A pilot study to evaluate urine LAM assay for diagnosis of pulmonary tuberculosis among non-HIV patients

Abstract

There is a great need to improve diagnostic tools for tuberculosis where the majority are without HIV co-infection in resource-poor settings and high-burden areas such as India. The urine LAM assay has not hitherto been studied and may have a role to play. Our study found that, as a stand-alone diagnostic tool, the assay was suboptimal when compared to Xpert MTB/Rif. However, a combination of LAM assay along with sputum microscopy may be useful in settings where molecular testing is unavailable.

Keywords

Point of care pulmonary tuberculosis lateral flow assay non-HIV

Introduction

In 2018, the World Health Organization (WHO) found that only 64% of the total estimated incidence of pulmonary tuberculosis (PTB) was actually reported. The highest discrepancy between estimated incidence and reported numbers, accounting for 26% of the total gap, was found in India.¹ In this context, the Determine TB-LAM Ag test (Abbott, Waltham, MA, USA; formerly Alere), which detects lipoarabinomannan (LAM) in urine, may be a promising assay. It is recommended by the WHO for detecting TB in patients with smear-negative HIV with low CD4 + counts.² These recommendations are based on a majority of studies done on the African continent. Moreover, sensitivity of LAM assay differs significantly between varying health settings.^3–6 In sub-Saharan Africa, 32% of the global TB burden is found in patients co-infected with HIV/AIDS, whereas in South-East Asia this figure is only 3.5%.⁷ This suggests that TB LAM assay could be useful in aiding diagnosis in settings where sputum smear microscopy remains the only available microbiological test. Thus, in our pilot study, we made an attempt to explore the performance of urine TB LAM test alone in combination with sputum microscopy, in the diagnosis of PTB.

Material and methods

A prospective cross-sectional study was carried out from January to June 2018 at a tertiary care centre in south coastal Karnataka after obtaining institutional ethical clearance (IEC No- 35/2018). A total of 56 patients suspected of PTB presenting with cough or fever of a duration >2 weeks, generalised weakness, recent weight loss, haemoptysis or history of contact with a patient with TB and being admitted in the pulmonary medicine ward, were enrolled for our study after obtaining written consent.

All sputum samples underwent fluorescent staining and were then examined using LED-FM (Zeiss Primo Star, Germany) according to Revised National TB Control Programme (RNTCP) guidelines.⁸ Cartridge-based nucleic acid amplification test (CB-NAAT), GeneXpert MTB/Rif (Cepheid, USA), was performed on all sputum samples. The Determine TB LAM test was performed by applying 60 µL of fresh urine to the sample pad. A positive result was indicated by the presence of a dark line in the assay test regions. The results were interpreted using the grading reference card according to the manufacturer's instructions. Statistical analysis was performed using SPSS Ver 16 (IBM Corp., Armonk, NY, USA).

Results

A total of 56 TB suspects comprising 35 men were enrolled for our study (35 men; mean age = 54.12 ± 14.9 years). Xpert MTB/Rif was positive in 27 (48.21%) patients.

On sputum smear microscopy, 18 (32.14%) samples were found positive (Table 1). In comparison with Xpert MTB/Rif, the overall sensitivity and specificity of sputum microscopy were found to be 57.1% and 92.9%, respectively (Table 2).

Table 1.

Results of smear microscopy of single spot sputum sample stratified according to urine TB-LAM results.

Sputum smear microscopy	Total (n = 56)	TB LAM positive (n = 18)	TB LAM negative (n = 38)	TB LAM grading				GeneXpert
Sputum smear microscopy	Total (n = 56)	TB LAM positive (n = 18)	TB LAM negative (n = 38)	1	2	3	4	GeneXpert
Negative	38 (100)	10 (26.3)	28 (73.6)	3 (7.8)	1 (2.6)	3 (7.8)	3 (7.8)	11 (29)
Scanty	6 (100)	4 (66.6)	2 (33.3)	1 (16.6)	1 (16.6)	–	2(33.3)	4 (66.6)
1+	4 (100)	2 (50)	2 (50)	1 (25)	–	1 (25)	–	4 (100)
2+	6 (100)	2 (33.3)	4 (66.6)	–	–	1 (16.6)	1(16.6)	6 (100)
3+	2 (100)	0	2 (100)	2 (100)	–	–	–	2 (100)

Values are given as n (%).

Table 2.

Performance of sputum smear microscopy and Determine TB LAM test in comparison with Xpert MTB/Rif assay (n = 56).

Xpert MTB/Rif	Sputum microscopy	Determine TB LAM test	Determine TB LAM test and sputum microscopy in combination
Positive 27 (48.21)	18 (32.1)	18 (32.1)	28 (50)
Negative 29 (51.78)	38 (67.9)	38 (67.9)	28 (50)
Sensitivity	57.1 (37.2–75.5)	48.2 (28.7–68.1)	85.2 (66.3–95.8)
Specificity	92.9 (76.5–99.1)	82.8 (64.2–94.2)	82.8 (64.2–94.2)
PPV	88.9 (66.9–96.9)	72.2 (51.7–86.3)	82.1 (67.1–91.2)
NPV	68.4 (58.4–77.1)	63.2 (53.5–71.9)	83.9 (71.7–92.4)

Values are given as n (%) or % (95% CI).

CI, confidence interval; NPV, negative predictive value; PPV, positive predictive value; TB, tuberculosis.

Urine TB LAM assay was positive in 18 (32.14%) cases. In comparison with GeneXpert, the overall sensitivity and specificity of LAM assay were found to be 48.2% and 82.8%, respectively (Table 2).

On comparing smear microscopy and LAM assay, 8 (14.28%) samples were positive with both the assays, whereas 20 (17.86%) samples each were positive with either of the assay (Table 1). The total number of both positive and negative final interpretation was found to be 50% (28/56). The overall sensitivity and specificity on this combination was found to be 85.2% and 82.8%, respectively (Table 2).

An increment in sensitivity of 37% and in negative predictive value of 20.7% was observed (Table 2). False negatives reduced from 14 (25%) in LAM assay alone to 4 (7.14%) on combination. The low mycobacterial load among these patients may be the cause for false negatives on both sputum smear microscopy and urinary LAM assay. False positives remained at 5 (8.92%) in LAM assay alone and in combination with smear microscopy. Among the five false-positive cases, two cases were diagnosed with non-tubercular mycobacterium complex and two cases were diagnosed with culture-positive Candidemia.

Discussion

Effective management of PTB depends on improved diagnosis; a sensitive and specific point of care test with such potential is highly desirable. Although many authors have evaluated the performance of LAM assay in HIV-positive patients, studies testing the performance in HIV-negative patients at various geographical locations are limited. In the past, sensitivity of urine LAM assay has been reported in the range of 13%–93% in patients with confirmed PTB.⁹ In the present study, LAM assay demonstrated the poor performance corresponding to other studies, though most of those studies were done in HIV-TB co-infection cases where the test has shown better sensitivity.¹ Xpert has been made available in most district centres under the Indian RNTCP but unfortunately installation and maintenance of Xpert MTB/Rif at rural outposts is difficult. LAM assay is inadequate to guide treatment alone, but a combination of smear microscopy and LAM assay may augment diagnosis. Other studies have reported significant additive value of other diagnostic tools thereby advocating the combined use of smear microscopy and LAM assay.^4,5 Both NTM and Candida are known agents of cross-reactivity in LAM assay.¹²

The Determine TB LAM assay works best on freshly collected urine samples and delays may compromise the results.¹² Proteinuria, haematuria or urinary tract infections may affect the assay results, but these factors were not taken into account in the present study. The small sample size of this pilot exercise remains a major limitation. Ease of sample collection, rapid results and minimal need of technical expertise, however, are in favour of use of the LAM assay. Performance needs to be improved before application in routine investigations and further evaluation of this test with larger numbers of participants is desirable, especially in high-burden countries.

Footnotes

Acknowledgements

The authors thank the Department of Pulmonary Medicine for their support during the study and all technical/nursing staff for their assistance during sample collection and processing. This study was presented at ASM Microbe 2019, San Francisco, CA, USA as a poster presentation.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received the following financial support for the research, authorship, and/or publication of this article: was received from Dr. TMA Pai Endowment Chair Fund for TB research.

ORCID iDs

Akshita Gupta

Asem Ali Ashraf

Haritha Madigubba

References

World Health Organization. Global Tuberculosis Report 2017, Geneva: WHO, 2017.

Huerga

Rucker

SCM

Bastard

, et al. Should Urine-LAM Tests Be Used in TB Symptomatic HIV-Positive Patients When No CD4 Count Is Available? A Prospective Observational Cohort Study from Malawi. J Acquir Immune Defic Syndr 2020; 83: 24–30.

Kerkhoff

Lawn

. A breakthrough urine-based diagnostic test for HIV-associated tuberculosis. Lancet 2016; 387: 1139–1141.

Lawn

Kerkhoff

Burton

, et al. 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. BMC Med 2017; 15: 67.

Sahle

Asress

Tullu

, et al. Performance of point-of-care urine test in diagnosing tuberculosis suspects with and without HIV infection in selected peripheral health settings of Addis Ababa, Ethiopia. BMC Res Notes 2017; 10: 74.

Peter

Theron

Chanda

, et al. Test characteristics and potential impact of the urine LAM lateral flow assay in HIV-infected outpatients under investigation for TB and able to self-expectorate sputum for diagnostic testing. BMC Infect Dis 2015; 15: 262.

Basnyat

Caws

Udwadia

. Tuberculosis in South Asia: A tide in the affairs of men. Multidiscip Respir Med 2018; 13: 10.

Bhadade

Mehta

Kanade

, et al. Utility of light-emitting diode microscopy for the diagnosis of pulmonary tuberculosis in HIV infected patients. Int J Mycobacteriology 2015; 4: 31–35.

Shah

Ssengooba

Armstrong

, et al. Comparative performance of urinary lipoarabinomannan assays and Xpert MTB/RIF in HIV-infected individuals. AIDS 2014; 28: 1307–1314.

10.

Bjerrum

Schiller

Dendukuri

, et al. Lateral flow urine lipoarabinomannan assay for detecting active tuberculosis in people living with HIV. Cochrane Database Syst Rev 2019; 10: CD011420.

11.

Sharma

Kohli

Yadav

, et al. Evaluating the diagnostic accuracy of Xpert MTB/RIF assay in pulmonary tuberculosis. PLoS ONE 2015; 10: e0141011.

12.

Cox

Lukande

Kalungi

, et al. Is urinary lipoarabinomannan the result of renal tuberculosis? Assessment of the renal histology in an autopsy cohort of Ugandan HIV-infected adults. PLoS ONE 2015; 10: e0123323.