Indian Journal of Medical Microbiology Medknow Publications on behalf of Indian Association of Medical Microbiology ISSN: 0255-0857 EISSN: 1998-3646 Vol. 28, Num. 4, 2010, pp. 281-289

Indian Journal of Medical Microbiology, Vol. 28, No. 4, October-December, 2010, pp. 281-289

Review Article

Diagnosis of tuberculosis in an era of HIV pandemic: A review of current status and future prospects

M Chaudhary¹, S Gupta², Shashi Khare², S Lal²

¹ Assistant Director, Microbiology Division, National Institute of Communicable Diseases, Delhi- 110054, India
² National Institute of Communicable Diseases, Delhi - 110 054, India
Correspondence Address: M Chaudhary, Assistant Director, Microbiology Division, National Institute of Communicable Diseases, Delhi- 110054, India
drmonica74@gmail.com

Date of Submission: 22-Oct-2009
Date of Acceptance: 29-Apr-2010

Code Number: mb10091

PMID: 20966555
DOI: 10.4103/0255-0857.71805

Abstract

Keywords: molecular diagnosis, HIV, Tuberculosis, HIV-TB co-infection

Current Scenario

Despite the discovery of the tubercle bacillus more than a hundred years ago, and all the advances in our knowledge of the disease made since then, tuberculosis still remains one of the major health problems facing mankind, particularly in the developing countries. Epidemiologically, tuberculosis (TB) is a disease that thrives in conditions of poverty, malnutrition and limited access to healthcare. Developing countries bear the brunt of the global burden. The World Health Organization (WHO) estimated 9.2 million new cases of TB globally in 2006 (139/100 000); of whom 709 000 (7.7%) were HIV positive. India, China, Indonesia, South Africa and Nigeria rank first to fifth, respectively, in terms of incident TB cases. ^[1] It is estimated that there are 14 million TB cases in India, of which 3.5 million are sputum positive. TB kills more than 2 million people annually and is the leading cause of death among adults (15-59 years) in Africa and Asia. ^[2] TB is the leading cause of death among HIV-infected persons. ^[3] This imposes a substantial burden on the economy in terms of current and future output losses because of premature deaths and ill health.

In India, there were 2.5 million people living with HIV and AIDS (PLWHA) at the end of 2007, while the incidence of TB was approximately 1.8 million cases per year. ^[4] In a survey carried out among new TB patients by the Revised National TB Control Program (RNTCP) in 2007, HIV sero-prevalence varied widely and ranged from 1 to 13.8% across 15 districts. ^[4] These two infections interact in fundamentally important ways: patho-physiologically, clinically and epidemiologically. There are several differences between HIV-infected and HIV-uninfected patients with TB that have practical diagnostic implications. In this article, we have reviewed the interactions between HIV and TB, the problems faced in diagnosing TB in HIV-infected population and some of the new tools and diagnostic modalities available to diagnose TB in a co-infected patient.

Interactions between HIV and TB

HIV fuels the TB epidemic in many ways. HIV promotes progression to active TB in both categories, that is, people with recently acquired TB and those with latent Mycobacterium tuberculosis infection (LTBI). HIV is the most powerful known risk factor for reactivation of LTBI to active disease. The lifetime risk of TB in immunocompetent persons is 5-10%, but in HIV+ve individuals, there is a 5-15% annual risk of developing active TB disease. ^[4] This increase in TB cases in PLHA (people living with HIV-AIDS) pose an increased risk of TB transmission to the general community, whether or not HIV-infected.

Clinically, the presentation of TB may be altered in an HIV-infected individual. All forms of TB in HIV+ve patients, except when cavitations occur, are paucibacillary in nature. ^[5] Extrapulmonary sites are more often involved, particularly when CD4 ⁺ counts are markedly depressed. Lymph nodes are the most common extrapulmonary sites, but studies also report higher incidence of tubercular meningitis, pleural TB and tubercular pericarditis, though pulmonary tuberculosis is present in over half of HIV-infected TB patients. ^[6] TB is more likely to be disseminated in nature and more difficult to diagnose by conventional diagnostic procedures as immunosuppression progresses. HIV also appears to increase the risk of paediatric TB, which is as such difficult to diagnose. Moreover, in the setting of HIV infection, the tuberculin skin test (TST) may give a low sensitivity, which is the mainstay of diagnosing TB in children.

Diagnosis of Tuberculosis in an HIV-infected Individual

Radiological features

The chest radiography is the cornerstone of diagnosis of pulmonary tuberculosis. The radiological findings in pulmonary TB may also be altered in the presence of HIV co-infection, to a degree proportional to the degree of immuno-suppression. With higher CD4+ T-cell counts (e.g. > 200 cells/μL), the radiographic pattern tends to be one of the reactivation disease with upper lobe infiltrates with or without cavities. ^[7] With greater immunosuppression (e.g. CD4+ count < 200 cells/μL), a pattern of primary disease with intra-thoracic lymphadenopathy and lower-lobe infiltrates can be seen. However, chest radiographs may appear normal in 7-14% of cases of HIV-TB co-infection. ^[7],[8]

Tuberculin skin test

An annual tuberculin skin test (TST) is highly recommended for all persons who are HIV+ve. If the skin test is positive, there is a strong suspicion of tuberculosis. Many HIV+ve patients will have a negative skin test despite TB infection or disease due to anergy, as the immuno-suppression progresses. ^[9]

Attempts to increase the sensitivity of the TST in this population through the use of a "two stage or booster test" have not been very successful. The two-stage tuberculin testing is not a substitute to anergy testing; however, it may have some utility in detecting M. tuberculosis infection in anergic HIV-TB co-infected patients. ^[10] Anergy is 4 times and 15 times more likely in persons with CD4+ counts of 200-400 cells /μL and <200 cells/μL, respectively, as compared to those with a CD4+ counts > 499 cells/μL. ^[10]

Alternative strategies

To overcome the poor specificity of existing skin test based on tuberculin, and to diagnose LTBI in profoundly immuno-suppressed patients such as HIV/AIDS and severe malnutrition, better assays are needed.

• TB MPB-64 skin patch test: One of these is MPB-64 skin patch test. MPB-64 is a specific mycobacterial antigen secreted by M. tuberculosis, M. bovis and some strains of M. bovis BCG. This patch test becomes positive in 3-4 days after patch application on skin, and induration on skin lasts for a week. However, the exact biological mechanism behind this skin response is unclear. In studies conducted in Japan and Philippines, the MPB-64 skin patch test was able to distinguish active TB from LTBI with a sensitivity of 98% and a specificity of 100% ^[11] Since this test is simple, non-invasive and does not require a laboratory or highly skilled personnel, it has the potential to make impact in developing countries especially, if shown to be accurate and unaffected by anergy in HIV-infected individuals. This test is being developed into a commercial test by Sequella Inc. (MD, USA) using recombinant rMPT64 gene and the trials are ongoing to establish accuracy, dose range and other operational characteristics. ^[12]
• IFN-γ release assays (IGRA): These in vitro blood assays are based on the principle that T cells of sensitized individuals produce IFN-γ when they re-encounter the antigens of M. tuberculosis. These assays use M. tuberculosis specific region-of-difference 1 (RD 1) antigens such as early secretory antigenic target 6 (ESAT 6) and culture filtrate protein 10 (CFP 10). These assays are available commercially in the ELISA format, for example QuantiFERON-TB(QFT) and the Enhanced QuantiFERON-TB Gold assay (Cellestis International, Carnegie, Australia) and ELISPOT format, for example T-SPOT-TB assay (Oxford Immunotec, Oxford, UK)]. ^[13] [Table - 1] ^[14] shows the differences in both the formats that are being used for IGRAs commonly.
• Institutions in North America and Europe are beginning to replace TST with IGRA. [Table - 2] compares various functional variables of the TST with IGRA. In December 2005, US CDC (Centre for Disease Control) published its interim guidelines on the US FDA (Food and Drug Administration) approved version of QFT assay. It states that QFT can replace the TST in all circumstances in which TST is currently being used, including serial testing. ^[15] Rapidly accumulating evidence from studies suggests that IGRAs have characteristics that are ideal for serial testing, are more specific than TST, can be repeated without concerns about sensitivity or boosting and the testing protocol requires fewer visits. They are also unaffected by previous BCG vaccination and eliminate the need for two-step testing.

A study in Zambia observed that ESAT-6/CFP-10-based ELISPOT assay had a high sensitivity in detecting active TB in HIV+ve individuals. ^[16] In a prospective blinded study of 293 African children with suspected TB in Kwazulu-Natal, a region with high HIV prevalence, it was seen that ELISPOT's sensitivity was not significantly affected in children below 3 years of age, HIV co-infection and with malnutrition. ^[17] A study of asymptomatic South African adults from a region of high prevalence for TB and HIV found that although the rates of TST positivity were much reduced in HIV+ve versus HIV-ve subjects, the rates of positive ELISPOT and ELISA results did not vary significantly by HIV status. ^[18]

Another recent study by Clark et al. from London, UK, showed that in 201 HIV-1 infected patients with risk factors for TB infection, the performance of ELISPOT assay to detect TB antigen-specific immune response is independent of the CD4+ T-cell counts. The sensitivity and specificity of this assay for the diagnosis of active TB does not differ significantly from values obtained in immunocompetent subjects with a positive predictive value of 86% and negative predictive value of 98.2%. They found this test to be a useful tool to exclude TB infection in immunodeficient HIV-1 patients. ^[19] An Indian study to evaluate the agreement between TST and IGRA (QFT-Gold) was conducted in Sevagram, Maharashtra on 726 healthcare workers. The researchers reported a high degree of agreement of 81% between TST and IGRA. ^[20]

However, IFN-γ assays are expensive, need a good laboratory infrastructure and trained personnel, which appear to limit their wider applicability; especially in resource-limited settings and developing countries where TB is rampant. In India, although no formal costing studies have been done, the material cost of IGRA may be 5-10 times higher than TST. ^[20] It also remains to be seen whether these initial encouraging results will translate into practically useful results in field.

Microscopy

For developing countries, the smear microscopy to detect Acid Fast Bacilli (AFB) in clinical specimens by ZN (Ziehl-Neelsen) staining / fluorescent staining is likely to remain the only cost-effective tool for diagnosing patients with TB and to monitor the progress of treatment. Some studies have found that sputum-smear examination is less sensitive in HIV-infected patients, ^[21] However, this observation has not been confirmed by others. ^[22] The AFB smear positivity rates have ranged from 31 to 90% in these studies, ^[21],[22] which is similar to results seen in immunocompetent adults. A zwitterionic detergent, CB-18 (carboxypropylbetaine 18), has been shown in both laboratory and clinical studies to enhance the sedimentation of mycobacteria during the concentration process and hence enabling better visualization of AFB. ^[23]

Culture

Culture remains the gold standard for diagnosis of M. tuberculosis infection. A positive blood culture for M. tuberculosis (uncommon in HIV-ve patients with TB) may be diagnostic of disseminated TB in patients with advanced HIV infection. However, due to alteration of the normal host immune response to M. tuberculosis in persons with HIV, cavitation and transfer of bacilli into respiratory secretions is markedly reduced, thereby hampering diagnosis by microscopy and culture. ^[24] In addition, the increased prevalence of extrapulmonary forms of tuberculosis in HIV-infected patients is a further challenge to the management of TB in resource poor settings, where access to histopathology and advance imaging tests are limited or absent. The sensitivity of culture positivity varies according to the nature of the specimen and ranges from 26 to 42%. ^[25] The traditional method of inoculating solid media such as the LJ medium or Middlebrook medium is sensitive but slow, as growth may not be visible until after 6-8 weeks of incubation. This results in delay in initiation of therapy, with detrimental effects on management of HIV+ve patients. Although a combination of traditional solid and liquid media is recommended for the primary isolation of mycobacteria, a few modern and rapid methods are also available now.

The introduction of the BACTEC TB 460 radiometric system (Becton Dickinson, Sparks, MD,USA) in 1980s was a breakthrough since it allowed the detection of M. tuberculosis in few days as compared with the conventional culture media. The introduction of these rapid and automated systems have increased the sensitivity of isolation of mycobacteria from clinical samples and has brought down the time required for positive culture substantially (9-10 days). Faster culture results in HIV-infected patients can result in evidence-based therapy and faster implementation of treatment. Other novel culture methods and techniques for faster isolation of mycobacteria are continuously being developed; however, a few techniques which have shown promise include:

• TK Medium^; Culture System (Salubris, Inc. MA, USA): A novel colorimetric culture system that indicates growth of mycobacteria by changing its colour. ^[26] This medium incorporates several colour dye indicators that change colour depending upon the metabolites and enzymes produced by different species, and the changes occur well before the colonies become visible. The original red colour of the TK medium turns yellow with mycobacterial growth and green with other bacterial or fungal species. TK medium promises to be a practical, low cost, simple test but published evidence is limited, and no studies are available showing its performance in HIV-TB co-infected individuals. Average time of detection was two weeks as compared to four weeks with LJ medium. ^[26],[27] It can also be used for drug-susceptibility testing and differentiating M. tuberculosis and NTM (non-tuberculous mycobacteria). This medium is currently not FDA approved. Larger studies are required to validate these results and answer the critical question of how this test will perform in smear-negative HIV patients, which will be a key indicator to its usefulness in high HIV-TB endemic populations.
• MODS: The microscopic observation drug susceptibility (MODS) assay relies on light microscopy to visualize the characteristic cording morphology of M. tuberculosis in culture. Decontaminated sputum samples are incubated with Middlebrook-7H9 broth, nutrients and an antibiotic cocktail in a standard 24-well culture plate. Cultures are studied daily for cording using an inverted light microscope in a standard laboratory. ^[24] In a recent large study from Peru. ^[28] MODS detected 94% of 1908 positive sputum cultures; whereas LJ medium detected only 87%. MODS also had shorter time to culture positivity (average 8 days) compared with LJ medium (average ~26 days). The cost effectiveness of MODS (~$2 per sample) compared favourably with LJ culture (~$6 per sample). Hence MODS appear to be a promising, novel and inexpensive tool that can rapidly detect TB and drug resistance directly from sputum samples. However, further studies are needed to address the performance of MODS in smear-negative cases, which is very common in HIV patients.

Bacteriophage-based assays

These assays rely on the ability of M. tuberculosis to support the growth of a specific infecting mycobacteriophage. The underlying principle is the amplification of bacteriophages after their infection of M. tuberculosis, followed by detection of progeny phages as lytic plaques on a lawn of M. smegmatis. Phage amplification assays are available as commercial kits from BIOTEC (Ipswich, Suffolk, UK). The FAST Plaque-TB assay can be used on sputum specimens; The FAST Plaque-TB-MDRi is designed to detect rifampicin resistance in culture isolates and FAST Plaque-TB-Response for direct use on clinical specimens. ^[12] This system appears robust and has the advantage of speed and the promise of high sensitivity. These kits are not currently FDA approved, but are CE (conformitι europιenne) marked for use in Europe.

An important finding of a recent study ^[29] was that the FAST Plaque-TB was able to detect mycobacteria in 50-65% of smear-negative specimens with a specificity of 98% and that a combination of the test with smear microscopy confirmed the presence of M. tuberculosis in 80-90% of culture positive specimens. In a meta-analysis ^[30] by Pai M, it was shown that, when performed on culture isolates, these assays have relatively high accuracy. A total of 11 out of 19 (58%) studies reported sensitivity and specificity estimates of at least 95%. Only two studies gave inconsistent results when this test was performed directly on sputum specimens. ^[30] During the evaluation of this kit at NICD, Delhi (unpublished data), though the specificity was 95%, the sensitivity was low (20%) especially in extrapulmonary cases of TB when compared with smear microscopy (25%) and culture by BACTEC TB 460 system (30%).

Another modified method, using bacteriophages is based on the principle that when recombinant mycobacteriophage containing firefly Luciferase gene enters mycobacteria, they multiply and express Luciferase gene leading to production of Luciferase enzyme. Final diagnosis is done by detection of fluorescence (using luminometry or photography) produced by action of Luciferase enzyme on Luciferin substrate. ^[31] A Luciferase reporter phage-based test called the "Bronx Box" is currently being developed as a commercial kit by Sequella Inc., ^[12] which is a semi-automated digital version. Whether such systems will eventually be useful in a TB endemic country, will depend on the reproducibility of the assay, the sensitivity and specificity of the test especially in extrapulmonary TB, the training/equipment required and the cost factors.

Molecular techniques

Nucleic acid amplification tests (NAAT), also called direct amplification tests (DAT), are designed to amplify nucleic acid regions specific for M. tuberculosis complex and can be used directly on the clinical samples. Tests include those that are "in-house", where they are based on a protocol developed in a non-commercial laboratory, and "commercial kits". Though all the NAATs rely on the amplification of specific DNA/RNA targets, their principles and techniques are different from each other. Commercial kits available in the market, their target gene and principle are shown in [Table - 3]. The Amplicor MTB^® and AMTD^® tests are currently FDA approved and are licensed for testing smear-positive specimens. FDA recently also approved a second generation AMTD (E-MTD) test for smear-negative specimens. The LCx, BD-Probe-Tec-ET and LAMP are currently not FDA approved. ^[32]

Commercial kits have the advantage of being well standardized, reproducible and have shown to be highly sensitive and specific in smear-positive samples. However, these values are much lower in smear negative samples or in extrapulmonary specimen (especially in HIV patients) where these assays are much needed. In summary, available NAATs have not realized their early promise. In addition to concerns about accuracy and reliability, their high cost and requirement for proper laboratory infrastructure and strict quality control procedures, reduce their applicability in resource-limited settings. Efforts are underway to simplify testing protocols and increase their accuracy. Few such developments are briefly described below:

• Loop-mediated isothermal amplification (LAMP): It is a novel, rapid and simplified NAT platform developed by Eiken Chemical Co. Ltd (Tokyo, Japan). ^[12] It has characteristics that may allow its use in less sophisticated settings. LAMP amplifies DNA with high efficiency under isothermal conditions using six set of primers specifically designed to recognize six different regions on the target gene. The large amount of DNA generated and the high specificity of the reaction makes it possible to detect amplification by visual inspection of fluorescence without the need for gel electrophoresis or instrument detection of the labeled probe. This allows development of a closed tube system that minimizes the risk of workplace contamination with an amplicon. ^[33] Its major advantages are speed, simplicity and lack of requirement for a thermal cycler, thus facilitating high throughput. The amplification efficiency is high and DNA can be amplified 10 ⁹ -10 ¹⁰ times in 15-60 min. ^[34] These features make it a promising platform for the molecular detection of TB in developing countries. The operational feasibility of this technique for diagnosis of pulmonary tuberculosis in microscopy centres of developing countries was evaluated in a study. ^[33] The researchers found the sensitivity of LAMP in smear and culture positive specimens to be 97.7% and the sensitivity in culture negative specimens to be 48.8%. The average hands on time for testing six samples and two controls was 54 min, similar to sputum microscopy and the optimal amplification time was 40 min. No indeterminate results were reported and technicians with no prior molecular experience could easily perform the test after a week of training. Though this technique still remains untested in the field of TB-HIV co-infection, the results appear promising in comparison to conventional techniques.
• Fluorescence in situ hybridization (FISH) using Peptide Nucleic Acid (PNA) probes: A commercial system designed by Dako. A/S Glostrup, Denmark has been proposed. It is based on the use of peptide nucleic acid (PNA) probes that bind to selected regions of mycobacterial 16s rRNA sequences. PNA is a novel DNA mimic in which the sugar-phosphate backbone of DNA has been replaced with polyamide backbone. The uncharged nature and the high conformational flexibility of PNA allow PNA probes to hybridize to DNA or RNA with excellent affinity and specificity. The test utilizes two fluorescin-labeled PNA probes: the MTB (M. tuberculosis) probe targeting MTBC (M. tuberculosis complex) species and the NTM (non-tuberculous mycobacteria) probe targeting NTM species for culture confirmation of isolated mycobacteria. They have also been used directly on sputum samples. ^[35] This allows distinction between TB and NTM and direct detection of MTB on smear positive samples and formalin-fixed paraffin-embedded biopsies. ^[36] Detection is done by microscopic observation. The diagnostic sensitivity of the MTBC probe for the LJ and MGIT cultures were 98 and 99%, respectively with 100% specificity in a study from Thailand. ^[35] Though this technique is rapid, simple and well suited for identification of MTBC and NTM from culture media, it needs to be adapted to direct identification of mycobacterial species in sputum samples.
• Line probe assays (LPA): These are a family of novel DNA strip based tests that use PCR and reverse hybridization methods for simultaneous detection, identification and drug resistance. Commercial assays include INNO-LiPA (Innogenetics NV, Gent, Belgium) and Geno. -Type mycobacterium assay (from Hain-Diagnostika, Nehren, Germany). ^[12],[37] This has been used for identification of mycobacteria from clinical samples and liquid cultures. It is based on amplification of the 16S-23S ribosomal DNA spacer region followed by hybridization with 16S-specific oligo-nucleotide probes. It has the advantage of detecting mixed mycobacterial infections. ^[38] Both the above kits are still not FDA approved.

A recent meta-analysis by Morgan and colleagues ^[39] demonstrated that the LPA had high-sensitivity and specificity when culture isolates are used (>95% sensitivity and 100% specificity). However, the results are less accurate when the test is applied directly on clinical specimens (e.g. sputum). In general, LPAs are expensive and require sophisticated laboratory infrastructure. Their role and utility in low-income, high-burden countries will be limited unless cost and technical issues are addressed.

Serological diagnosis of tuberculosis

• Detection of antibodies: Immune-based tests for the detection of antibodies, antigen and immune complexes have been attempted for decades and their performances have been extensively reviewed. ^[40],[41] Despite the long history of serological tests and persistent attempts at improving them, no assay is currently accurate enough to replace microscopy and culture. The HIV epidemic and its impact on immunity further threatens the progress in this area. In cases of HIV co-infection, most of these assays perform very poorly, detecting less than one-third of patients with active disease. ^[42] A recent review by Wanchu A ^[10] focuses on the advances in serology-based diagnostic aspects of tuberculosis in HIV-infected patients. ELISA techniques employing various mycobacterial antigens, e.g. glycolipids from M. bovis BCG, antigen 5 and 6 from M. tuberculosis, 64 kDa protein from M. bovis BCG, 32 kDa protein antigen (P32) and antigen 60 have been attempted for serological diagnosis of tuberculosis with variable success rates. Promising research developments in serology include: i) the development of multi-antigen tests that are expected to provide high specificity and sensitivity; ii) the characterization of a number of non-protein antigens, some of which appear promising in HIV-infected patients and; iii) the development of improved and simplified test formats such as those accepting whole blood or non-invasively collected specimens, for example urine. TB STAT-PAK and Insta Test TB are commercial immuno-chromatographic tests (ICT) based on lateral flow principle. ^[43] The limitation of these tests is their extremely low sensitivity in smear-negative patients, HIV ^+ve cases and in disease-endemic countries with high-infection rate. Several authors have attempted to analyze reasons for the apparent failure of serological tests. ^[40] TB represents a wide spectrum of disease ranging from exposure, through latent TB infection (LTBI) and active disease, to severe disease. ^[44] Several other factors such as BCG vaccination, exposure to NTM, M. tuberculosis strain and HIV co-infection also affect the performance of immune-based tests. Immune response in mycobacterial disease appears to be associated with HLA class II allotypes and different patients appear to recognize different antigens. ^[43] Moreover, many serological assays use single antigens (e.g. 38 kDa protein) that may not be recognized by the host immune response during all stages. The development of cocktails of multiple specific antigens may partially address this problem. ^[45] It has been suggested that each TB state is characterized by a bacterial antigen signature that resemble barcodes. ^[46] Certain combinations of the presence and absence of antigen-specific immune response may indicate specific disease states. This may then be used to distinguish between various stages of TB infection and diseases. ^[46],[47] In recent studies, efforts have been made to identify specific antibody responses among HIV-infected individuals. Antibodies against sulfo-lipid antigen (SL-IV) could be detected in 12/13 patients with TB and HIV with a specificity of 96.0%. ^[10] Another study showed that antibodies to 88 kDa secreted antigen were present in the sera of 74% of HIV/TB patients for up to six years prior to manifestation of active, clinical TB and 96% were positive by the time TB was diagnosed. ^[48]
• Detection of antigen: Efforts are underway to develop immune-based assays that focus on antigen, rather than antibody detection, in samples such as sputum, urine etc. Capture ELISA test is available to detect lipoarabinomanan (LAM) in urine specimens. Another test being used in field trials is the dip-stick method (semi-quantitative) for the detection of LAM in both pulmonary and extrapulmonary specimens. Preliminary studies have shown a sensitivity and specificity of 93 and 95%, respectively. ^[43] Free mycobacterial antigen at a concentration of 3-20 ng/mL can be detected in biological fluids such as CSF or pleural fluid. The formats used for antigen detection are: sandwich ELISA, inhibition ELISA, latex agglutination and reverse passive HA tests. ^[43] Overall, the lessons learnt from previous failures should facilitate the development of new generation of immune-based tests, including ones that detect antigen and/or circulating immune complexes, especially if they can be packaged as point-of-care strip tests that may be used in primary care settings.

Other non-microbiological diagnostic techniques

The detection of adenosine deaminase (ADA) has received most attention as an indirect method of diagnosis of TB. An ADA is an enzyme present in almost all mammalian cells, principally in the lymphocytes, being directly related to lymphocyte activation. As a result, in diseases presenting greater lymphocyte activation and participation, elevated levels of ADA are usually detectable. This enzyme has mainly been studied in the diagnosis of pleural TB, peritoneal TB and TB meningitis where elevated levels of this enzyme are present. However, the main drawback is that in other disease conditions that cause pleural effusion with predominance of lymphocytes such as SLE, lymphoma etc, high levels of ADA may also be seen. Determination of ADA levels has been praised as a promising marker since it can be performed easily, rapidly and at a low cost by a colorimetric method.

In a study from Brazil, it was shown that in cases of pleural TB, an ADA cutoff value of 35U/L gave a sensitivity and specificity of 92.8 and 93.3%, respectively. Mean ADA levels in pleural TB group was 84.7 ± 43.1 U/L versus 15.9 ± 11.1 U/L in non-pleural TB group. There was no significant difference in ADA levels between patients with and without HIV co-infection in both the groups. ^[49] Though these results appear promising, more studies need to be done to evaluate its utility and applicability in HIV-TB co-infected patients.

Challenges for the Future

In a recent study, ^[50] the potential impact of enhanced TB diagnostic techniques as a TB control strategy in an adult population with high HIV prevalence was studied and compared with other TB control interventions including active TB case finding, treatment of LTBI and treatment of HIV ^+ve adults with HAART. They found that implementation of a rapid molecular test or culture-based system for TB diagnosis could avert 20% or more of all TB deaths, an impact equivalent to that of successfully extending existing diagnostic techniques to 33% of the general community, who otherwise would not seek care for symptoms of TB. When existing diagnostic practices are poor, smear-negative cases (especially HIV+ve) will represent an increasing proportion of total TB prevalence with higher relative infectiousness. In the above model, ^[50] 76% of prevalent TB cases are smear negative at baseline; these patients are thus responsible for over 40% of all transmission events despite being only one-fifth as infectious as smear-positive individuals. This model suggested that diagnosis of smear-negative TB must be a priority for TB control efforts in HIV endemic regions.

Conclusions

As TB rates continue to increase in HIV endemic regions, improved diagnostic techniques merit further consideration as TB control strategies. One concern with the development of new tools is that they may involve expensive technologies beyond the reach of developing countries affected by the dual TB/HIV epidemic. Such high-tech tools are unlikely to make an impact in these countries even if they are accurate. By contrast, simpler, friendly formats (such as MPB-64 Skin patch test), rapid, point-of-care immunodiagnostics (lateral flow formats), innovative use of microscopy (MODS) and rapid simplified cultures (such as TK medium) may have greater applicability.

Finally, any new method or approach, sophisticated or not, commercial or in house, should be evaluated in well-designed and controlled clinical trials and tested in a high-TB/HIV-endemic, low-resource settings where the implementation and use of these methods are more needed to contribute to the improvement of tuberculosis control.^[51]

References

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