Tuberculosis (TB) is a leading cause of morbidity and mortality in antiretroviral therapy (ART) services in sub-Saharan Africa [1–3]. These congregate clinical settings are also associated with a substantial risk of nosocomial TB transmission [4,5]. As a result, there is increasing recognition of the importance of intensified TB case finding in this patient group [4,6,7]. However, clinical screening algorithms have limited sensitivity and specificity  and this is further compounded by the progressively lower sensitivity of sputum microscopy with advancing HIV [8,9]. In addition, culture is slow and the practical challenges to the widespread expansion of sputum culture facilities in Africa are daunting. New, simple diagnostic screening tests are urgently needed.
Lipoarabinomannan (LAM) is a major lipopolysaccharide constituent of the cell wall of Mycobacterium tuberculosis  and a number of studies have explored the detection of this antigen within blood, sputum, pleural fluid and urine as a means for TB diagnosis [11–17]. Assays used in earlier studies required extensive sample preparation, limiting the potential for use in the field [11–15]. However, a more recently developed, commercially available enzyme-linked immunosorbent assay (ELISA) detects LAM in urine with little or no sample processing and showed promising performance characteristics in a field evaluation in Tanzania .
We hypothesized that this urine LAM ELISA might be particularly useful among HIV-infected patients with advanced immunodeficiency in whom the use of sputum microscopy and culture has substantial limitations. Since such patients are more likely to have mycobacterial dissemination, we hypothesized that renal clearance of antigen from blood would frequently result in detectable antigenuria. In the present study, we have evaluated the utility of this assay as a routine diagnostic screening test among HIV-infected patients enrolling for ART in a community-based service in a poor community in South Africa.
Antiretroviral therapy service
We have previously described in detail the ART service in Gugulethu township in Cape Town where both HIV prevalence and the TB notification rate are high [1,18,19]. The national ART programme provided treatment for those with World Health Organization (WHO) stage 4 disease or a blood CD4 cell count more than 200 cells/μl. The extraordinarily high burden of TB diagnosed during routine clinical practice in this service has been previously reported [1,20].
Patients, laboratory investigations and follow-up
Eligible patients were ART-naïve adults (≥18 years of age) referred to start ART and without a current diagnosis of TB. Unselected eligible patients completed a symptom screen and had chest radiographs that underwent unblinded reporting by a specialist. All patients provided two sputum samples, with one or both being induced using nebulised hypertonic (3%) saline. Urine samples were also obtained and stored at −20°C for later analysis for LAM. All patients provided their written informed consent and the study was approved by the Research Ethics Committee of the Faculty of Health Sciences of the University of Cape Town.
Sputum samples were analysed within accredited laboratories using standardized protocols and quality control procedures. Following decontamination with N-acetyl-L-cysteine sodium hydroxide, centrifuged sputum deposits were examined for acid-fast bacilli (AFB) using auramine O fluorescent stain and cultured using mycobacterial growth indicator tubes (MGIT, Becton-Dickinson, Sparks, Maryland, USA). Cultures positive for AFB were identified as M. tuberculosis complex by inhibition of growth by p-nitrobenzoic acid or by PCR. Routine local laboratory procedures are described elsewhere . Stored isolates underwent spoligotyping to assess possible cross-contamination. Deaths and development of TB immune reconstitution disease were ascertained up to 16 weeks of ART.
Cases of TB were defined by one or more positive cultures of M. tuberculosis. Patients defined as being at risk of paradoxical TB immune reconstitution disease were those who initiated ART during a successful response to antituberculosis therapy. Such events were defined according to consensus case definitions .
Lipoarabinomannan enzyme-linked immunosorbent assay
Stored urine samples were analysed for the presence of LAM using a commercially available polyclonal antibody LAM ELISA in a 96-well plate format (Chemogen Inc., South Portland, Maine, USA), with strict adherence to the manufacturer's instructions. Urine samples were prepared by heating to 95–100°C for 30 min and, after cooling, were centrifuged at 10 000 revolutions per minute for 15 min. Supernatants were analysed in duplicate together with positive and negative controls, generating results within approximately 2 h. An optical density of at least 0.1 above the negative control value was scored as positive in accordance with the manufacturer's instructions.
In a further evaluation, samples were also concentrated by centrifugation through a 10 K Nanosep molecular filter (Merck, Whitehouse Station, New Jersey, USA) and reanalysed in the LAM ELISA. This concentration procedure is simple and takes only a few minutes. In further experiments, LAM antigen supplied by the ELISA manufacturer was used to form standard curve, permitting quantification of LAM within positive samples.
The sensitivity, specificity and predictive values of different assays were calculated together with 95% confidence intervals (CI). Proportions were compared using chi-squared or Fisher's exact tests as appropriate. The associations between LAM and age, sex, CD4 cell count, WHO stage and outcomes were analysed using contingency tables and logistic regression. All statistical tests are two-sided at alpha = 0.05.
Patients and culture-positive tuberculosis diagnoses
Of 235 eligible patients who were screened for TB, the median age was 33 years [interquartile range (IQR) = 29–39 years], 73% were female and 10% were pregnant. The median CD4 cell count was 125 cells/μl (IQR = 67–185; range 1–402) and 53% had WHO stage 3 or 4 disease.
Sputum culture-positive TB was diagnosed in 58 patients, giving a disease prevalence of 0.25 (95% CI = 0.20–0.31). Spoligotyping of paired isolates from TB cases were identical whereas those from consecutively recruited patients were different, providing no evidence of cross-contamination. All TB cases were referred to a TB treatment service and, following re-evaluation, all were commenced on TB treatment according to national guidelines.
TB cases and patients who were TB-free did not differ with regard to median age (32 versus 34 years) or the proportion who were female (79% versus 71%). However, TB cases had a lower median CD4 cell count (78 versus 144 cells/μl, P < 0.001) and a greater proportion of them had pre-existing WHO stage 3 or 4 disease (71% versus 48%; P < 0.01).
One or more symptoms of cough for at least 2 weeks, night sweats, fever, or significant recent weight loss were reported by 46 (79%) of TB patients and 109 (62%) of patients who were TB-free. Reporting of chest radiographs for any abnormality consistent with TB had a sensitivity of 71% and a specificity of 48%. A combination of a positive symptom screen and abnormal chest radiograph had a sensitivity of 64% and specificity of just 39%.
Sputum microscopy versus lipoarabinomannan enzyme-linked immunosorbent assay
Smear microscopy for AFB was positive in eight cases, with a sensitivity of just 0.14 (95% CI = 0.07–0.25) and a specificity of 1.00 (0.98–1.00). The sensitivity was similar across CD4 cell count strata less than 50, 50–100 and more than 100 cells/μl (Table 1). In contrast, the sensitivity of the LAM ELISA was substantially greater using either unconcentrated urine (0.33, 95% CI = 0.22–0.46) or using concentrated urine (0.38, 95% CI = 0.27–0.51). The positive predictive value of the LAM assay was 1.00 (0.82–1.00) and the negative predictive value was 0.83 (0.77–0.88). All internal quality control criteria for each of the ELISA runs were fully satisfied. Among those with detectable LAM, the median absolute urine antigen concentration was 1.82 ng/ml (IQR = 0.67–4.70).
Among TB cases, an increasing likelihood of a positive LAM assay was strongly associated with decreasing CD4 cell counts (Table 1). The difference in the sensitivity between the LAM assay and microscopy was evident below a CD4 cell count threshold of 100 cells/μl. At CD4 cell counts below this level, the sensitivity of smear microscopy (0.14, 95% CI = 0.06–0.30) was significantly lower than that obtained using the LAM ELISA analysing either unconcentrated urine (0.51, 95% CI = 0.36–0.67; P = 0.002) or concentrated urine (0.54, 95% CI = 0.38–0.70; P < 0.001). A small increase in overall sensitivity was observed when the LAM assay and sputum microscopy results were combined (Table 1).
We next examined the factors in TB patients that were associated with detectable urine LAM (Table 2). In multivariate analyses, a positive result was not associated with age or gender but was very strongly associated with lower CD4 cell counts and advanced clinical disease (WHO stage 3 or 4) prior to TB diagnosis.
Time to laboratory diagnosis
The time to culture of M. tuberculosis was prolonged (mean, 24 days; SD, 9.2 days). Since smear microscopy was so insensitive, the use of this test did not significantly reduce the overall mean time to diagnosis. The mean time to a positive smear or a positive mycobacterial culture (time to first positive result) was 22 days (SD 12 days).
Outcomes and tuberculosis immune reconstitution disease
We ascertained deaths and TB immune reconstitution disease events from enrolment up to 16 weeks of ART. Although there was a trend towards a higher mortality in cases with detectable LAM, this was not statistically significant (Table 2). However, of 22 patients at risk of paradoxical TB immune reconstitution disease, five cases were diagnosed and all five had detectable LAM in their urine (Table 2). These patients had a median CD4 cell count of 28 cells/μl (IQR = 21–33) and three of them had WHO stage 4 disease.
In the present study, we evaluated a commercially available LAM ELISA as a diagnostic screening assay for TB among HIV-infected patients with very advanced immunodeficiency just prior to initiating ART. The assay was found to be highly specific and to have a sensitivity that, although modest, was substantially superior to sputum smear microscopy in those patients with CD4 cell counts less than 100 cells/μl. In such patients, rapid detection of LAM in urine may reduce the time to diagnosis by a mean of approximately 3 weeks compared with culture.
TB is a leading cause of high early mortality in ART programmes in sub-Saharan Africa and an important cause of nosocomial disease transmission. Rapid means of diagnosis are urgently needed. Routine microbiological screening on unselected patients revealed a very high prevalence of culture-positive disease, as found elsewhere in South Africa . Spoligotyping of isolates provided no evidence of sample cross-contamination. Such a high prevalence supports the need for routine active screening for TB. However, symptom screening and chest radiology performed poorly and the sensitivity of sputum fluorescence microscopy was extremely low (<0.20) as was found in a similar study elsewhere in South Africa . Very low bacillary burden in sputum samples would also explain the prolonged time to culture positivity (mean, 24 days).
Although the sensitivity of the LAM ELISA was limited, it far exceeded that of sputum microscopy, reaching more than 0.50 in those with CD4 cell counts less than 100 cells/μl. Moreover, the specificity and positive predictive value were 1.00, increasing the potential utility of the test. A simple urine concentration procedure was associated with a modest increase in sensitivity without compromising specificity. The negative predictive value of the assay (0.83) was limited in this cohort, but will vary according to disease prevalence. Thus, the LAM assay has potential as a rapid diagnostic but could not be used to exclude a diagnosis of TB in this clinical setting.
Patients with TB diagnosed by active screening typically have substantially less advanced disease compared with those diagnosed during passive case funding [24,25]. This may explain why the LAM assay sensitivity was lower in the present study compared with that observed in a study of TB patients in Tanzania who typically had advanced symptomatic disease . The sensitivity in patients with smear-negative culture positive disease we found is similar to that in a study in Zimbabwe . However, the assay specificity was substantially greater in our study, possibly indicating the reliability of our culture-based gold standard. In a further study from Cape Town, this assay was not found to be useful in the diagnosis of pleural TB .
In marked contrast to existing TB diagnostics in which test performance declines with advancing immunodeficiency , the sensitivity of the LAM assay was greatest among those with the lowest CD4 cell counts and those with pre-existing advanced WHO stage of disease. We suspect that this reflects a greater propensity for such patients to have high mycobacterial burden and systemic dissemination, with greater LAM antigenaemia resulting in detectable LAM concentrations in urine.
We have previously characterized TB immune reconstitution disease and high early mortality as key outcomes in this ART cohort [19,20]. We hypothesized that, if urine LAM reflects systemic mycobacterial antigen load, it may also be of prognostic value with regard to these complications. Although there was no significant association with mortality, all five patients who developed TB immune reconstitution disease had detectable LAM in the urine. This is consistent with the assertion that high systemic antigen load predisposes to this complication . In addition, LAM was also a marker of advanced immunodeficiency, which in turn is also a risk factor for TB immune reconstitution disease. Whether by direct or indirect association, LAM antigenuria may potentially serve as a useful prognostic marker for development of TB immune reconstitution disease and these initial findings warrant further evaluation.
In summary, this assay shows promise as a rapid TB diagnostic assay among patients with advanced HIV, such as those entering ART services or hospital in-patients. The assay may greatly shorten the time to TB diagnosis in this group of patients in whom diagnosis is most challenging. In addition, this assay should be further evaluated as a prognostic marker for TB immune reconstitution disease. Should a point of care urine dip-stick format of this assay have comparable sensitivity and specificity, this potentially represents a valuable diagnostic tool in this clinical setting.
S.D.L. and K.K. are funded by the Wellcome Trust, London, UK. RW and LGB are funded in part by the National Institutes of Health (NIH) through a CIPRA grant 1U19AI53217-01 and RO1 grant (A1058736-01A1). D.J.E. is funded by the NIH through the International Clinical Research Fellows Program at Vanderbilt (R24 TW007988). We are grateful to Chemogen Inc. who kindly donated the LAM ELISA kits and LAM antigen. The funding sources played no role in the decision to publish these data. The authors gratefully acknowledge the dedicated staff of the Hannan Crusaid ART clinic and the Desmond Tutu HIV Centre.
S.D.L. designed the study, analysed the data and wrote the paper with input from R.W. L.G.B. and R.W. were responsible for the field site. D.J.E. was responsible for data collection and data management. K.K. assisted with the statistical analysis. M.V. did the laboratory assays. All authors approved the final version of the paper.
There are no conflicts of interests.
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