/features_archive/policy_statement. pdf. 26. Ling DI, Zwerling AA, Pai M. GenoType MTBDR assays for the diagnosis of multidrug-resistant tuberculosis: a meta- analysis. Eur Respir J. 2008;32:1165e1174. 27. Weetjens BJ, Mgode GF, Machang'u RS, et al. African pouched rats for the detection of pulmonary tuberculosis in sputum samples. Int J Tuberc Lung Dis. 2009;13(6):737e743. 28. Kolk Arend, Hoelscher Michael, Maboko Leonard, et al. Electronic-nose technology using sputum samples in diagnosis of patients with tuberculosis. J Clin Microbiol. November 2010;48(11):4235e4238. 29. Perkins MD, Cunningham J. Facing the crisis: improving the diagnosis of tuberculosis in the HIV era. J Infect Dis. 2007;196(suppl 1):S15eS27. 30. World Health Organization and Stop TB Partnership. New laboratory diagnostic tools for tuberculosis control. http:// www.apps.who.int/tdr; Accessed 30.06.09. 31. Drobniewski F, Nikolayevsky V, Balabanova Y, et al. Diagnosis of tuberculosis and drug resistance: what can new tools bring us? Int J Tuberc Lung Dis 16:860e870. a p o l l o m e d i c i n e x x x ( 2 0 1 4 ) 1 e5 5 Please cite this article in press as: Narreddy S, Muthukuru S, Newer diagnostic methods in tuberculosis detection, Apollo Medicine (2014), http://dx.doi.org/10.1016/j.apme.2014.05.008
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