Evaluation of genoquick MTB dipstick assay for rapid detection of mycobacterium tuberculosis in sputum

Abstract

Tuberculosis, although a curable disease, continues to be one of the most important infectious causes of deaths worldwide (WHO, 2010). Globally, an estimated 1.4 million deaths occurred in 2011 as a result of infection with tuberculosis, one-fourth of the deaths were associated with HIV infections and most of it occurred in resource-limited settings (DeCock et al., 2013) where the burden of HIV infection is high. In Africa, tuberculosis (TB) is the leading cause of deaths among persons with HIV infections as the continent harbors 80% of the world HIV-TB cases (Corbett et al., 2003) and Uganda is ranked 20th of the TB high burden countries (WHO, 2006) The alarming increase in morbidity and mortality highlights the need to strengthen control measures. Diagnostic delay for TB is still considerable especially in developing countries which results in increased morbidity and prolonged transmission. Though it remains to be demonstrated, more sensitive, more rapid, and more patient-friendly diagnostic tools might have a significant impact on disease control by abbreviating diagnostic delay and reducing the period of transmission. Currently, TB diagnosis relies on traditional tests which include clinical and X-ray screening, conventional laboratory techniques including microscopy and culture, and of recent MTB/RIF GeneXpert, which are currently employed in countries like Uganda. Clinical and X-ray screening largely depend on the experience of the clinicians. Although sputum smear microscopy remains a laboratory diagnostic tool of choice for tuberculosis in resource limited settings (RLS) due to its simplicity, inexpensiveness and predictive power, it is affected by low sensitivity (60%) and its performance is often affected by poor equipment, trained manpower and quality assurance system in settings where resources are scarce (Parsons et al., 2011). Culture techniques are highly sensitive and specific, but the cost, technical complexity and time delay before results are available make culture not scalable for rapid detection and treatment of tuberculosis. Recent advances in TB have made it possible to use molecular technology like Xpert MTB/RIF (Cepheid) assay to detect mycobacterial DNA with great accuracy. This newly introduced molecular assay has shortened the turnaround time of the patient results and has the potential to detect Rifampicin resistance in the sample but the test is expensive and the costs can only be maintained by donations. This necessitates the need for more sensitive, specific and inexpensive point of care TB diagnostic tool(s). GenoQuick® MTB assay (Hain Lifescience, Nehrem, Germany), a test based on PCR and GenoQuick® technology has demonstrated its usefulness for the identification of other micro-organisms (Cohen-Bacrie et al., 2010) and has been adopted for the rapid diagnosis of TB (GQ-MTB) (Weizenegger, 2010). It enables the detection of Mycobacterium tuberculosis complex from decontaminated pulmonary and extra pulmonary clinical specimens. The principle of this new method involves sputum processing with 1% Nacetyl-l-cysteine (NALC), 4% sodium hydroxide and 2.9% sodium citrate (NALC-NaOH) in a biosafety cabinet to obtain a pellet, extraction of Mycobacterial DNA using Genolyse (HainLifescience, Nehrem, Germany) method followed by selective DNA amplification of IS6110 gene by polymerase chain reaction (PCR). Resultant single-stranded amplicons hybridize with specific MTBC probe that are included in the primer-nucleotide mix. This complex (MTBC probeamplicon) then binds selectively to the test band on the dipstick and is visualized by gold labeling on a lateral flow assay. When detectable adduct levels are reached, a golden color band is formed indicating a positive result. It is a fast, reliable and easy to use TB diagnostic method with the potential for becoming a point of care assay especially in high burden countries. Though highly promising, its diagnostic performance is yet to be evaluated especially in resource-limited settings like Uganda where TB is a burden. The present study aimed at evaluation of this assay using -20oC frozen NALC-NaOH processed sputum samples and directly on fresh sputum processed with a heat method that excludes a biosafety cabinet. The results of this study may have potential applicability in peripheral laboratory settings, with limited biosafety facilities.