Rapid diagnosis of tuberculosis through the detection of mycobacterial DNA in urine by nucleic acid amplification methods

Summary

Tuberculosis kills over 1·7 million people worldwide every year and nearly 40% of patients with active tuberculosis remain undiagnosed because of the poor sensitivity of the current, century old diagnostic method: sputum microscopy. Sputum microscopy is not able to easily detect paediatric, extrapulmonary, or HIV-associated tuberculosis, which are now important causes of morbidity and mortality in developing countries. Newer diagnostic methods for tuberculosis remain less sensitive than sputum microscopy. Alternative strategies to diagnose tuberculosis by use of nucleic acid amplification methods to detect fragments of mycobacterial DNA in urine have been developed over the past decade with varying sensitivities and specificities. Methods using quantitative PCR on urine samples to detect transrenal mycobacterial DNA are under development. The detection of transrenal DNA makes it possible to assay the total body burden of mycobacterial infection in any age group and in extrapulmonary tuberculosis with urine samples, which can be collected non-invasively. This Review discusses the developments and application of nucleic acid amplification of mycobacterial transrenal DNA for improved tuberculosis diagnostics.

Introduction

Worldwide, tuberculosis is a health problem of enormous proportions. Every year about 9 million people develop tuberculosis and over 1·7 million die as a consequence. Sub-Saharan Africa has the greatest tuberculosis burden, with 363 people per 100 000 of the population with active disease, and 63% of the worldwide number of people with tuberculosis that are HIV positive.¹ Case-detection rates remain low, despite widespread implementation of the WHO directly observed treatment, short-course strategy over the past decade (the case-detection rate in Africa is 46% compared with the 70% Millennium Development Goals global target²). Although this low case-detection rate is largely because of unsatisfactory national tuberculosis programme activities, it is compounded by the fact that diagnostic algorithms in use at present in countries with a high tuberculosis burden are based on inaccurate tests that have been in clinical use for many decades.³ Bacteriological testing for tuberculosis in the majority of laboratories in disease-endemic countries is restricted to microscopic examination of the acid-fast stained sputum smear, a method that was introduced over a century ago. Even in resource-rich settings where a variety of diagnostics are more readily available, a delay in tuberculosis diagnosis increases mortality.⁴

A major shortcoming of conventional microscopy is its relatively low sensitivity compared with culture. Furthermore, sputum microscopy is not sensitive enough for the detection of tuberculosis in children, who frequently cannot produce sputum, or in patients with HIV-associated⁵ or extrapulmonary tuberculosis. HIV coinfection tends to reduce caseating necrosis and lower the numbers of acid-fast bacilli in the airway.⁶ In some settings, HIV might also reduce the specificity of sputum microscopy by increasing the number of patients infected with non-tuberculous mycobacteria.⁷ Alternative diagnostic methods commonly used for non-pulmonary forms of tuberculosis include culture or nucleic acid amplification techniques on tissue biopsies,⁸ blood, urine,⁹ gastric aspirates,¹⁰ or stool.10, 11 These examinations are done if tuberculosis is suspected. Autopsy studies have shown the presence of Mycobacterium tuberculosis DNA in various organs including the kidney in adults with a known pulmonary tuberculosis diagnosis.¹² It is likely that extrapulmonary tuberculosis is under-reported because of its non-specific presentation and the difficulty of its diagnosis. A cross-sectional study of 525 nephrology patients in Spain found tuberculosis in 259 per 100 000 people, substantially higher than the national average of 35 per 100 000 people, of which 86% had extrapulmonary involvement.¹³

The lack of accurate and rapid diagnostic testing for tuberculosis is an important impediment to worldwide tuberculosis control.¹⁴ There is a great need to develop alternative rapid-diagnostic methods—appropriate for use in both developing and developed countries—that are more sensitive and specific than sputum microscopy and better able to detect tuberculosis disease anywhere in the body.