Standard Diagnostic Procedure for Tuberculosis: A Review

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1 ISSN: Standard Diagnostic Procedure for Tuberculosis: A Review Suresh Jaiswal*, Jay Prakash Sah, Bhoopendra Sharma Department of Medical Lab Science, School of Health and Allied Sciences, Pokhara University, Lekhnath, Kaski, Nepal Abstract Despite continuous effort in monitoring and treatment of tuberculosis, the disease remains a major public health issue. Rapid diagnosis and appropriate therapies become the first priorities in controlling the growing epidemics. The bedside decision on the initiation of anti-tuberculous drug therapy are based on epidemiologic, clinical, radiolographic, and/or histological findings, which can generally be supported by a rapid microbiologic test, commonly a positive acid-fast bacilli (AFB) smear result. However, AFB smear is positive in only half of patients with subsequently culture positive for Mycobacterium tuberculosis. Although the sensitivity of the smear is improved by fluorescent staining, the test fails to distinguish between tuberculous and nontuberculous mycobacteria. Diagnosis of Tuberculosis (TB) is mainly based on the culture method and non-culture methods. Molecular techniques are becoming more advanced and confirmatory diagnostic procedure of TB. Recent surveys also reveal that drug-resistant tuberculosis is still ubiquitous and alarmingly high in several countries. The situation is further complicated by the emergence of multi-drug resistant tuberculosis (MDR-TB). MDR-TB results from improper administration of antibiotics in chemotherapies of TB patients and is recognised as Mycobacterium tuberculosis resistant to at least isoniazid (INH) and rifampin (RIF), the two most common first-line antituberculosis drugs. Keywords: Tuberculosis, AFB smear, Multi Drug Resistant (MDR), LJ medium *Author for Correspondence: suuress@gmail.com INTRODUCTION Tuberculosis (TB) remains the leading cause of death from a curable infectious disease, despite the availability of short-course therapy that can be both inexpensive and effective. Clinical management of cases in developing countries is hampered by the lack of a simple and effective diagnostic test [1]. Active tuberculosis (TB) is diagnosed by detecting Mycobacterium tuberculosis complex bacilli in specimens from the respiratory tract (pulmonary TB) or in specimens from other bodily sites (extra pulmonary TB). Although many new (molecular) diagnostic methods have been developed, acid fast bacilli (AFB) smear microscopy and culture on Lowenstein-Jensen medium are still the gold standards for the diagnosis of active TB and, especially in lowresource countries, the only methods available for confirming TB in patients with a clinical presumption of active disease. AFB smear microscopy is rapid and inexpensive and thus is a very useful method to identify highly contagious patients. Culture is used to detect cases with low mycobacterial loads and is also requested in cases at risk of drug-resistant TB for drug susceptibility testing, or in cases where disease due to another member of the Mycobacterium genus is suspected. AFB smear microscopy and culture can also be used to monitor the effectiveness of treatment and can help to determine when a patient is less likely to be infectious. Two manuals are recommended for the laboratory diagnosis of TB [2, 3]. Correct diagnosis of TB is needed to improve treatment, reduce transmission, and control development of drug resistance. In patients with active pulmonary TB, only an estimated 45% of infections are detected by sputum microscopy. This test, first developed in the RRJoLS(2013) 1-10 STM Journals All Rights Reserved Page 1

2 Standard Diagnostic Procedure for Tuberculosis Jaiswal et al. 1880s and basically unchanged today, has the advantage of being simple, but is hampered by very low sensitivity: it may only detect half of all cases with active infection. It is also very dependent on the skill of the technician, and a single technician can only process a relatively small number of slides per day [3]. Furthermore, a staggering three million people who present annually with suspected TB may not be properly diagnosed, because their infection (so-called smear-negative disease) cannot be detected by sputum microscopy [4]. There are specific epidemiological factors that present additional challenges to TB diagnosis. HIV infection is thought to be a major contributor to the increase in TB incidence across the world [2]. An estimated 9% of adults globally with newly diagnosed TB are HIV positive. HIV co-infection with TB presents challenges to effective diagnosis of TB and diagnosis can also be more difficult in children. The rapid rise of drug-resistant (DR) TB has further complicated TB diagnosis [6]. Tests that measure drug susceptibility are essential to monitor the spread of resistant TB strains, and ensure that patients are given effective treatment. New diagnostic tests that are simple and robust enough to be used in the field, accurate enough to diagnose all infected individuals, and able to identify drug resistance are desperately needed, and represent an essential complement to new drug development efforts and to effective control and treatment programmes. An individual who is suspected of having TB disease requires a complete medical evaluation, including the following [4]: 1. Medical history, including exposure, symptoms, previous treatment for TB, and risk factors. 2. Human immunodeficiency virus (HIV) screening. 3. Physical examination. 4. Tuberculin skin test (TST) or interferon gamma release assay (IGRA). 5. Chest radiography. 6. Bacteriologic examination. MEDICAL HISTORY Clinicians should ask about the patient s history of TB exposure, infection, or disease. It is also important to consider demographic factors, (e.g., country of origin, age, ethnic or racial group, occupation) that may increase the patient s risk for exposure to TB or to drugresistant TB. Also, clinicians should determine whether the patient has medical conditions, especially HIV infection, that increases the risk of latent TB infection progressing to TB disease [5]. HUMAN IMMUNODEFICIENCY VIRUS SCREENING Voluntary counseling and testing for HIV is recommended for all patients with TB. HIV counseling and testing has also been recommended for contacts of persons with TB [6]. The Centers for Disease Control and Prevention (CDC) recommends the following: Routine HIV screening for all patients ages seeking health care for any reason, without regard to any patient s known risks for HIV infection. Annual HIV screening of patients known to be at high risk [7]. PHYSICAL EXAMINATION A physical exam can provide valuable information about the patient s overall condition and other factors that may affect how TB is treated, such as HIV infection or other illnesses. TEST FOR TB INFECTION The Mantoux tuberculin skin test (TST) or the special TB blood test can be used to test for M. tuberculosis infection. Additional tests are required to confirm TB disease. The Mantoux tuberculin skin test is performed by injecting a small amount of fluid called tuberculin into the skin in the lower part of the arm. The test is read within 48 to 72 h by a trained health care worker, who looks for a reaction (induration) on the arm (Figure 1). The special TB blood test measures the patient s immune system reaction to M. tuberc ulosis. RRJoLS (2013) 1-10 STM Journals All Rights Reserved Page 2

3 Volume 3, Issue 3, ISSN: Fig. 1: Mauntax Tuberculin Test and its Reading in h. CHEST RADIOGRAPH A posterior-anterior chest radiograph is used to detect chest abnormalities (Figure 2). Lesions may appear anywhere in the lungs and may differ in size, shape, density, and cavitation. These abnormalities may suggest TB, but cannot be used to definitively diagnose TB. However, a chest radiograph may be used to rule out the possibility of pulmonary TB in a person who has had a positive reaction to a TST or special TB blood test and no symptoms of disease [8]. Fig. 2: Showing Chest Radiograph with Lesions and Chest Abnormalities. DIAGNOSTIC MICROBIOLOGY The presence of acid-fast-bacilli (AFB) on a sputum smear or other specimen often indicates TB disease. Acid-fast microscopy is easy and quick, but it does not confirm a diagnosis of TB because some acid-fast-bacilli are not M. tuberculosis. Therefore, a culture is done on all initial samples to confirm the diagnosis. (However, a positive culture is not always necessary to begin or continue treatment for TB). A positive culture for M. tuberculosis confirms the diagnosis of TB disease. Culture examinations should be completed on all specimens, regardless of AFB smear results. Laboratories should report positive results on smears and cultures within 24 h by telephone or fax to the primary health care provider and to the state or local TB control program, as required by law. MATERIALS AND METHODS Specimens The successful isolation of the pathogen requires that the best specimen be properly collected, promptly transported and carefully processed. Many different types of clinical specimens may be obtained for the microbiological diagnosis. If pulmonary TB is suspected, specimens originating from the respiratory tract should be collected, i.e., sputum, induced sputum, broncho alveolar lavage or a lung biopsy. For the diagnosis of pulmonary TB, three first-morning sputum RRJoLS (2013) 1-10 STM Journals All Rights Reserved Page 3

4 Standard Diagnostic Procedure for Tuberculosis Jaiswal et al. specimens (not saliva) obtained after a deep, productive cough on non-consecutive days are usually recommended. Several studies have shown, however, that the value of the third sputum is negligible for the diagnosis of TB, as virtually all cases are identified from the first and/or the second specimen (Yassin 2003, Nelson 1998, Dorronsoro 2000, Finch 1997). Specimens to be collected for the diagnosis of extrapulmonary disease depend on the site of the disease. The most common specimens received in the laboratory are biopsies, aspirates, pus, urine, and normally sterile body fluids, including cerebrospinal fluid, synovial, pleural, pericardial, and peritoneal liquid. Stool can be collected when intestinal TB is suspected and also in the case of suspected Mycobacterium avium infection in AIDS patients. AFB Smear AFB smear microscopy plays an important role in the early diagnosis of mycobacterial infections because most mycobacteria grow slowly and culture results become available only after weeks of incubation (Table 1). In addition, AFB smear microscopy is often the only available diagnostic method in developing countries. Smear staining is based on the high lipid content of the cell wall of mycobacteria which makes them resistant to decolorization by acid-alcohol after the primary staining. To determine that a clinical specimen contains AFB, the specimen is spread onto a microscope slide, heat-fixed, stained with a primary staining, decolorized with acid-alcohol solution and counterstained with a contrasting dye in order to obtain better differentiation between the microorganism and the background. The slide is observed under the microscope for the detection of AFB (Figure 3). Several methods can be used for determining the acid-fast nature of an organism. Fig. 3: A and B showing Myctobacterim Bacilli in Sputum and Slide Smear. Table 1: Showing the Count of Mycobacterium Bacilli in per Fields of 100x. Count on Ziehl-Neelsen/Kinyoun stain (1000x) Report 0 Non AFB observed 1 9/100 fields Exact count 10 99/100 fields /field 2+ > 10/field 3+ Diagnostic methods in detection of Mycobacterium Tuberculosis are as follows: 1. Culture based methods 2. Non culture based methods. Culture Based Methods Culture of Mycobacterium tuberculosis remains the gold standard for both diagnosis and drug sensitivity testing. This section reviews culture tests currently in use, and newly developed techniques. Conventional culture methods using Lowenstein-Jensen (LJ) or 7H11 medium, while cheap and simple, have the major disadvantage of being very slow. LJ cultures take days for diagnosis and four to six weeks after initial RRJoLS (2013) 1-10 STM Journals All Rights Reserved Page 4

5 Volume 3, Issue 3, ISSN: culture for drug sensitivity testing. 7H11 medium slightly accelerates the process, but requires antibiotics in the medium to prevent contamination and a CO 2 incubator. Diagnosis with 7H11 medium takes days, Daylight Saving Time (DST) information is available three to six weeks later. Some more rapid culture methods have been developed and are commercially available, most of which are difficult to implement in the field due to the complexity of the technique or the required equipment. There are also some emerging simplified culture techniques that can reduce time to diagnosis or DST that seem more appropriate for use in resource-limited settings. The sensitivity of culture is limited by the need to have bacilli present in the sample to be cultured. HIV positive patients and children have difficulty in producing sputum and sputum culture will not detect extrapulmonary (EP) forms of TB. EP TB is very common in HIV positive patients and is rapidly fatal. Even in patients with active pulmonary TB, the bacilli may be protected in lung cavities or not present in a particular sputum sample, or may be lost in the decontamination treatment required to process sputum for mycobacterial culture. All these factors limit the usefulness of the technique. New Rapid Commercial Methods for Diagnosis and DST A number of commercial systems are available for culture and DST, some of which may have slight advantages in certain settings. However, none of the tests are easily adaptable to the realities of field projects, given the difficulties in setting up and running culture laboratories. Phage-based Tests Phage-based tests require limited culture facilities and promise rapid results (~2 days). However, MSF field evaluations shown that it is very hard to implement in non-culture facilities in the field, even in relatively wellsupported urban settings. Dedicated areas are required, careful control of access to the rooms is needed to reduce contamination, and even seemingly simple requirements, like a stable power supply and a functioning biosafety hood, are very difficult and often enormously expensive to ensure. Metanalyses comparing phage based tests to culture in field settings have shown that in most cases they are no more informative that smear microscopy. Non-Culture Methods A number of strategies to detect and report the presence of M. Tuberculosis have been developed. Serology (detection of antibodies) has not produced any reliable, informative tests despite decades of work. Detection of antigens is a more promising approach, as it detects the presence of the organism and thus may be able to diagnose active infection. The use of nucleic acid amplification (NAA) tests in non-specialised laboratories is technically challenging. These tests have been shown to be highly specific, but sensitive if starting from patient samples, low and highly variable and is difficult to assess 28. These tests can also be used from primary culture. Although this improves the sensitivity, the technique is then very slow. For this reason, we have decided to include NAA tests in the nonculture section of this report, in order to focus on the tests use on direct clinical samples. Here we also look at some polymerase chain reaction (PCR) based techniques which are being validated for use on patient samples for rapid detection of rifampicin/isoniazid resistance. There are also some tests being developed that detect immunological responses (interferon gamma assays). These tests are rather expensive and complicated to perform, and still need to be validated in endemic areas, and their interpretation is not clear. Techniques using Antibody Detection In 2005, WHO/TDR performed an evaluation of commercially available rapid diagnostic tests (RDTs). Twenty-seven manufacturers were invited to submit their products for evaluation, but of the 19 who agreed, only six provided information on the antigen used. All tests detect antibodies in serum. Test samples came from the TDR specimen bank. The WHO study found that TB rapid diagnostic tests currently available on the market vary widely in performance, with some products showing a high lot-to-lot and readerto-reader variability. At less than 80%, the specificity was poor in the majority of products when tested in TB suspected cases RRJoLS (2013) 1-10 STM Journals All Rights Reserved Page 5

6 Standard Diagnostic Procedure for Tuberculosis Jaiswal et al. from endemic settings. Those tests with a better specificity (over 90%) had poor sensitivity, detecting fewer than 40% of TB patients. The tests performed even worse in HIV co-infected samples. The conclusion of the study was that none of the assays perform well enough even to replace microscopy [9]. Based on this and other information, it seems that antibody detection is unlikely to be a good strategy for the development of a reliable diagnostic test for TB. It is important to note that the tests named in the above table are only those that agreed to participate in the study: absence from the list does not imply that the test works. We found no convincing evidence supporting the use of any existing antibody detection tests [10]. Techniques using Antigen Detection Several tests using antigen detection are currently commercialised or under development. Nucleic Acid Amplification (NAA) NAA techniques require strong laboratory capacities, good quality control procedures, and remain relatively expensive. In recent years, some improvements have been made, such as isothermal amplification steps, the inclusion of internal amplification controls to ensure that inhibitors (resulting in false negatives) are not present, the design of single-tube reactions to reduce contamination and the development of detection by emitted light or by dipstick. The use of NAA techniques remains technically challenging. Despite being usually highly specific, NAA tests have lower (and greatly variable) sensitivity. A positive NAA test is considered good evidence of infection but a negative result is not informative enough. Use of NAA tests has not been recommended for sputum negative patients. As these tests cannot distinguish live from dead bacteria, they cannot be used for patients receiving treatment. One study considered that current NAA tests cannot replace microscopy or culture, and should be used only in conjunction with these tests. While some NAA assays reported seem to work quite well (sometimes sensitivities near 90% were reported), there is very wide variability, even from very resource rich laboratories, making their use in the field uncertain. Their difficulty of use and quality control issues combined with the variability and lack of sensitivity in sputum negative and extra pulmonary TB do not support their use [11, 12]. DRUG RESISTANCE For all patients, the initial M. tuberculosis isolate should be tested for drug resistance. It is crucial to identify drug resistance as early as possible to ensure effective treatment. Drug susceptibility patterns should be repeated for patients who do not respond adequately to treatment or who have positive culture results despite three months of therapy. Susceptibility results from laboratories should be promptly reported to the primary health care provider and the state or local TB control program [9, 13]. CONCLUSION AND RECOMMENDATION This overview and development pipeline for tuberculosis diagnostics and drug sensitivity testing gives a mixed picture. On the one hand, the pipeline is unquestionably active, with a number of different tracks being explored, culture or otherwise, and some improvements have been made. However, if it is now possible to increase throughput, or obtain results faster than can be achieved with traditional culture methods, these improvements often come at the expense of sensitivity and simplicity. In part this is due to the complexity of the problem for TB diagnostics today. REFERENCES 1. World Health Organization. The World Health Report 2003: Changing History. Geneva, Switzerland, PT Kent, GP Kubica. Public Health Mycobacteriology: A Guide for the Level III Laboratory. US Department of Health and Human Services, CDC, Atlanta, Georgia. 3. Master, R.N. Section Editor. "Mycobacteriology". Clinical Microbiology Procedures Handbook, 1: ASM, Washington D.C In the WAC, see Chapter (Notifiable Conditions) in the Title 246 (Department of Health) at te= RRJoLS (2013) 1-10 STM Journals All Rights Reserved Page 6

7 Volume 3, Issue 3, ISSN: American Thoracic Society (ATS) and CDC. Diagnostic standards and classification of tuberculosis in adults and children. Am. J. Respir. Crit. Care. Med. 2000; 161: ll/161/4/ ATS, CDC, IDSA. Controlling Tuberculosis in the United States: Recommendations from the American Thoracic Society, CDC, and the Infectious Diseases Society of America. MMWR 2005; 54(No. RR-12): 51p. 7. CDC. Revised Recommendations for HIV Testing of Adults, Adolescents, and Pregnant Women in Health-Care Settings. MMWR 2006; 55(No. RR-14): 1 17p. 8. CDC. Medical Evaluation. In: Chapter 5: diagnosis of TB. Core Curriculum on Tuberculosis (2000) [Division of Tuberculosis Elimination Web site]. Updated November Available at: index.htm. Accessed March 11, J.Cunningham, Presented at 36th Union World Conference on Lung Health of the International Union Against Tuberculosis and Lung Disease, Paris ATS, CDC, and Infectious Diseases Society of America. Treatment of Tuberculosis. MMWR 2003; 52 (No. RR- 11) pdf 11. CDC. Guidelines for using the QuantiFERON -TB Gold test for Detecting Mycobacterium Tuberculosis Infection, United States. MMWR 2005; 54(No. RR-15): 52p. 12. Updated Guidelines for the Use of Nucleic Acid Amplification Tests in the Diagnosis of Tuberculosis. MMWR 2009; 58(1): html/mm5801a3.htm?s_cid=mm5801a3_e 13. Centers for Disease Control and Prevention. Guidelines for the Investigation of Contacts of Persons with Infectious Tuberculosis and Guidelines for using the QuantiFERON -TB Gold test for detecting Mycobacterium Tuberculosis Infection, United States. MMWR 2005; 54 (No. RR-15). df 14. Sources: ATS, CDC, IDSA. Controlling Tuberculosis in the United States: Recommendations from the American Thoracic Society, CDC, and the Infectious Diseases Society of America. MMWR 2005; 54(No. RR-12): 19p; and R.Tenover, et al. The Resurgence of Tuberculosis: is your Laboratory Ready? J. Clin. Microbiol. 1993; p. 15. Y Akselband, C Cabral, DS Shapiro, et al. Rapid Mycobacteria Drug Susceptibility Testing using Gel Microdrop (GMD) Growth Assay and Flow Cytometry. J. Microbiol. Meth. 2005; 62: p. RRJoLS (2013) 1-10 STM Journals All Rights Reserved Page 7

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