Gounder, Celine R MD, ScM*; Kufa, Tendesayi MBChB, MPH†; Wada, Nikolas I MHS‡; Mngomezulu, Victor MBBCh, FCRadDiag, MBA§; Charalambous, Salome MBBCh, MSc†; Hanifa, Yasmeen MRCGP, MSc‖; Fielding, Katherine PhD¶; Grant, Alison MBBS, PhD#; Dorman, Susan MD*; Chaisson, Richard E MD*; Churchyard, Gavin J MBBCh, PhD†‖
From the *Center for TB Research, Division of Infectious Diseases, Department of Medicine, School of Medicine, Johns Hopkins University, Baltimore, MD; †Aurum Institute for Health Research, Johannesburg, South Africa; ‡Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD; §Division of Diagnostic Radiology, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa; ‖Queen Mary, University of London, London, United Kingdom; ¶Department of Infectious Disease Epidemiology, Faculty of Epidemiology and Population Health; and #Department of Clinical Research, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom.
Received for publication April 28, 2011; accepted June 28, 2011.
Dr Celine Gounder was funded by National Institues of Health grant T32AI007291. This study was funded in part by a grant from the United States Agency for International Development (USAID).
All authors contributed to the conception and design of the study, interpretation of the data, and reviewing drafts of the article. T.K. was responsible for managing the field site. C.R.G. and N.I.W. analyzed the data. C.R.G. drafted and revised the article.
The authors have no conflict of interests to disclose.
The contents of this report are the sole responsibility of the authors and do not necessarily reflect the views of United States Agency for International Development or the United States Government.
Correspondence to: Dr Celine R. Gounder, MD, ScM, Center for Tuberculosis Research, Johns Hopkins University, CRB-2, Room M1.06, 1550 Orleans Street, Baltimore, MD 21287 (e-mail: firstname.lastname@example.org).
Tuberculosis (TB) in HIV-infected persons is predominantly smear negative, necessitating a multistep diagnostic algorithm to confirm or exclude TB in smear-negative TB suspects. Completing this algorithm is onerous and costly for patients and providers alike.1 Moreover, considerable uncertainty remains even when the full diagnostic algorithm is followed.2 A rapid, sensitive, point-of-care test for TB, allowing TB disease status to be accurately assigned on the day of initial presentation could transform TB diagnosis and screening, allowing for prompt initiation of TB treatment and for intensified case finding, isoniazid preventive therapy, and antiretroviral therapy (ART) to be readily integrated into the first days of HIV care.
The ideal TB diagnostic test would be accurate, rapid, point-of-care, safe, robust, widely applicable to different populations and forms of TB; affordable and acceptable to clinicians, laboratory technicians, and patients; and should either replace sputum smear microscopy or increase microbiologic confirmation of TB among sputum smear-negative cases.3
Lipoarabinomannan (LAM) is a cell wall lipopolysaccharide only found in mycobacteria4 and can be detected in urine of patients with mycobacterial infections.5 Enzyme-linked immunosorbent assays (ELISAs) have been developed to detect LAM in urine.6
The aims of this study were to assess the sensitivity and positive predictive value of urine LAM to screen HIV-infected persons for TB and the specificity and negative predictive value of the urine LAM to exclude TB among HIV-infected persons before initiation of ART and/or isoniazid preventive therapy.
Study Site and Population
This study was conducted at the Tembisa Main Clinic in Ekurhuleni, South Africa. Consecutive patients presenting to the clinic between October 2009 and May 2010 for HIV-related care were asked to participate in the study. Inclusion criteria were age ≥18 years, HIV infection (testing was provided by routine clinical services, not the study), ability to communicate in English, Zulu, or Sepedi, and written informed consent. Patients who were already on treament for TB or who had discontinued TB treatment in the past 3 months, patients on dialysis, and prisoners were excluded.
Demographic information and clinical history were obtained through interview and medical record review. All study participants provided 2 sputum, 1 blood, and 1 urine specimen regardless of symptoms. Participants unable to expectorate spontaneously underwent percussion, and if necessary, sputum induction with 3% hypertonic saline. All participants were examined for lymphadenopathy and a fine needle aspiration was performed on all superficial easily accessible lymph nodes ≥1 cm in size.
Additional diagnostic work up and all treatment was provided at the discretion of the treating clinicians who were given the results of all study-related diagnostic testing except the urine LAM test.
Urine specimens were refrigerated after collection, transported to the laboratory in cooler boxes at 2-8°C on the day of collection, heat inactivated, spun down, and stored at −20°C until testing was performed. All other specimens were transported to the laboratory at ambient temperature and processed on the day of collection.
Sputum smears were decontaminated with N-acetyl-L-cysteine-sodium hydroxide. A Ziehl-Neelsen-stained smear was prepared, examined under 100× magnification using a light microscope and graded according to the World Health Organization grading system. Processed sputum sediment from both specimens was cultured using the BACTEC mycobacteria growth indicator tube 960 system (BD Diagnostics, Sparks, MD).
Blood was sent for complete blood count and CD4+ T-cell counts and inoculated directly into BACTEC Myco-F/Lytic bottles at the time of collection. Smears of lymph node aspirates were prepared at the time of collection and sent for histopathology.
Mycobacterial species identification was performed using the Capilia MPB64 monoclonal antibody test (TAUNS, Numazu, Japan).
Urine was tested for LAM using the Clearview TB ELISA (Inverness Medical Innovations, Waltham, MA) according to the manufacturer's instructions in batches.6 The laboratory technicians reading the urine LAM ELISA optical densities were blinded to the participants' histories and other diagnostic test results.
A rapid urine pregnancy test was also performed on all female participants not already known to be pregnant. All participants who were not pregnant underwent a chest x-ray. The chest x-ray was read by a single reader (V.M.) for active TB.
Participants were classified as having pulmonary TB (PTB) if they had at least 1 sputum culture that was positive for Mycobacterium tuberculosis, or at least 1 sputum smear that was acid-fast bacilli (AFB) positive, but with culture not performed or contaminated. Participants were classified as having extrapulmonary TB (ETB) if they had at least 1 blood culture that was positive for M. tuberculosis, or at least 1 fine needle aspiration from an enlarged lymph node that was AFB positive and/or histologically consistent with TB.
Data Management and Statistical Analyses
We assumed that the urine LAM test would have a sensitivity of 50%-80%,7,8 and thus that we would be able to estimate the sensitivity of the test with 95% confidence intervals (CIs) of ±15% if we enrolled at least 400 participants with a TB prevalence of 10%-15%. We assumed that the urine LAM test would have a specificity of 85%-100%, and thus that we would be able to estimate the specificity of the test with 95% CIs of ±5% if we enrolled at least 400 participants with a TB prevalence of 10%-15%.
The statistical software package SAS 9.2 (SAS Institute Inc, Cary, NC) was used for data analysis. The performance characteristics of the urine LAM as compared with the gold-standard case definitions, and the prevalence of TB was calculated along with 95% CIs using the exact binomial method.
The study was approved by the Gauteng Province Department of Health, the Ekhuruleni Metropolitan Municipality Executive Director for Health, the Ekhuruleni Metropolitan Municipality Ethics Committee, the University of the Witwatersrand Human Research Ethics Committee, the Johns Hopkins University School of Medicine Institutional Review Board, and the London School of Hygiene and Tropical Medicine Ethics Committee.
Between October 2009 and May 2010, 443 persons presenting to the study clinic for routine HIV-related care were invited and consented to participate and were screened for eligibility; 21 (5%) were ineligible [HIV-uninfected (8), currently being treated for TB (12), and completion of TB treatment <3 months before eligibility screening (1)] (Fig. 1). One hundred fourty-four (34%) of participants were male, the median age was 37 years [interquartile range (IQR): 31-44 years], median CD4+ T-cell count was 215 cells per microliter (IQR: 107-347 cells/μL), and 212 (50%) were receiving ART. The median CD4+ T-cell count among those not receiving ART was 160 cells per microliter (IQR: 65-315 cells/μL). Participants receiving ART had a median CD4+ T-cell count of 258 cells per microliter (IQR: 165-357 cells/μL) and had been receiving ART for a median duration of 247 days (IQR: 99-848 days) excluding those who has just been initiated on ART the day of enrollment in the study.
Three hundred sixty-one (86%) of participants reported having any duration of cough, fever, night sweats, or weight loss. A total of 30 TB cases (7%, 95% CI: 5% to 10%) was identified, 18 (60%) had only PTB, 7 (23%) had PTB and ETB, and 5 (17%) had only ETB (Fig. 1). Seven PTB cases were diagnosed on the basis of a positive sputum smear and 25 on the basis of a positive sputum culture. Of the 12 cases with ETB, 7 had a positive blood culture, 3 had lymph node histopathology consistent with TB, and 2 had both. Twenty-seven percent (95% CI: 12% to 48%) of TB cases were sputum AFB positive.
The performance characteristics of symptoms, body mass index, routine laboratorie,s and urine LAM are shown in Table 1. The sensitivity, specificity, positive predictive value, and negative predictive value of the urine LAM test were 32% (95% CI: 16% to 52%), 98% (95% CI: 96% to 99%), 53% (95% CI: 28% to 77%), and 95% (95% CI: 93% to 97%), respectively. The urine LAM test had sensitivities of 40% (5% to 85%) and 20% (95% CI: 6% to 44%) among sputum AFB-positive and AFB-negative TB cases. The urine LAM test was more sensitive in TB cases with night sweats (42%, 95% CI: 20% to 67%), weight loss (36%, 95% CI: 18% to 57%), body mass index <18.5 kg/m2 (67%, 95% CI: 30% to 93%), anemia (43%, 95% CI: 22% to 66%), CD4+ T-cell counts ≤50 cells per microliter (56%, 95% CI: 21% to 86%), moderate/advanced disease on chest x-ray (38%, 95% CI: 14% to 68%), or ETB (64%, 95% CI: 31% to 89%), but none of these differences achieved statistical significance.
Having either a positive urine LAM test or sputum smear was 36% sensitive (95% CI: 18% to 57%), 98% specific (95% CI: 95% to 99%), and had positive and negative predictive values of 64% (95% CI: 35% to 87%) and 93% (95% CI: 89% to 96%).
Four of 7 participants who grew non-tuberculous mycobacteria and did not grow TB from sputum and/or blood had positive urine LAM tests.
The sensitivity of the urine LAM among HIV-infected ambulatory patients in this study was only 50% among sputum AFB-positive and 16% among AFB-negative TB cases, respectively, making it inadequate to replace sputum smear microscopy in AFB-positive TB cases or mycobacterial culture in AFB-negative TB cases, even if the urine LAM is formatted into a rapid lateral flow assay.
Our study differed from prior studies in that we studied the urine LAM test to screen a high-risk population for TB rather than to confirm a diagnosis of TB among symptomatic persons; our participants were ambulatory patients presenting for routine HIV-related care, who were less immunosuppressed than populations in which this test has previously been studied; and a highly sensitive standard diagnostic work up for TB was performed in all study participants regardless of symptoms reported.
The prevalence of active TB among ambulatory HIV-infected adults in our study was 7%, which was lower than previously found by Bassett et al and Houlihan et al in KwaZulu Natal and by Lawn et al in the Western Cape, South Africa, where the prevalence of TB was 20%-30% among HIV-infected ambulatory patients presenting for initiation of ART; however, the HIV-infected patients in these prior studies had a lower median CD4+ T-cell counts (100-125 cells/μL) than the median CD4+ T-cell count of participants in our study (215 cells/μL) and were not receiving ART in contrast to the 50% in our study who were on ART.9-11
The sensitivity of the urine LAM in our study (32%, 95% CI: 16% to 52%) was comparable with or lower than that reported in previous studies (20%-80%),7,8,12-16 likely because our study participants were ambulatory, relatively less sick persons with lower bacillary burdens of TB. Prior studies have demonstrated that the urine LAM has greater sensitivity for detecting TB among patients with a higher bacillary burden: sputum AFB-positive versus AFB-negative TB patients;7,8,13,15-19 patients with definite rather than probable or possible TB;7,8,15,16 HIV-infected versus HIV-uninfected TB patients;7,14-16 HIV-infected patients with lower CD4+ T-cell counts;7,12,14,17 PTB versus ETB patients;17 and TB patients with mycobacteremia versus TB patients without mycobacteremia.7,17
The sensitivity of the urine LAM alone did not exceed 64% in any subgroup, and thus the urine LAM was not a useful test to rule out TB in any subgroup. The specificity of the urine LAM exceeded 90% in all subgroups and may be a useful test for ruling in TB among sick hospitalized HIV-infected persons with a high burden of TB, but in this out-patient population, the positive predictive value reached only 57%, even among those with CD4+ T-cell counts ≤200 cells per microliter.
In the past year, there has been much excitement about the Cepheid GeneXpert MTB/RIF assay, a molecular test for TB that is run within 2 hours, could be used at the point of care and which has >98% and >72% sensitivity for sputum smear-positive and smear-negative TB, respectively, and >98% specificity, significantly better performance characteristics than that of the urine LAM.20
One of the main benefits of active TB case finding is to detect patients early in the natural history of disease, and such programs will have limited impact if they rely on TB diagnostics with poor sensitivity in this group. Development of TB diagnostics should focus on rapid point-of-care technologies that have significantly higher sensitivity, specificity, and predictive value among patients with both early and advanced TB disease.
We thank the staff and patients of Tembisa Main Clinic and the staff of the Aurum Institute for Health Research and the Bioanalytical Research Corporation who made this study possible. We would especially like to recognize our study coordinator Marcia Masevhe.
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