There is increasing concern over TB/HIV co-infection. An estimated one third of the 40 million people living with HIV/AIDS worldwide are co-infected with TB and, without proper treatment, approximately 90% of those living with HIV die within months of contracting TB. Since not all existing diagnostic methods for TB are suitable for TB/HIV co-infected cases, The Global Plan to Stop TB has prioritised the development of simple, accurate and affordable tests for use in low resource settings for TB detection in both HIV+ and HIV- patients.
by Dr C. Smit

The effective control of tuberculosis (TB) is impeded by a lack of rapid, accurate and affordable diagnostic methods. As an airborne infectious disease, approximately one third [1] of the world’s population is infected with TB. Infection is most prevalent in developing regions such as Africa, South America and Asia [2] [Figure 1]. However, a rising number of people in the developed world are contracting TB due to the increased prevalence of immunosuppression as a consequence of therapeutic drugs, substance abuse or HIV/AIDS.

There is increasing concern over TB/HIV co-infection. With a suppressed immune system, individuals with HIV are more likely to acquire a new TB infection, and previously treated patients are likely to suffer a relapse of the disease. TB is the leading cause of death amongst HIV-infected patients, with HIV promoting the conversion of latent TB infection to active disease, whilst TB infection accelerates the progression to AIDS in HIV+ patients. An estimated one third of the 40 million people living with HIV/AIDS worldwide are co-infected with TB. Furthermore, without proper treatment, approximately 90% of those living with HIV die within months of contracting TB [3].

Existing diagnostic methods for TB are not all suitable for TB/HIV co-infected cases. The tuberculin skin test and chest X-rays are often inconclusive or atypical in HIV+ patients. The Global Plan to Stop TB has prioritised the development of simple, accurate and affordable tests for use in low resource health settings for TB detection in both HIV+ and HIV- patients [4].

Diagnostic methods
Two diagnostic tests predominate in the routine diagnosis of active TB. Smear microscopy is the most commonly used diagnostic testing method worldwide, which has been available for over 100 years. It can detect the most infectious cases and is highly specific in high prevalence settings. Most importantly, smear microscopy is inexpensive. However, as well as being difficult to carry out in the field, the method lacks sensitivity in those who are HIV+, detecting less than half of HIV/TB co-infected cases [5]. It also requires repeat patient visits (two to three) before diagnosis is reached.

Mycobacterial culture is the current, preferred method for diagnosis of active TB. It is more sensitive than smear microscopy, enables TB species to be identified and allows for drug susceptibility testing. However the method is not rapid, taking two to six weeks, rather than days, to reach a result. Additionally it is not suitable for use in resource limited settings, as it requires specialist personnel, equipment, water and electricity.

The development of new diagnostic methods for the detection of active-TB, particularly for populations in which HIV/TB co-infection is high, has proved challenging. However, new commercially available Lipoarabinomannan (LAM) antigen tests, such as Clearview TB ELISA (Inverness Medical Inc.), have proved very useful as diagnostic tools for the detection of TB in those co-infected with HIV.

LAM is a major lipopolysaccharide constituent of the cell wall of Mycobacterium tuberculosis. During the degradation of bacterial cells, LAM antigen enters the bloodstream and is filtered out by the kidneys, passing into the urine. The detection of LAM in urine has the advantage of being able to provide a diagnostic result independent of the location of infection. A sputum based sample is only useful for the diagnosis of pulmonary TB, whereas urine-based LAM antigen testing may provide an indication of both pulmonary and extrapulmonary TB.

With the availability of ELISA-based LAM antigen detection assays, researchers have sought to establish the effectiveness of the diagnostic test in people suspected of having TB. Increasingly, studies have revealed that when used as a general screening tool, the LAM-ELISA test sensitivity is significantly lower in subjects who are HIV- when compared to HIV+ subjects. This is not surprising, given that levels of TB bacteria in subjects who are HIV+ are likely to be higher, resulting in an increase in LAM antigen in the urine.

In addition, in two recent studies [6,7], LAM-ELISA results were correlated against CD4 count. CD4 count is commonly used as a method of monitoring the status of the immune system in HIV+ subjects. CD4 count decreases as HIV infection proceeds through the clinical stages towards AIDS. When CD4 count reaches 200 cells/µL or fewer, the patient’s immune response begins to fail resulting in an increased chance of contacting an opportunistic infection such as TB. For those subjects who are co-infected with TB/HIV, as CD4 count decreases, resulting in an immunosuppression and an increase in TB bacteria in the body, the potential for LAM concentration to meet detectable levels is increased. One would therefore expect a trend of increased LAM test sensitivity as CD4 count decreases.

In a recent paper by Lawn et al [6], the LAM assay was found to be highly specific and to have a sensitivity substantially superior to sputum smear microscopy in patients with CD4 cell counts <100 cells/µL being screened for TB prior to the initiation of HAART. Lawn concluded that for such patients, rapid detection of LAM in urine could reduce the time to diagnosis by approximately three weeks when compared with bacterial culture.

In the most extensive study to date, the Johns Hopkins University Center for Tuberculosis Research enrolled 499 hospitalised South African adults with signs and/or symptoms of active TB [7]. The diagnostic tests carried out included AFB smear microscopy, mycobacterial culture, serum HIV antibody testing (plus CD4 cell count for HIV+ patients) and LAM urine testing. Additionally, patients were interviewed two months after enrolment on the research study to assess clinical status. This process ensured that the true clinical outcome of the patient could be traced and that the exact percentage of patients who developed extrapulmonary or pulmonary TB could be confirmed. Overall the study reported a LAM test sensitivity of 59% in confirmed TB/HIV cases, and a specificity of 96% among individuals classified as “not TB” when the test was used to diagnose active TB in hospitalised patients in a high HIV prevalent setting. Sensitivity was again observed to be higher in HIV+ TB patients than in those uninfected with HIV, and was highest in the subgroup of HIV+ patients with a CD4 cell count <50 cells/µL, in agreement with Lawn that LAM sensitivity is highest in patients with the lowest CD4 counts. 

Summary
Based on these findings, the true utility of LAM antigen diagnostics is becoming clear, that is, the use of LAM antigen testing for TB suspects who present a smear negative microscopy result and who are HIV+ with a CD4 count < 200 cells/µL. It is in this very population of TB/HIV co-infected cases that conventional TB diagnostics often fail to yield a result, and in which hospitalisation rates and mortality are highest. Current LAM antigen tests such as the Clearview TB ELISA can provide results in <3 hours, enabling rapid follow up and treatment. Meeting a need in HIV prevalent and resource limited settings, the assay could be an additional test to be carried out in laboratories already equipped for ELISA-based HIV testing in both the developing and developed world [7].

As diagnostic testing moves closer to the patient, development of a point-of-care (POC) LAM-based rapid test appears to be the next objective. Characteristically, immunochromatic rapid tests are developed in dip-stick or cassette based formats and provide qualitative result in 10-20 minutes. Should a LAM test be available in either format, the testing algorithm for HIV positive TB suspects [Figure 2] might be
developed further.

For HIV+ TB suspects with low CD4 counts, LAM testing may be better than sputum smear microscopy. However, a combination of sputum smear and LAM testing provides good overall sensitivity. Thus, LAM appears to be a good rule-in test in HIV-infected patients suspected of having TB who have low CD4 T cell counts.

References
1. WHO 2009. Tuberculosis Facts. www.who.int/tb/publications/2009/tbfactsheet_2009_one_page.pdf
2. WHO 2009. Tuberculosis factsheet [online].
Available at www.who.int/mediacentre/factsheets/fs104/en/index.html
3. WHO 2009 HIV Factsheet [online]. Available at www.who.int/tb/hiv/faq/en/
4. Stop TB partnership and WHO. Global plan to stop TB 2006-2015. WHO 2006.
5. Getahun H et al. Diagnosis of smear negative pulmonary tuberculosis in people with HIV infection or AIDS in resource-constrained settings. Lancet 2007; 369:2042-2049.
6. Lawn S et al. Urine lipoarabinomannan assay for tuberculosis screening before antiretroviral therapy diagnostic yield and association with immune reconstitution disease. AIDS 2009; 23: 1875-1880.
7. Shah M et al. Diagnostic accuracy of a urine lipoarabinomannan assay for active tuberculosis in hospitalized patients in a high HIV prevalence setting. Journal of Acquired Immune Deficiency Syndrome 2009; 52 (2): 145-151.

The author
Dr Chris Smit
Blood Borne Pathogens Product Manager
Inverness Medical
Bedford, UK
email: professionaldiagnostics@invmed.com
Website: www.invernessmedical.com