Downloaded from http://pmj.bmj.com/ on December 19, 2014 - Published by group.bmj.com
Review
Application of lipoarabinomannan antigen in tuberculosis diagnostics: current evidence Pronoti Sarkar,1 Debasis Biswas,1,2 Girish Sindhwani,3 Jagdish Rawat,3 Aarti Kotwal,1 Barnali Kakati1 ▸ Additional material is published online only. To view please visit the journal online (http://dx.doi.org/10.1136/ postgradmedj-2013-132053). 1
Department of Microbiology, Himalayan Institute of Medical Sciences, HIHT University, Dehradun, Uttarakhand, India 2 Department of Microbiology, All India Institute of Medical Sciences Bhopal, Bhopal, India 3 Department of Pulmonary Medicine, Himalayan Institute of Medical Sciences, HIHT University, Dehradun, Uttarakhand, India Correspondence to Dr Debasis Biswas, Department of Microbiology, Himalayan Institute of Medical Sciences, HIHT University, Swami Ram Nagar, Jolly Grant, Dehradun, Uttarakhand 248140, India; [email protected] Received 16 April 2013 Revised 13 December 2013 Accepted 1 January 2014 Published Online First 15 January 2014
ABSTRACT Tests based on the detection of mycobacterial lipoarabinomannan (LAM) antigen in urine have emerged as potential point-of-care tests for tuberculosis (TB). We aimed to assimilate the current evidence regarding the diagnostic performance of LAM assays and to ascertain their clinical indication in settings with high and low prevalence of HIV-TB co-infection. Owing to suboptimal sensitivity, the urinary LAM assays are unsuitable as general screening tests for TB. However, unlike traditional diagnostic methods, they demonstrate improved sensitivity in HIV-TB co-infection which further increases with low CD4 counts. Accordingly, these assays are indicated as rule-in tests for TB in patients with advanced HIV-induced immunosuppression, and facilitate the early initiation of antituberculous treatment in them. They also offer incremental sensitivity and specificity when used as adjunct tests to smear microscopy and chest radiography in HIV-TB co-infection. They obviate the biohazards associated with sputum samples and provide an alternative diagnostic tool in sputum-scarce patients. Notwithstanding these advantages, the specificity of these assays is variable, which is mostly attributable to misclassification bias and cross-reactivity with nontuberculous mycobacteria or other commensal flora. Furthermore, the inability to detect low titres of antigen in HIV-uninfected patients makes these assays unsuitable for use in settings with a low HIV prevalence. Future research targeted towards inclusion of specific monoclonal antibodies and more sensitive immunoassay platforms might help to improve the diagnostic performance of these assays and extend their applicability to the general population of patients with TB.
INTRODUCTION
To cite: Sarkar P, Biswas D, Sindhwani G, et al. Postgrad Med J 2014;90:155–163.
Tuberculosis (TB) continues to remain a major cause of mortality and morbidity throughout the world, with the WHO estimating figures of 8.6 million incident cases and 1.3 million deaths occurring in the year 2012.1 The challenges of multidrug resistant and extensively drug resistant TB and the ongoing epidemic of HIV-TB co-infection have further complicated effective TB control. In line with the Millennium Development Goals, the WHO has developed a six-point ‘Stop TB Strategy’ to dramatically reduce the global burden of TB by 2015. Early case detection and providing standardised treatment under supervision constitute the first component of this strategic initiative.2 In spite of the importance of early diagnosis, none of the diagnostic modalities currently available offer a simple, affordable, rapid and reliable test that can be easily accessed by patients in low- and middle-income countries, which account for over
Sarkar P, et al. Postgrad Med J 2014;90:155–163. doi:10.1136/postgradmedj-2013-132053
90% of the global TB burden. Sputum-smear microscopy, though considered the cornerstone of the Directly Observed Treatment Short-Course (DOTS) strategy, fails to detect smear-negative cases of pulmonary TB,3 which may account for 24–61% of all pulmonary cases in HIV-infected individuals.4 Chest radiography has the limitations of poor specificity, infrastructure dependence and sub-optimal sensitivity in HIV-TB co-infection.5 6 Mycobacterial culture, which is regarded as the diagnostic gold standard, has a long turn-around time and demands elaborate infrastructure and trained manpower.7 Newer diagnostic tests based on nucleic acid amplification are too expensive and complex for routine use at peripheral locations in resource-constrained settings.8 In this context, the potential benefits of a reliable point-of-care test with adequate sensitivity in HIV-TB co-infected patients cannot be overemphasised, and antigen detection tests have been tested as potential solutions to address this diagnostic lacuna. Of the various antigens that have recently been tested for their diagnostic potential, the most promising results have been obtained with lipoarabinomannan (LAM) antigen, a major lipopolysaccharide component of the mycobacterial cell wall. The use of LAM as a diagnostic target has two very attractive advantages. First, unlike the alternative diagnostic modalities such as smear microscopy and chest radiography, LAM antigen detection tests deliver increased sensitivity in HIV-TB co-infected patients.9 10 Second, being detectable in urine, these tests obviate the potential biohazards associated with the handling of sputum samples and provide an alternative diagnostic tool in sputum-scarce patients.10 11 As a result of these inherent advantages, the LAM antigen has been extensively targeted for the development of diagnostic assays and has lately been incorporated into commercial ELISA and point-of-care lateral flow immunochromatography tests. The diagnostic performance of these assays has been evaluated in several studies based on both urinary and nonurinary samples, involving patients with both pulmonary and extrapulmonary TB and encompassing a diverse range of clinical settings ranging from tertiary referral hospitals to community clinics. In view of the increasing literature on the subject, we aimed to assimilate the current evidence regarding the diagnostic performance and test characteristics of these assays in both urinary and non-urinary samples and to ascertain their clinical indication in TB diagnostic practice. We also explored the potential utility of their usage, alongside the existing diagnostic tools for TB, in settings with a high and relatively low prevalence of HIV-TB co-infection. 155
Downloaded from http://pmj.bmj.com/ on December 19, 2014 - Published by group.bmj.com
Review BIOLOGY OF LAM ANTIGEN LAM, a 19 kDa glycolipid, is the most extensively studied immunomodulatory lipid in the mycobacterial cell wall. Being a heat-stable molecule, it does not degrade readily in clinical samples and, hence, is suitable as a target for diagnostic application in peripheral centres.12 However, its alkali-labile nature leads to the denaturation of antigenic epitopes on using sodium hydroxide for the pretreatment of sputum samples,13 and necessitates alternative decontamination protocols using N-acetyl L-cysteine-proteinase K or DTT in assays designed to detect LAM antigen in sputum.11 14 LAM has been classified into three structural types depending on the nature of the capping motif attached to it, with each structural type carrying a distinct functional overtone. While the pathogenic mycobacterial species such as Mycobacterium tuberculosis, M leprae, M avium and M kansasii have a mannose cap (ManLAM),15–17 the fast-growing non-pathogenic species M fortuitum and M smegmatis have an inositol phosphate cap (PILAM)18 and a third family devoid of both these types of caps (AraLAM) has recently been identified in M chelonae.19 These structural variations, schematically depicted in the online supplement, are associated with differential roles in evading or activating host immunity. While AraLAM, extracted from avirulent mycobacteria, elicits proinflammatory cytokine responses from macrophages,20 21 ManLAM is predominantly associated with downregulation of host immunity through a number of pathways including suppression of M tuberculosis-induced apoptosis,22 23 interleukin 12 secretion24 and phagolysosomal fusion25 26 in macrophages, maturation of dendritic cells27 and expansion of regulatory T cells.28 Unlike its structural analogues in avirulent mycobacteria, ManLAM thus contributes to the pathogenicity of virulent mycobacteria by undermining a protective host response and evading host immunity. Unlike other antigens in diagnostic use (eg, capsular polysaccharides of Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis and Cryptococcus neoformans which are composed of repeating carbohydrate motifs), the LAM antigen evokes a potent humoral immune response and is present in the systemic circulation in an immune-complexed form.29 Although studies on the pharmacokinetics, tissue distribution and mechanism of urinary excretion of LAM antigen have not been undertaken in humans, animal studies in mice intraperitoneally injected with a crude cell wall extract of M tuberculosis have shown the urinary excretion of LAM antigen within 17 h.30 However, in experiments designed to mimic the systemic circulation of LAM antigen in the immune-complexed state (by injecting purified LAM into mice pretreated with anti-LAM IgM antibody), tissue distribution of LAM antigen has been observed in the liver and localisation in the kidneys and excretion in urine has not been demonstrated.31 In view of these findings, the underlying basis of urinary detection of LAM antigen in patients with TB remains unclear. It is likely that, of the two forms of this antigen present in the systemic circulation of patients with TB (ie, free and immune-complexed), only the former is excreted in urinary samples.32
LAM ANTIGEN: TEST FORMATS IN DIAGNOSTIC APPLICATION Three types of assay formats have been tested in recent years for the detection of LAM antigen. A sandwich ELISA technique using urine samples has been employed in the commercially available Clearview TB ELISA test (Inverness Medical Innovations, USA) which now supersedes the pre-commercial 156
prototype (MTB LAM ELISA Test, Chemogen, Portland, Oregon, USA) first tested in Tanzania in 2005.33 Although the same polyclonal antibody has been used in the commercial and pre-commercial versions of the ELISA test, differences in the manufacturing technology and protocols might lead to differences in test performance between the two variants.34 Nevertheless, the use of urine samples in these tests presents a remarkable advantage, as mentioned above. Notwithstanding these merits, the ELISA tests require a basic laboratory set-up owing to the need for initial incubation of the urine samples at 95–100°C for 30 min, high-speed centrifugation, trained manpower and a functioning ELISA reader.10 These limitations, inherent with the ELISA format, were overcome with the advent of the Determine TB-LAM Ag test (Alere, Waltham, Massachusetts, USA) in which unprocessed urine samples could be tested on commercialised test strips in a lateral flow immunochromatography format and results could be read after incubation for 25 min (figure 1). This test did not require trained laboratory personnel or refrigerated storage of test strips and hence could be easily adopted as a point-of-care test in remote settings. Moreover, the test strips provided sensitivity and specificity that were comparable with ELISA results and, hence, this technology represented a significant advancement in the use of LAM as a diagnostic antigen.10 35–38 The most recent test format using the LAM antigen was an immunoswab platform that used a bispecific monoclonal antibody (bsMAb) capable of simultaneously binding LAM and the enzyme, horseradish peroxidise.39 The use of bsMAb allowed the avoidance of enzyme addition and subsequent washing steps, observed in a typical ELISA format, and thus made the assay more reproducible and less time-consuming.40 The assay was initially tested in animal samples spiked with synthetic LAM and was found to have a limit of detection of 5.0 ng/mL (bovine urine), 0.5 ng/mL (rabbit serum) and 0.005 ng/mL (saline). With native LAM from M tuberculosis H37Rv, the limit of detection was subsequently found to be 0.5 ng/mL (rabbit serum). The assay was also evaluated with 21 stored clinical serum samples following a preprocessing step with urea at 60°C in which it showed 100% specificity and 64% sensitivity and the end point was visually readable within 2 h of sample collection.39 The assay, however, remains to be prospectively evaluated for diagnostic usefulness and field adaptability with larger numbers of clinical samples.
DIAGNOSTIC PERFORMANCE AND TEST CHARACTERISTICS OF LAM ASSAYS The diagnostic performance of the commercial LAM-based assays has been recently evaluated in several independent studies (table 1). In a recent meta-analysis on the use of urinary LAM assays, the sensitivity and specificity of these assays was shown to range from 13% to 93% and from 87% to 99%, respectively.41 Although their performance was similar in smearpositive ( pooled sensitivity 54% (95% CI 18% to 86%); pooled specificity 90% (95% CI 83% to 95%)) and smear-negative (pooled sensitivity 51% (95% CI 18% to 83%); pooled specificity 90% (95% CI 79% to 96%)) patients, HIV status was found to have a significant impact on the test outcome. Among studies on HIV-uninfected patients with TB, the pooled sensitivity of urinary LAM assays was 14% (95% CI 4% to 38%) and the pooled specificity was 97% (95% CI 86% to 100%). Although relatively higher in HIV-infected patients with TB, the pooled sensitivity was still suboptimal (47%; 95% CI 26% to 68%) and pooled specificity was similar to that in HIV-uninfected patients (96%; 95% CI 81% to 100%).8
Sarkar P, et al. Postgrad Med J 2014;90:155–163. doi:10.1136/postgradmedj-2013-132053
Downloaded from http://pmj.bmj.com/ on December 19, 2014 - Published by group.bmj.com
Review Figure 1 Determine TB-lipoarabinomannan (LAM) test strip.
Low sensitivity has been described as one of the major shortcomings of the currently available urinary LAM assays.37 42 Nature of capture antibodies is an important determinant of assay sensitivity, with polyclonal antibodies being capable of reacting with multiple antigenic epitopes compared with monoclonal antibodies that recognise a single epitope.43 The currently available commercial assays employ polyclonal antibodies, which may be responsible for increased batch-to-batch variation during the manufacturing process and thus account for the wide variation in assay sensitivity among the reported studies.44 Variations in urine concentration, HIV status of recruited patients, proportions of free versus immune-complexed antigen in circulation and in urine samples can also influence the sensitivity of the assay.42
predominantly in an immune-complexed form that is too large to be filtered through the normal glomerulus.47 As a result, the increased sensitivity of urinary LAM assays in HIV-TB co-infected patients has been hypothesised to reflect local renal involvement with TB42 or glomerular podocyte dysfunction resulting from HIV-associated nephropathy.35 48 The former possibility has been substantiated by studies showing the detection of mycobacteruria in urine Xpert MTB/RIF assay (Cepheid) in approximately half of urine LAM-positive patients and none of urine LAM-negative patients with HIV-TB co-infection.32 49 50 The latter hypothesis, based on the role of renal dysfunction, was proposed following the reported association of LAM antigenuria with the presence of proteinuria, as demonstrated through simple urinalysis stick tests51 and elevated urinary protein:creatinine ratios.32
Improved sensitivity in HIV-TB co-infection
Variable specificity
Unlike alternative diagnostics such as smear microscopy and chest radiography which lose sensitivity with worsening immunosuppression, the LAM assays deliver enhanced sensitivity in HIV-TB co-infected patients which increases further with increasing immunodeficiency.9 10 45 However, the underlying causes accounting for this outstanding feature remain to be adequately understood. It could be attributable to higher mycobacterial replication and consequently to higher levels of circulating LAM in HIV-infected patients, and particularly so in those with low CD4 counts.36 Lack of cavity formation in immunocompromised patients might also enable increased bacillary replication in tissues and thereby facilitate the diffusion of shed LAM into the circulation.41 Dissemination of M tuberculosis directly into the bloodstream, especially in patients with advanced immunosuppression, might also be responsible for systemic antigenaemia.37 46 With a molecular weight similar to myoglobin (17 kDa) which is freely filterable through the glomerular basement membrane, detection of LAM in urine might be a function of systemically circulating LAM exuded from actively replicating mycobacterial cells at the site of active disease. However, being a strong immunogen, LAM is likely to circulate
In contrast to the consistent observation regarding the increased sensitivity of urinary LAM assays in HIV-TB co-infected patients, the specificity of these assays has varied between studies and the reasons for this have remained largely unresolved.11 33 36 37 51–53 Misclassification bias and non-tuberculous mycobacteria have emerged as the two principal causes for the variable specificity. The misclassification bias could result from the inadequate sensitivity of the microbiological gold standard assay used in the study. For example, in the study reported by Mutetwa et al, use of solid rather than liquid culture medium for case detection is likely to have limited the diagnostic sensitivity and led to apparently false positive results in urinary LAM assays.52 Suboptimal sensitivity of the gold standard assays might also result from the collection of improper quality or inadequate quantity of sputum samples or from their over-decontamination. Weak cough strength and infection control concerns might also jeopardise proper sputum collection in very sick hospitalised patients and result in negative culture results.12 Reliance on sputum samples alone for gold standard diagnosis in HIV-TB co-infected patients might also lead to misclassification bias in those who do not have significant pulmonary involvement and thus yield negative
Suboptimal sensitivity
Sarkar P, et al. Postgrad Med J 2014;90:155–163. doi:10.1136/postgradmedj-2013-132053
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158
Table 1
Summary of published studies reporting performance characteristics of commercially available urinary LAM-based diagnostic tests Number of patients screened
Number of Microbiological TB cases reference method
Overall sensitivity Sensitivity in HIV+ve of LAM assay % % (95% CI) (95% CI)
334
132
LJ culture
80.3
81.2 (71 to 88) –
200
45
17.8 (8.5 to 32.6)
20 (1 to 70.1)
–
87.7 (81.3 to 92.3)
291
69
LJ and BACTEC 460 culture LJ and MGIT culture
50.7 (38.4 to 63.0) 62 (48 to 74)
–
87.8 (78.7 to 94)
235
58
MGIT culture
37.9 (22 to 46)
499
193
MGIT culture
59 (52 to 66)
Zimbabwe Ambulatory OPD attendees South Ambulatory OPD Africa attendees
397
195
LJ culture
44 (36 to 52)
37.9 (22 to 46) CD4>100: 13 (4 to 33) CD4 50–100: 41 (22 to 64) CD4200: 55 (41 to 69) CD4150–200: 14 (4 to 58) CD4150–200: 56 (30 to 80) CD450–100: 71 (51 to 87) CD4
Review
Application of lipoarabinomannan antigen in tuberculosis diagnostics: current evidence Pronoti Sarkar,1 Debasis Biswas,1,2 Girish Sindhwani,3 Jagdish Rawat,3 Aarti Kotwal,1 Barnali Kakati1 ▸ Additional material is published online only. To view please visit the journal online (http://dx.doi.org/10.1136/ postgradmedj-2013-132053). 1
Department of Microbiology, Himalayan Institute of Medical Sciences, HIHT University, Dehradun, Uttarakhand, India 2 Department of Microbiology, All India Institute of Medical Sciences Bhopal, Bhopal, India 3 Department of Pulmonary Medicine, Himalayan Institute of Medical Sciences, HIHT University, Dehradun, Uttarakhand, India Correspondence to Dr Debasis Biswas, Department of Microbiology, Himalayan Institute of Medical Sciences, HIHT University, Swami Ram Nagar, Jolly Grant, Dehradun, Uttarakhand 248140, India; [email protected] Received 16 April 2013 Revised 13 December 2013 Accepted 1 January 2014 Published Online First 15 January 2014
ABSTRACT Tests based on the detection of mycobacterial lipoarabinomannan (LAM) antigen in urine have emerged as potential point-of-care tests for tuberculosis (TB). We aimed to assimilate the current evidence regarding the diagnostic performance of LAM assays and to ascertain their clinical indication in settings with high and low prevalence of HIV-TB co-infection. Owing to suboptimal sensitivity, the urinary LAM assays are unsuitable as general screening tests for TB. However, unlike traditional diagnostic methods, they demonstrate improved sensitivity in HIV-TB co-infection which further increases with low CD4 counts. Accordingly, these assays are indicated as rule-in tests for TB in patients with advanced HIV-induced immunosuppression, and facilitate the early initiation of antituberculous treatment in them. They also offer incremental sensitivity and specificity when used as adjunct tests to smear microscopy and chest radiography in HIV-TB co-infection. They obviate the biohazards associated with sputum samples and provide an alternative diagnostic tool in sputum-scarce patients. Notwithstanding these advantages, the specificity of these assays is variable, which is mostly attributable to misclassification bias and cross-reactivity with nontuberculous mycobacteria or other commensal flora. Furthermore, the inability to detect low titres of antigen in HIV-uninfected patients makes these assays unsuitable for use in settings with a low HIV prevalence. Future research targeted towards inclusion of specific monoclonal antibodies and more sensitive immunoassay platforms might help to improve the diagnostic performance of these assays and extend their applicability to the general population of patients with TB.
INTRODUCTION
To cite: Sarkar P, Biswas D, Sindhwani G, et al. Postgrad Med J 2014;90:155–163.
Tuberculosis (TB) continues to remain a major cause of mortality and morbidity throughout the world, with the WHO estimating figures of 8.6 million incident cases and 1.3 million deaths occurring in the year 2012.1 The challenges of multidrug resistant and extensively drug resistant TB and the ongoing epidemic of HIV-TB co-infection have further complicated effective TB control. In line with the Millennium Development Goals, the WHO has developed a six-point ‘Stop TB Strategy’ to dramatically reduce the global burden of TB by 2015. Early case detection and providing standardised treatment under supervision constitute the first component of this strategic initiative.2 In spite of the importance of early diagnosis, none of the diagnostic modalities currently available offer a simple, affordable, rapid and reliable test that can be easily accessed by patients in low- and middle-income countries, which account for over
Sarkar P, et al. Postgrad Med J 2014;90:155–163. doi:10.1136/postgradmedj-2013-132053
90% of the global TB burden. Sputum-smear microscopy, though considered the cornerstone of the Directly Observed Treatment Short-Course (DOTS) strategy, fails to detect smear-negative cases of pulmonary TB,3 which may account for 24–61% of all pulmonary cases in HIV-infected individuals.4 Chest radiography has the limitations of poor specificity, infrastructure dependence and sub-optimal sensitivity in HIV-TB co-infection.5 6 Mycobacterial culture, which is regarded as the diagnostic gold standard, has a long turn-around time and demands elaborate infrastructure and trained manpower.7 Newer diagnostic tests based on nucleic acid amplification are too expensive and complex for routine use at peripheral locations in resource-constrained settings.8 In this context, the potential benefits of a reliable point-of-care test with adequate sensitivity in HIV-TB co-infected patients cannot be overemphasised, and antigen detection tests have been tested as potential solutions to address this diagnostic lacuna. Of the various antigens that have recently been tested for their diagnostic potential, the most promising results have been obtained with lipoarabinomannan (LAM) antigen, a major lipopolysaccharide component of the mycobacterial cell wall. The use of LAM as a diagnostic target has two very attractive advantages. First, unlike the alternative diagnostic modalities such as smear microscopy and chest radiography, LAM antigen detection tests deliver increased sensitivity in HIV-TB co-infected patients.9 10 Second, being detectable in urine, these tests obviate the potential biohazards associated with the handling of sputum samples and provide an alternative diagnostic tool in sputum-scarce patients.10 11 As a result of these inherent advantages, the LAM antigen has been extensively targeted for the development of diagnostic assays and has lately been incorporated into commercial ELISA and point-of-care lateral flow immunochromatography tests. The diagnostic performance of these assays has been evaluated in several studies based on both urinary and nonurinary samples, involving patients with both pulmonary and extrapulmonary TB and encompassing a diverse range of clinical settings ranging from tertiary referral hospitals to community clinics. In view of the increasing literature on the subject, we aimed to assimilate the current evidence regarding the diagnostic performance and test characteristics of these assays in both urinary and non-urinary samples and to ascertain their clinical indication in TB diagnostic practice. We also explored the potential utility of their usage, alongside the existing diagnostic tools for TB, in settings with a high and relatively low prevalence of HIV-TB co-infection. 155
Downloaded from http://pmj.bmj.com/ on December 19, 2014 - Published by group.bmj.com
Review BIOLOGY OF LAM ANTIGEN LAM, a 19 kDa glycolipid, is the most extensively studied immunomodulatory lipid in the mycobacterial cell wall. Being a heat-stable molecule, it does not degrade readily in clinical samples and, hence, is suitable as a target for diagnostic application in peripheral centres.12 However, its alkali-labile nature leads to the denaturation of antigenic epitopes on using sodium hydroxide for the pretreatment of sputum samples,13 and necessitates alternative decontamination protocols using N-acetyl L-cysteine-proteinase K or DTT in assays designed to detect LAM antigen in sputum.11 14 LAM has been classified into three structural types depending on the nature of the capping motif attached to it, with each structural type carrying a distinct functional overtone. While the pathogenic mycobacterial species such as Mycobacterium tuberculosis, M leprae, M avium and M kansasii have a mannose cap (ManLAM),15–17 the fast-growing non-pathogenic species M fortuitum and M smegmatis have an inositol phosphate cap (PILAM)18 and a third family devoid of both these types of caps (AraLAM) has recently been identified in M chelonae.19 These structural variations, schematically depicted in the online supplement, are associated with differential roles in evading or activating host immunity. While AraLAM, extracted from avirulent mycobacteria, elicits proinflammatory cytokine responses from macrophages,20 21 ManLAM is predominantly associated with downregulation of host immunity through a number of pathways including suppression of M tuberculosis-induced apoptosis,22 23 interleukin 12 secretion24 and phagolysosomal fusion25 26 in macrophages, maturation of dendritic cells27 and expansion of regulatory T cells.28 Unlike its structural analogues in avirulent mycobacteria, ManLAM thus contributes to the pathogenicity of virulent mycobacteria by undermining a protective host response and evading host immunity. Unlike other antigens in diagnostic use (eg, capsular polysaccharides of Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis and Cryptococcus neoformans which are composed of repeating carbohydrate motifs), the LAM antigen evokes a potent humoral immune response and is present in the systemic circulation in an immune-complexed form.29 Although studies on the pharmacokinetics, tissue distribution and mechanism of urinary excretion of LAM antigen have not been undertaken in humans, animal studies in mice intraperitoneally injected with a crude cell wall extract of M tuberculosis have shown the urinary excretion of LAM antigen within 17 h.30 However, in experiments designed to mimic the systemic circulation of LAM antigen in the immune-complexed state (by injecting purified LAM into mice pretreated with anti-LAM IgM antibody), tissue distribution of LAM antigen has been observed in the liver and localisation in the kidneys and excretion in urine has not been demonstrated.31 In view of these findings, the underlying basis of urinary detection of LAM antigen in patients with TB remains unclear. It is likely that, of the two forms of this antigen present in the systemic circulation of patients with TB (ie, free and immune-complexed), only the former is excreted in urinary samples.32
LAM ANTIGEN: TEST FORMATS IN DIAGNOSTIC APPLICATION Three types of assay formats have been tested in recent years for the detection of LAM antigen. A sandwich ELISA technique using urine samples has been employed in the commercially available Clearview TB ELISA test (Inverness Medical Innovations, USA) which now supersedes the pre-commercial 156
prototype (MTB LAM ELISA Test, Chemogen, Portland, Oregon, USA) first tested in Tanzania in 2005.33 Although the same polyclonal antibody has been used in the commercial and pre-commercial versions of the ELISA test, differences in the manufacturing technology and protocols might lead to differences in test performance between the two variants.34 Nevertheless, the use of urine samples in these tests presents a remarkable advantage, as mentioned above. Notwithstanding these merits, the ELISA tests require a basic laboratory set-up owing to the need for initial incubation of the urine samples at 95–100°C for 30 min, high-speed centrifugation, trained manpower and a functioning ELISA reader.10 These limitations, inherent with the ELISA format, were overcome with the advent of the Determine TB-LAM Ag test (Alere, Waltham, Massachusetts, USA) in which unprocessed urine samples could be tested on commercialised test strips in a lateral flow immunochromatography format and results could be read after incubation for 25 min (figure 1). This test did not require trained laboratory personnel or refrigerated storage of test strips and hence could be easily adopted as a point-of-care test in remote settings. Moreover, the test strips provided sensitivity and specificity that were comparable with ELISA results and, hence, this technology represented a significant advancement in the use of LAM as a diagnostic antigen.10 35–38 The most recent test format using the LAM antigen was an immunoswab platform that used a bispecific monoclonal antibody (bsMAb) capable of simultaneously binding LAM and the enzyme, horseradish peroxidise.39 The use of bsMAb allowed the avoidance of enzyme addition and subsequent washing steps, observed in a typical ELISA format, and thus made the assay more reproducible and less time-consuming.40 The assay was initially tested in animal samples spiked with synthetic LAM and was found to have a limit of detection of 5.0 ng/mL (bovine urine), 0.5 ng/mL (rabbit serum) and 0.005 ng/mL (saline). With native LAM from M tuberculosis H37Rv, the limit of detection was subsequently found to be 0.5 ng/mL (rabbit serum). The assay was also evaluated with 21 stored clinical serum samples following a preprocessing step with urea at 60°C in which it showed 100% specificity and 64% sensitivity and the end point was visually readable within 2 h of sample collection.39 The assay, however, remains to be prospectively evaluated for diagnostic usefulness and field adaptability with larger numbers of clinical samples.
DIAGNOSTIC PERFORMANCE AND TEST CHARACTERISTICS OF LAM ASSAYS The diagnostic performance of the commercial LAM-based assays has been recently evaluated in several independent studies (table 1). In a recent meta-analysis on the use of urinary LAM assays, the sensitivity and specificity of these assays was shown to range from 13% to 93% and from 87% to 99%, respectively.41 Although their performance was similar in smearpositive ( pooled sensitivity 54% (95% CI 18% to 86%); pooled specificity 90% (95% CI 83% to 95%)) and smear-negative (pooled sensitivity 51% (95% CI 18% to 83%); pooled specificity 90% (95% CI 79% to 96%)) patients, HIV status was found to have a significant impact on the test outcome. Among studies on HIV-uninfected patients with TB, the pooled sensitivity of urinary LAM assays was 14% (95% CI 4% to 38%) and the pooled specificity was 97% (95% CI 86% to 100%). Although relatively higher in HIV-infected patients with TB, the pooled sensitivity was still suboptimal (47%; 95% CI 26% to 68%) and pooled specificity was similar to that in HIV-uninfected patients (96%; 95% CI 81% to 100%).8
Sarkar P, et al. Postgrad Med J 2014;90:155–163. doi:10.1136/postgradmedj-2013-132053
Downloaded from http://pmj.bmj.com/ on December 19, 2014 - Published by group.bmj.com
Review Figure 1 Determine TB-lipoarabinomannan (LAM) test strip.
Low sensitivity has been described as one of the major shortcomings of the currently available urinary LAM assays.37 42 Nature of capture antibodies is an important determinant of assay sensitivity, with polyclonal antibodies being capable of reacting with multiple antigenic epitopes compared with monoclonal antibodies that recognise a single epitope.43 The currently available commercial assays employ polyclonal antibodies, which may be responsible for increased batch-to-batch variation during the manufacturing process and thus account for the wide variation in assay sensitivity among the reported studies.44 Variations in urine concentration, HIV status of recruited patients, proportions of free versus immune-complexed antigen in circulation and in urine samples can also influence the sensitivity of the assay.42
predominantly in an immune-complexed form that is too large to be filtered through the normal glomerulus.47 As a result, the increased sensitivity of urinary LAM assays in HIV-TB co-infected patients has been hypothesised to reflect local renal involvement with TB42 or glomerular podocyte dysfunction resulting from HIV-associated nephropathy.35 48 The former possibility has been substantiated by studies showing the detection of mycobacteruria in urine Xpert MTB/RIF assay (Cepheid) in approximately half of urine LAM-positive patients and none of urine LAM-negative patients with HIV-TB co-infection.32 49 50 The latter hypothesis, based on the role of renal dysfunction, was proposed following the reported association of LAM antigenuria with the presence of proteinuria, as demonstrated through simple urinalysis stick tests51 and elevated urinary protein:creatinine ratios.32
Improved sensitivity in HIV-TB co-infection
Variable specificity
Unlike alternative diagnostics such as smear microscopy and chest radiography which lose sensitivity with worsening immunosuppression, the LAM assays deliver enhanced sensitivity in HIV-TB co-infected patients which increases further with increasing immunodeficiency.9 10 45 However, the underlying causes accounting for this outstanding feature remain to be adequately understood. It could be attributable to higher mycobacterial replication and consequently to higher levels of circulating LAM in HIV-infected patients, and particularly so in those with low CD4 counts.36 Lack of cavity formation in immunocompromised patients might also enable increased bacillary replication in tissues and thereby facilitate the diffusion of shed LAM into the circulation.41 Dissemination of M tuberculosis directly into the bloodstream, especially in patients with advanced immunosuppression, might also be responsible for systemic antigenaemia.37 46 With a molecular weight similar to myoglobin (17 kDa) which is freely filterable through the glomerular basement membrane, detection of LAM in urine might be a function of systemically circulating LAM exuded from actively replicating mycobacterial cells at the site of active disease. However, being a strong immunogen, LAM is likely to circulate
In contrast to the consistent observation regarding the increased sensitivity of urinary LAM assays in HIV-TB co-infected patients, the specificity of these assays has varied between studies and the reasons for this have remained largely unresolved.11 33 36 37 51–53 Misclassification bias and non-tuberculous mycobacteria have emerged as the two principal causes for the variable specificity. The misclassification bias could result from the inadequate sensitivity of the microbiological gold standard assay used in the study. For example, in the study reported by Mutetwa et al, use of solid rather than liquid culture medium for case detection is likely to have limited the diagnostic sensitivity and led to apparently false positive results in urinary LAM assays.52 Suboptimal sensitivity of the gold standard assays might also result from the collection of improper quality or inadequate quantity of sputum samples or from their over-decontamination. Weak cough strength and infection control concerns might also jeopardise proper sputum collection in very sick hospitalised patients and result in negative culture results.12 Reliance on sputum samples alone for gold standard diagnosis in HIV-TB co-infected patients might also lead to misclassification bias in those who do not have significant pulmonary involvement and thus yield negative
Suboptimal sensitivity
Sarkar P, et al. Postgrad Med J 2014;90:155–163. doi:10.1136/postgradmedj-2013-132053
157
Review
158
Table 1
Summary of published studies reporting performance characteristics of commercially available urinary LAM-based diagnostic tests Number of patients screened
Number of Microbiological TB cases reference method
Overall sensitivity Sensitivity in HIV+ve of LAM assay % % (95% CI) (95% CI)
334
132
LJ culture
80.3
81.2 (71 to 88) –
200
45
17.8 (8.5 to 32.6)
20 (1 to 70.1)
–
87.7 (81.3 to 92.3)
291
69
LJ and BACTEC 460 culture LJ and MGIT culture
50.7 (38.4 to 63.0) 62 (48 to 74)
–
87.8 (78.7 to 94)
235
58
MGIT culture
37.9 (22 to 46)
499
193
MGIT culture
59 (52 to 66)
Zimbabwe Ambulatory OPD attendees South Ambulatory OPD Africa attendees
397
195
LJ culture
44 (36 to 52)
37.9 (22 to 46) CD4>100: 13 (4 to 33) CD4 50–100: 41 (22 to 64) CD4200: 55 (41 to 69) CD4150–200: 14 (4 to 58) CD4150–200: 56 (30 to 80) CD450–100: 71 (51 to 87) CD4
