Determine TB-LAM point-of-care tuberculosis assay predicts poor outcomes in outpatients during their first year of antiretroviral therapy in South Africa.

BACKGROUND: Determine TB-LAM is the first point-of-care test (POC) for HIV-associated tuberculosis (TB) and rapidly identifies TB in those at high-risk for short-term mortality. While the relationship between urine-LAM and mortality has been previously described, the outcomes of those undergoing urine-LAM testing have largely been assessed during short follow-up periods within diagnostic accuracy studies. We therefore sought to assess the relationship between baseline urine-LAM results and subsequent hospitalization and mortality under real-world conditions among outpatients in the first year of ART. METHODS: Consecutive, HIV-positive adults with a CD4 count < 100 cells/uL presenting for ART initiation were enrolled. TB diagnoses and outcomes (hospitalization, loss-to-follow and mortality) were recorded during the first year following enrolment. Baseline urine samples were retrospectively tested using the urine-LAM POC assay. Kaplan Meier survival curves were used to assess the cumulative probability of hospitalization or mortality in the first year of follow-up, according to urine-LAM status. Cox regression analyses were performed to determine independent predictors of hospitalization and mortality at three months and one year of follow-up. RESULTS: 468 patients with a median CD4 count of 59 cells/uL were enrolled. There were 140 patients (29.9%) with newly diagnosed TB in the first year of follow-up of which 79 (56.4%) were microbiologically-confirmed. A total of 18% (n = 84) required hospital admission and 12.2% (n = 57) died within a year of study entry. 38 out of 468 (8.1%) patients retrospectively tested urine-LAM positive - including 19.0% of those with microbiologically-proven TB diagnoses (n = 15/79) and 23.0% (n = 14/61) of those with clinical-only TB diagnoses; 9 of 38 (23.7%) of patients retrospectively testing LAM positive were never diagnosed with TB under routine program conditions. Among all patients (n = 468) in the first year of follow-up, a positive urine-LAM result was strongly associated with all-cause hospitalization and mortality with a corresponding adjusted hazard ratio (aHR) of 3.7 (95%CI, 1.9-7.1) and 2.6 (95%, 1.2-5.7), respectively. CONCLUSIONS: Systematic urine-LAM testing among ART-naïve HIV-positive outpatients with CD4 counts < 100 cells/uL detected TB cases that were missed under routine programme conditions and was highly predictive for subsequent hospitalization and mortality in the first year of ART.

Diagnostic accuracy, incremental yield and prognostic value of Determine TB-LAM for routine diagnostic testing for tuberculosis in HIV-infected patients requiring acute hospital admission in South Africa: a prospective cohort.

BACKGROUND: We previously reported that one-third of HIV-positive adults requiring medical admission to a South African district hospital had laboratory-confirmed tuberculosis (TB) and that almost two-thirds of cases could be rapidly diagnosed using Xpert MTB/RIF-testing of concentrated urine samples obtained on the first day of admission. Implementation of urine-based, routine, point-of-care TB screening is an attractive intervention that might be facilitated by use of a simple, low-cost diagnostic tool, such as the Determine TB-LAM lateral-flow rapid test for HIV-associated TB. METHODS: Sputum, urine and blood samples were systematically obtained from unselected HIV-positive adults within 24 hours of admission to a South African township hospital. Additional clinical samples were obtained during hospitalization as clinically indicated. TB was defined by the detection of Mycobacterium tuberculosis in any sample using Xpert MTB/RIF or liquid culture. The diagnostic yield, accuracy and prognostic value of urine-lipoarabinomannan (LAM) testing were determined, but urine-LAM results did not inform treatment decisions. RESULTS: Consecutive HIV-positive adult acute medical admissions not already receiving TB treatment (n = 427) were enrolled regardless of clinical presentation or symptoms. TB was diagnosed in 139 patients (TB prevalence 32.6%; median CD4 count 80 cells/µL). In the first 24 hours of admission, sputum (spot and/or induced) samples were obtained from 37.0% of patients and urine samples from 99.5% of patients (P < 0.001). The diagnostic yields from these specimens were 19.4% (n = 27/139) for sputum-microscopy, 26.6% (n = 37/139) for sputum-Xpert, 38.1% (n = 53/139) for urine-LAM and 52.5% (n = 73/139) for sputum-Xpert/urine-LAM combined (P < 0.01). Corresponding yields among patients with CD4 counts <100 cells/µL were 18.9%, 24.3%, 55.4% and 63.5%, respectively (P < 0.01). The diagnostic yield of urine-LAM was unrelated to respiratory symptoms, and LAM assay specificity (using a grade-2 cut-off) was 98.9% (274/277; 95% confidence interval [CI] 96.9-99.8). Among TB cases, positive urine-LAM status was strongly associated with mortality at 90 days (adjusted hazard ratio 4.20; 95% CI 1.50-11.75). CONCLUSIONS: Routine testing for TB in newly admitted HIV-positive adults using Determine TB-LAM to test urine provides major incremental diagnostic yield with very high specificity when used in combination with sputum testing and has important utility among those without respiratory TB symptoms and/or unable to produce sputum. The assay also rapidly identifies individuals with a poor prognosis.

Relationship Between Blood Concentrations of Hepcidin and Anemia Severity, Mycobacterial Burden, and Mortality Among Patients With HIV-Associated Tuberculosis.

BACKGROUND: Anemia is very common in patients with human immunodeficiency virus (HIV)-associated tuberculosis, and hepcidin may be key in mediating this. We explored the relationship between blood hepcidin concentrations and anemia severity, mycobacterial burden and mortality in patients with HIV-associated tuberculosis. METHODS: Consecutive unselected HIV-infected adults in South Africa were systematically investigated for tuberculosis. Three groups were studied: 116 hospitalized inpatients with HIV infection and tuberculosis (hereafter, "hospitalized patients"), 58 ambulatory outpatients with HIV infection and newly diagnosed tuberculosis (hereafter, "ambulatory patients with tuberculosis"), and 58 ambulatory outpatients with HIV infection and without tuberculosis (hereafter, "ambulatory patients without tuberculosis"). Blood hepcidin concentrations were determined for all patients. Vital status at 3 months was determined, and independent predictors of mortality were identified. RESULTS: Median hepcidin concentrations were 38.8 ng/mL among hospitalized patients, 19.1 ng/mL among ambulatory patients with tuberculosis, and 5.9 ng/mL among ambulatory patients without tuberculosis (P < .001). In both groups with HIV-associated tuberculosis, hepcidin concentrations were strongly associated with greater anemia severity. Additionally, strong, graded associations were observed between hepcidin and composite indices of mycobacterial burden and dissemination. Patients dying within 3 months had significantly higher hepcidin concentrations, which independently predicted mortality. CONCLUSIONS: High hepcidin concentrations were strongly associated with disseminated disease, anemia, and poor prognosis in patients with HIV-associated tuberculosis. Hepcidin may be a mechanistically important mediator underlying the high prevalence of severe anemia in these patients.

Rapid microbiological screening for tuberculosis in HIV-positive patients on the first day of acute hospital admission by systematic testing of urine samples using Xpert MTB/RIF: a prospective cohort in South Africa.

BACKGROUND: Autopsy studies of HIV/AIDS-related hospital deaths in sub-Saharan Africa reveal frequent failure of pre-mortem diagnosis of tuberculosis (TB), which is found in 34-64 % of adult cadavers. We determined the overall prevalence and predictors of TB among consecutive unselected HIV-positive adults requiring acute hospital admission and the comparative diagnostic yield obtained by screening urine and sputum samples obtained on day 1 of admission with Xpert MTB/RIF (Xpert). METHODS: To determine overall TB prevalence accurately, comprehensive clinical sampling (sputum, urine, blood plus other relevant samples) was done and TB was defined by detection of Mycobacterium tuberculosis in any sample using Xpert and/or mycobacterial liquid culture. To evaluate a rapid screening strategy, we compared the diagnostic yield of Xpert testing sputum samples and urine samples obtained with assistance from a respiratory study nurse in the first 24 h of admission. RESULTS: Unselected HIV-positive acute adult new medical admissions (n = 427) who were not receiving TB treatment were enrolled irrespective of clinical presentation or symptom profile. From 2,391 cultures and Xpert tests done (mean, 5.6 tests/patient) on 1,745 samples (mean, 4.1 samples/patient), TB was diagnosed in 139 patients (median CD4 cell count, 80 cells/µL). TB prevalence was very high (32.6 %; 95 % CI, 28.1-37.2 %; 139/427). However, patient symptoms and risk factors were poorly predictive for TB. Overall, ≥1 non-respiratory sample(s) tested positive in 115/139 (83 %) of all TB cases, including positive blood cultures in 41/139 (29.5 %) of TB cases. In the first 24 h of admission, sputum (spot and/or induced samples) and urine were obtainable from 37.0 % and 99.5 % of patients, respectively (P <0.001). From these, the proportions of total TB cases (n = 139) that were diagnosed by Xpert testing sputum, urine or both sputum and urine combined within the first 24 h were 39/139 (28.1 %), 89/139 (64.0 %) and 108/139 (77.7 %) cases, respectively (P <0.001). CONCLUSIONS: The very high prevalence of active TB and its non-specific presentation strongly suggest the need for routine microbiological screening for TB in all HIV-positive medical admissions in high-burden settings. The incremental diagnostic yield from Xpert testing urine was very high and this strategy might be used to rapidly screen new admissions, especially if sputum is difficult to obtain.

HIV-associated tuberculosis: relationship between disease severity and the sensitivity of new sputum-based and urine-based diagnostic assays.

BACKGROUND: Reducing mortality from HIV-associated tuberculosis (TB) requires diagnostic tools that are rapid and have high sensitivity among patients with poor prognosis. We determined the relationship between disease severity and the sensitivity of new sputum-based and urine-based diagnostic assays. METHODS: Consecutive ambulatory patients enrolling for antiretroviral treatment in South Africa were screened for TB regardless of symptoms using diagnostic assays prospectively applied to sputum (fluorescence smear microscopy, Xpert MTB/RIF and liquid culture (reference standard)) and retrospectively applied to stored urine samples (Determine TB-LAM and Xpert MTB/RIF). Assay sensitivities were calculated stratified according to pre-defined indices of disease severity: CD4 count, symptom intensity, serum C-reactive protein (CRP), hemoglobin concentration and vital status at 90 days. RESULTS: Sputum culture-positive TB was diagnosed in 15% (89/602) of patients screened and data from 86 patients were analyzed (median CD4 count, 131 cells/µL) including 6 (7%) who died. The sensitivity of sputum microscopy was 26.7% overall and varied relatively little with disease severity. In marked contrast, the sensitivities of urine-based and sputum-based diagnosis using Determine TB-LAM and Xpert MTB/RIF assays were substantially greater in sub-groups with poorer prognosis. Rapid diagnosis from sputum and/or urine samples was possible in >80% of patients in sub-groups with poor prognosis as defined by either CD4 counts <100 cells/µL, advanced symptoms, CRP concentrations >200 mg/L or hemoglobin <8.0 g/dl. Retrospective testing of urine samples with Determine TB-LAM correctly identified all those with TB who died. CONCLUSIONS: The sensitivities of Xpert MTB/RIF and Determine TB-LAM for HIV-associated TB were highest among HIV-infected patients with the most advanced disease and poorest prognostic characteristics. These data provide strong justification for large-scale intervention studies that assess the impact on survival of screening using these new sputum-based and urine-based diagnostic approaches.

Characteristics and early outcomes of patients with Xpert MTB/RIF-negative pulmonary tuberculosis diagnosed during screening before antiretroviral therapy.

BACKGROUND: A proportion of patients with tuberculosis diagnosed by sputum culture during screening before antiretroviral therapy (ART) have false-negative Xpert MTB/RIF assay results (Xpert-negative tuberculosis). We determined the characteristics and early ART program outcomes of such patients. METHODS: Adult patients who enrolled in a South African township ART service were systematically screened for pulmonary tuberculosis regardless of symptoms by testing paired sputum samples with Xpert MTB/RIF and liquid culture. The ART service provided follow-up for all patients, and early (90-day) programmatic outcomes were determined. RESULTS: Among 602 patients screened, 523 had ≥1 Xpert and culture result, yielding 89 culture-positive tuberculosis diagnoses. Of these, 37 (42%) of the patients with tuberculosis were Xpert-negative when a single sputum sample was tested, compared with 25 (28%) when 2 samples were tested. Compared with patients with Xpert-positive tuberculosis, those with Xpert-negative tuberculosis (using either definition) had substantially higher CD4 cell counts, lower plasma viral loads, higher hemoglobin concentrations, and higher body mass index. Their tuberculosis was also less advanced, with a lower frequency of prolonged cough (≥2 weeks), less extensive radiographic abnormalities, and a lower frequency of detectable lipoarabinomannan antigenuria and mycobacteriuria. Xpert-negative cases were all sputum smear negative with prolonged time to culture positivity (median, 21 days). Despite greater delays in starting tuberculosis treatment, Xpert-negative patients were less likely to die during follow-up. CONCLUSIONS: Compared to patients with Xpert-positive tuberculosis diagnosed during pre-ART screening, Xpert-negative cases had less advanced immunosuppression and less advanced tuberculosis and did not have adverse outcomes despite substantial delays in starting tuberculosis treatment.

Lipoarabinomannan in urine during tuberculosis treatment: association with host and pathogen factors and mycobacteriuria.

BACKGROUND: Detection of lipoarabinomannan (LAM), a Mycobacterium tuberculosis (Mtb) cell wall antigen, is a potentially attractive diagnostic. However, the LAM-ELISA assay has demonstrated variable sensitivity in diagnosing TB in diverse clinical populations. We therefore explored pathogen and host factors potentially impacting LAM detection. METHODS: LAM-ELISA assay testing, sputum smear and culture status, HIV status, CD4 cell count, proteinuria and TB outcomes were prospectively determined in adults diagnosed with TB and commencing TB treatment at a South African township TB clinic. Sputum TB isolates were characterised by IS61110-based restriction fragment length polymorphism (RFLP) and urines were tested for mycobacteriuria by Xpert® MTB/RIF assay. RESULTS: 32/199 (16.1%) of patients tested LAM-ELISA positive. Median optical density and proportion testing LAM positive remained unchanged during 2 weeks of treatment and then declined over 24 weeks. LAM was associated with positive sputum smear and culture status, HIV infection and low CD4 cell counts but not proteinuria, RFLP strain or TB treatment outcome. The sensitivity of LAM for TB in HIV-infected patients with CD4 counts of ≥ 200, 100-199, 50-99, and < 50 cells/µl, was 15.2%, 32%, 42.9%, and 69.2% respectively. Mycobacteriuria was found in 15/32 (46.9%) of LAM positive patients and in none of the LAM negative controls. CONCLUSIONS: Urinary LAM was related to host immune factors, was unrelated to Mtb strain and declined steadily after an initial 2 weeks of TB treatment. The strong association of urine LAM with mycobacteriuria is a new finding, indicating frequent TB involvement of the renal tract in advanced HIV infection.

Screening for HIV-associated tuberculosis and rifampicin resistance before antiretroviral therapy using the Xpert MTB/RIF assay: a prospective study.

BACKGROUND: The World Health Organization has endorsed the Xpert MTB/RIF assay for investigation of patients suspected of having tuberculosis (TB). However, its utility for routine TB screening and detection of rifampicin resistance among HIV-infected patients with advanced immunodeficiency enrolling in antiretroviral therapy (ART) services is unknown. METHODS AND FINDINGS: Consecutive adult HIV-infected patients with no current TB diagnosis enrolling in an ART clinic in a South African township were recruited regardless of symptoms. They were clinically characterised and invited to provide two sputum samples at a single visit. The accuracy of the Xpert MTB/RIF assay for diagnosing TB and drug resistance was assessed in comparison with other tests, including fluorescence smear microscopy and automated liquid culture (gold standard) and drug susceptibility testing. Of 515 patients enrolled, 468 patients (median CD4 cell count, 171 cells/µl; interquartile range, 102-236) produced at least one sputum sample, yielding complete sets of results from 839 samples. Mycobacterium tuberculosis was cultured from 81 patients (TB prevalence, 17.3%). The overall sensitivity of the Xpert MTB/RIF assay for culture-positive TB was 73.3% (specificity, 99.2%) compared to 28.0% (specificity, 100%) using smear microscopy. All smear-positive, culture-positive disease was detected by Xpert MTB/RIF from a single sample (sensitivity, 100%), whereas the sensitivity for smear-negative, culture-positive TB was 43.4% from one sputum sample and 62.3% from two samples. Xpert correctly identified rifampicin resistance in all four cases of multidrug-resistant TB but incorrectly identified resistance in three other patients whose disease was confirmed to be drug sensitive by gene sequencing (specificity, 94.1%; positive predictive value, 57%). CONCLUSIONS: In this population of individuals at high risk of TB, intensive screening using the Xpert MTB/RIF assay increased case detection by 45% compared with smear microscopy, strongly supporting replacement of microscopy for this indication. However, despite the ability of the assay to rapidly detect rifampicin-resistant disease, the specificity for drug-resistant TB was sub-optimal.

Screening for cryptococcal antigenemia in patients accessing an antiretroviral treatment program in South Africa.

BACKGROUND: Cryptococcal meningitis is a leading cause of death in patients with acquired immunodeficiency syndrome and contributes substantially to the high early mortality in antiretroviral treatment (ART) programs in low-resource settings. Screening for cryptococcal antigen in patients who enroll in ART programs may identify those at risk of cryptococcal meningitis and permit targeted use of preemptive therapy. METHODS: In this retrospective study, cryptococcal antigen was measured in stored plasma samples obtained from patients when they enrolled in a well-characterized ART cohort in South Africa. The predictive value of screening for cryptococcal antigen before initiation of ART for development of microbiologically confirmed cryptococcal meningitis or death during the first year of follow-up was determined. RESULTS: Of 707 participants with a baseline median CD4 cell count of 97 cells/microL (interquartile range, 46-157 cells/microL), 46 (7%) were positive for cryptococcal antigen. Antigenemia was 100% sensitive for predicting development of cryptococcal meningitis during the first year of ART, and in multivariate analysis, it was an independent predictor of mortality (adjusted hazard ratio, 3.2; 95% confidence interval, 1.5-6.6). Most cases (92%) of cryptococcal meningitis developed in patients with a CD4 cell count or 1:8 was 100% sensitive and 96% specific for predicting incident cryptococcal meningitis during the first year of ART in those with no history of the disease. CONCLUSIONS: Cryptococcal antigen screening before initiation of ART in patients with a CD4 cell count

Plasma levels of soluble urokinase-type plasminogen activator receptor (suPAR) and early mortality risk among patients enrolling for antiretroviral treatment in South Africa.

BACKGROUND: Serum concentrations of soluble urokinase-type plasminogen activator receptor (suPAR) have a strong independent association with HIV-1-related mortality. The practical utility of plasma suPAR in assessing short-term all-cause mortality risk was evaluated in patients with advanced immunodeficiency enrolling in an antiretroviral treatment (ART) programme in South Africa. METHODS: An enzyme-linked immunosorbent assay (ELISA) was used to measure plasma concentrations of suPAR in patients at the time of enrollment to the ART programme. The association between plasma suPAR concentrations, baseline patient characteristics and cohort outcomes after 4 months of ART were determined. RESULTS: Patients (n = 293, 70% female) had a median age of 33 years and were followed up for a median of 5 months from enrollment. The median CD4 cell count was 47 cells/mul (IQR = 22-72) and 38% of patients had WHO stage 4 disease. 218 (74%) patients remained alive after 4 months of ART; 39 (13%) died and 36 (12%) were lost to the programme for other reasons. Patients who died had significantly higher plasma suPAR concentrations compared to those who either survived (P < 0.01) or left the programme for other reasons (P < 0.043). In multivariate analysis, higher log10 suPAR concentrations were significantly associated with lower CD4 cell counts, WHO clinical stage 4 disease and male sex. In multivariate analysis to identify factors associated with death, log10 suPAR concentration was the most strongly associated variable (P < 0.001). However, examination of sensitivity and specificity characteristics using receiver operating characteristic (ROC) analysis revealed that results from this assay did not have a discriminatory cut-point to provide clinically useful information. CONCLUSION: Plasma suPAR concentration was the strongest independent predictor of short-term mortality risk among patients with advanced immunodeficiency enrolling in this ART programme. However, lack of a discriminatory threshold did not permit this marker to be used to triage patients according to short-term mortality risk.

Utility of interferon-gamma ELISPOT assay responses in highly tuberculosis-exposed patients with advanced HIV infection in South Africa.

BACKGROUND: Interferon-gamma (IFN-gamma) ELISPOT assays incorporating Mycobacterium tuberculosis-specific antigens are useful in the diagnosis of tuberculosis (TB) or latent infection. However, their utility in patients with advanced HIV is unknown. We studied determinants of ELISPOT responses among patients with advanced HIV infection (but without active TB) living in a South African community with very high TB notification rates. METHODS: IFN-gamma responses to ESAT-6 and CFP-10 in overnight ELISPOT assays and in 7-day whole blood assays (WBA) were compared in HIV-infected patients (HIV+, n = 40) and healthy HIV-negative controls (HIV-, n = 30) without active TB. Tuberculin skin tests (TSTs) were also done. RESULTS: ELISPOTs, WBAs and TSTs were each positive in >70% of HIV- controls, reflecting very high community exposure to M. tuberculosis. Among HIV+ patients, quantitative WBA responses and TSTs (but not the proportion of positive ELISPOT responses) were significantly impaired in those with CD4 cell counts <100 cells/mul compared to those with higher counts. In contrast, ELISPOT responses (but not WBA or TST) were strongly related to history of TB treatment; a much lower proportion of HIV+ patients who had recently completed treatment for TB (n = 19) had positive responses compared to those who had not been treated (11% versus 62%, respectively; P < 0.001). Multivariate analysis confirmed that ELISPOT responses had a strong inverse association with a history of recent TB treatment (adjusted OR = 0.06, 95%CI = 0.10-0.40, P < 0.01) and that they were independent of CD4 cell count and viral load. Among HIV+ individuals who had not received TB treatment both the magnitude and proportion of positive ELISPOT responses (but not TST or WBA) were similar to those of HIV-negative controls. CONCLUSION: The proportion of positive ELISPOT responses in patients with advanced HIV infection was independent of CD4 cell count but had a strong inverse association with history of TB treatment. This concurs with the previously documented low TB risk among patients in this cohort with a history of recent treatment for TB. These data suggest ELISPOT assays may be useful for patient assessment and as an immuno-epidemiological research tool among patients with advanced HIV and warrant larger scale prospective evaluation.

Prognostic value of a quantitative analysis of lipoarabinomannan in urine from patients with HIV-associated tuberculosis.

BACKGROUND: Detection of the mycobacterial cell wall antigen lipoarabinomannan (LAM) in urine can be used to diagnose HIV-associated tuberculosis (TB) using a qualitative (positive/negative) read-out. However, it is not known whether the quantity of LAM present in urine provides additional prognostic information. METHODS/FINDINGS: Consecutively recruited adult outpatients initiating antiretroviral therapy (ART) in South Africa were investigated for TB regardless of clinical symptoms using sputum smear microscopy and liquid culture (reference standard). Urine samples were tested using the Clearview TB-ELISA for LAM and the Xpert MTB/RIF assay. The ELISA optical densities (OD) were used as a quantitative assessment of urine LAM. Among 514 patients with complete sputum and urine LAM OD results, culture-confirmed TB was diagnosed in 84 patients. Twenty-three (27.3%) were LAM-positive with a median LAM OD of 0.68 (IQR 0.16-2.43; range, 0.10-3.29) and 61 (72.6%) were LAM negative (LAM OD <0.1 above background). Higher LAM ODs were associated with a range of prognostic indices, including lower CD4 cell counts, lower haemoglobin levels, higher blood neutrophil counts and higher mycobacterial load as assessed using both sputum and urine samples. The median LAM OD among patients who died was more than 6.8-fold higher than that of patients who remained alive at 3 months (P<0.001). The small number of deaths, however, precluded adequate assessment of mortality risk stratified according to urine LAM OD. CONCLUSIONS: In patients with HIV-associated TB, concentrations of LAM in urine were strongly associated with a range of poor prognostic characteristics known to be associated with mortality risk. Urine LAM assays with a semi-quantitative (negative vs. low-positive vs. high-positive) read-out may have improved clinical utility over assays with a simple binary result.

Predictive value of anemia for tuberculosis in HIV-infected patients in Sub-Saharan Africa: an indication for routine microbiological investigation using new rapid assays.

BACKGROUND: The relationship between anemia and undiagnosed tuberculosis (TB) in patients living with HIV in sub-Saharan Africa is incompletely defined. We assessed the prevalence of TB among those with HIV-related anemia and evaluated new means of rapid TB diagnosis. METHODS: Blood hemoglobin levels were measured in unselected antiretroviral treatment-naive patients in Cape Town, South Africa, and anemia was classified according to World Health Organization criteria. All patients were screened for TB by testing paired sputum samples using liquid culture (reference standard), fluorescence microscopy, and Xpert MTB/RIF. Urine samples were tested for lipoarabinomannan (LAM) using the Determine TB-LAM diagnostic assay. RESULTS: Of 602 adults screened, 485 had complete results. Normal hemoglobin levels were found in 44.5% (n = 216) of patients, and mild, moderate, or severe anemia were present in 24.9% (n = 121), 25.4% (n = 123) and 5.2% (n = 25) of patients, respectively. Culture-confirmed pulmonary TB was diagnosed in 8.8% (19/216) of those without anemia compared with 16.5% (20/121), 26.0% (32/123), and 40.0% (10/25) among those with mild, moderate, or severe anemia, respectively (P < 0.001). Anemia was a strong independent predictor of TB. The sensitivities of diagnostic assays were much higher among those with moderate/severe anemia compared with those with no/mild anemia using sputum microscopy (42.9% vs 15.4%), urine LAM (54.8% vs 0%), sputum microscopy plus urine LAM (71.4% vs 15.4%), and sputum Xpert (73.8% vs 41.0%) (P < 0.01 for all). CONCLUSIONS: A very high prevalence of undiagnosed TB was found in patients with moderate or severe anemia. Such patients should be prioritized for routine microbiological investigation using rapid diagnostic assays.

Blood neutrophil counts in HIV-infected patients with pulmonary tuberculosis: association with sputum mycobacterial load.

BACKGROUND: Increasing evidence suggests that neutrophils play a role in the host response to Mycobacterium tuberculosis. We determined whether neutrophil counts in peripheral blood are associated with tuberculosis (TB) and with mycobacterial load in sputum in HIV-infected patients. METHODOLOGY/PRINCIPAL FINDINGS: Adults enrolling in an antiretroviral treatment (ART) clinic in a Cape Town township were screened for TB regardless of symptoms. Paired sputum samples were examined using liquid culture, fluorescence microscopy, and the Xpert MTB/RIF assay. Absolute neutrophil counts (ANC) were measured in blood samples. Of 602 HIV-infected patients screened, 523 produced one or more sputum samples and had complete results available for analysis. Among these 523 patients, the median CD4 count was 169×10(9)/L (IQR, 96-232) and median ANC was 2.6×10(9)/L (IQR, 1.9-3.6). Culture-positive pulmonary tuberculosis was diagnosed in 89 patients. Patients with TB had a median ANC of 3.4×10(9)/L (IQR, 2.4-5.1) compared to 2.5×10(9)/L (IQR, 1.8-3.4) among those who were culture negative (p<0.0001). In multivariable analyses, having pulmonary TB was associated with an adjusted risk ratio (aRR) of 2.6 (95%CI, 1.5-4.5) for having an ANC level that exceeded the median value (ANC ≥2.6×10(9)/L; p = 0.0006) and an aRR of 6.8 (95%CI, 2.3-20.4) for having neutrophilia defined by a neutrophil count exceeding the upper limit of the normal range (ANC >7.5×10(9)/L; p = 0.0005). Patients were then classified into four mutually exclusive groups with increasing sputum mycobacterial load as defined by the results of culture, Xpert MTB/RIF and sputum smear microscopy. Multivariable analyses demonstrated that increasing sputum mycobacterial load was positively associated with blood ANC ≥2.6×10(9)/L and with neutrophilia. CONCLUSIONS/SIGNIFICANCE: Increased blood neutrophil counts were independently associated with pulmonary TB and sputum mycobacterial burden in this HIV-infected patient group. This observation supports the growing body of literature regarding the potential role for neutrophils in the host response to TB.

Diagnostic accuracy of a low-cost, urine antigen, point-of-care screening assay for HIV-associated pulmonary tuberculosis before antiretroviral therapy: a descriptive study.

BACKGROUND: The diagnostic accuracy of sputum smear microscopy and routine chest radiology for HIV-associated tuberculosis is poor, and culture-based diagnosis is slow, expensive, and is unavailable in most resource-limited settings. We assessed the diagnostic accuracy of a urine antigen test Determine TB-LAM Ag (Determine TB-LAM; Alere, Waltham, MA, USA) for screening for HIV-associated pulmonary tuberculosis before antiretroviral therapy (ART). METHODS: In this descriptive study, consecutive adults referred to a community-based ART clinic in Gugulethu township, South Africa, were all screened for tuberculosis by obtaining sputum samples for fluorescence microscopy, automated liquid culture (gold-standard test), and Xpert MTB/RIF assays (Cepheid, Sunnyvale, CA, USA) and urine samples for the Clearview TB-ELISA (TB-ELISA; Alere, Waltham, MA, USA) and Determine TB-LAM test. Patients with Mycobacterium tuberculosis cultured from one or more sputum samples were defined as cases of tuberculosis. The diagnostic accuracy of Determine TB-LAM used alone or combined with sputum smear microscopy was compared with that of sputum culture and the Xpert MTB/RIF assay for all patients and subgroups of patients stratified by CD4 cell count. FINDINGS: Patients were recruited between March 12, 2010, and April 20, 2011. Of 602 patients enrolled, 542 were able to provide one or more sputum samples, and 94 had culture-positive tuberculosis (prevalence 17·4%, 95% CI 14·2-20·8). Complete results from all tests were available for 516 patients (median CD4 count, 169·5 cells per µL; IQR 100-233), including 85 culture-positive tuberculosis, 24 of whom (28·2%, 95% CI 19·0-39·0) had sputum smear-positive disease. Determine TB-LAM test strips provided results within 30 min. Agreement was very high between two independent readers of the test strips (κ=0·97) and between the test strips and TB-ELISA (κ=0·84). Determine TB-LAM had highest sensitivity at low CD4 cell counts: 66·7% (95% CI 41·0-86·7) at <50 cells per µL, 51·7% (32·5-70·6) at <100 cells per µL, and 39·0% (26·5-52·6) at <200 cells per µL; specificity was greater than 98% for all strata. When combined with smear microscopy (either test positive), sensitivity was 72·2% (95% CI 46·5-90·3) at CD4 counts less than 50 cells per µL, 65·5% (45·7-82·1) at less than 100 cells per µL, and 52·5% (39·1-65·7) at less than 200 cells per µL, which did not differ statistically from the sensitivities obtained by testing a single sputum sample with the Xpert MTB/RIF assay. INTERPRETATION: Determine TB-LAM is a simple, low-cost, alternative to existing diagnostic assays for tuberculosis screening in HIV-infected patients with very low CD4 cell counts and provides important incremental yield when combined with sputum smear microscopy. FUNDING: Wellcome Trust.

Clinical significance of lipoarabinomannan detection in urine using a low-cost point-of-care diagnostic assay for HIV-associated tuberculosis.

OBJECTIVE: A low-cost point-of-care urine assay for lipoarabinomannan (LAM) used for screening patients prior to antiretroviral therapy (ART) rapidly diagnoses a proportion of tuberculosis (TB) cases. We determined the characteristics and outcomes of such patients. METHODS: Adults enrolling in a South African township ART clinic were systematically screened for pulmonary TB by testing paired sputum samples using microscopy, liquid culture and Xpert MTB/RIF in a centralized laboratory. Stored urine samples were retrospectively tested for LAM using the Determine TB-LAM assay, but results did not inform treatment. Patients were followed up in the routine ART service and early (90-day) programmatic outcomes were determined. Analysis was restricted to those with CD4 cell counts below 200 cells/µl. RESULTS: Of patients with CD4 cell counts below 200 cells/µl and complete results (n=325), 59 (18.2%) had culture-positive TB. Of these, 23 (39%) patients tested urine LAM-positive and 36 (61%) urine LAM-negative. Patients with LAM-positive TB had much lower CD4 cell counts, higher plasma viral loads, lower haemoglobin concentrations and lower BMIs compared to those with LAM-negative TB. They also had evidence of higher mycobacterial load, more frequently testing sputum smear-positive, Xpert-positive (sputum and urine) and having a shorter time to sputum culture positivity. Of five (8.5%) patients who died, four did so before TB treatment was started. All five retrospectively tested LAM-positive. CONCLUSIONS: A low-cost point-of-care urine test for LAM rapidly diagnoses a sub-group of cases with advanced HIV-associated TB and poor prognosis. If used in combination with laboratory-based diagnostics, treatment delays would decrease and survival might be improved.

High diagnostic yield of tuberculosis from screening urine samples from HIV-infected patients with advanced immunodeficiency using the Xpert MTB/RIF assay.

We determined the diagnostic yield of the Xpert MTB/RIF assay for tuberculosis (TB) when testing small volumes of urine from ambulatory HIV-infected patients before starting antiretroviral therapy in South Africa. Compared with a gold standard of sputum culture, the sensitivity of urine Xpert among those with CD4 cell counts of <50, 50-100, and >100 cells per microliter were 44.4%, 25.0%, and 2.7% (P = 0.001), respectively. Urine Xpert testing provides a means of rapid TB diagnosis in patients with advanced immunodeficiency and poor prognosis. These data are indicative of high rates of TB dissemination and renal involvement in this clinical population.

Urine lipoarabinomannan assay for tuberculosis screening before antiretroviral therapy diagnostic yield and association with immune reconstitution disease.

OBJECTIVE: To assess the utility of urine lipoarabinomannan (LAM) detection as a diagnostic screening test for tuberculosis (TB) in HIV-infected patients with advanced immuno deficiency and high prevalence of sputum smear-negative pulmonary disease. DESIGN: Cross-sectional survey. METHODS: Unselected adults enrolling for antiretroviral therapy (ART) in a South African clinic were screened for TB with two sputum samples for fluorescence microscopy and mycobacterial liquid culture. LAM was measured in urine samples using a commercially available enzyme-linked immunosorbent assay. RESULTS: Sputum culture-positive TB was diagnosed in 58 patients (median CD4 cell count=78 cells/microliter) out of 235 patients screened (TB prevalence=0.25). Cultures were identified as positive after a mean of 24 days (SD=9 days). The sensitivity of sputum microscopy was just 0.14 (specificity=1.00), whereas that of LAM in concentrated urine was 0.38 (P<0.01; specificity=1.00). In those with CD4 cell counts of less than 50, 50-100 and more than 150 cells/microliter, the LAM assay sensitivities were 0.67, 0.41 and 0.13, respectively. Corresponding values for the combined use of the LAM assay and microscopy were 0.67, 0.53 and 0.21, respectively. Among TB patients, detectable LAM was very strongly associated with low CD4 cell counts and advanced clinical stage. All patients who developed TB immune reconstitution disease (n=5) had detectable urinary LAM at baseline. CONCLUSION: The LAM assay has substantially superior sensitivity to sputum microscopy as a routine diagnostic TB screening test among patients with CD4 cell counts less than 100 cells/microliter. In one half of such patients, this assay could reduce the mean time to diagnosis by approximately 3 weeks. Furthermore, detectable urinary LAM may predict the development of TB immune reconstitution disease.

Quantitative and functional differences between peripheral blood myeloid dendritic cells from patients with pleural and parenchymal lung tuberculosis.

Dendritic cells (DCs) play a pivotal role in generating protective host immunity to Mycobacterium tuberculosis. Few studies have addressed DC function in the context of active tuberculosis (TB), largely due to technical constraints in obtaining adequate numbers of DC from sick patients. We quantitated peripheral blood myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) in the whole blood of patients with active TB and show that blood from patients with pleural TB was characterized by high circulating levels of mDCs. We also developed and optimized a novel whole-blood assay to study mDC production of the Th1-promoting cytokine interleukin 12 (IL-12) and upregulation of the maturation marker CCR7 after incubation with mycobacteria. We found that pleural TB was associated with increased IL-12 production and CCR7 expression compared to lung parenchymal disease. Our findings suggest important differences in innate immunity between patients with different forms of active TB, and this may contribute to the differences in natural history observed between the two groups.