A new screening tool for TB diagnosis

Tuberculosis (TB) declared as a global public health emergency in early 1990s still ranks second as a leading cause of death worldwide. In India, the situation is alarming with a World Health Organization (WHO) report stating that India accounts for a quarter of the total global incidence of TB. India also accounts for 60 per cent of multi-drug resistant (MDR)-TB cases worldwide along with China and the Russian Federation.

A recent report from the nation’s financial capital, on the emergence of total-drug resistant TB strains, highlights the grave nature of this problem and the hurdles in effective patient management. In addition, increasing reports of medical and paramedical professionals being diagnosed with TB (or MDR-TB) has further worsened the problem. This situation highlights the need for better therapeutic and diagnostic approaches.

Diagnosis of TB: Journey from microscopy to molecular assays
Robert Koch’s recognition in 1882 of Mycobacterium tuberculosis, led to the visual identification of stained mycobacteria in clinical specimens. This introduced the first-ever diagnostic assay widely used till date - “smear microscopy”. But unfortunately, the TB diagnostic pipeline has always lagged behind the virulent nature of this mycobacterial bacillus.

In the wake of the human immuno-deficiency virus (HIV)-TB pandemic and multi-drug resistant (MDR) forms of the disease, the advances in TB diagnostics seemed inadequate. This worsening scenario highlighted the existence of inadequate tools and weak systems of the laboratory-based diagnosis of active TB. This led to a large proportion of sputum-smear negative patients being left undiagnosed, fuelling TB transmission within the community. Culture of MTB in clinical specimens is substantially more sensitive than smear microscopy. Culture can be performed using solid media, such as Lowenstein-Jensen, or liquid media, commercially available in automated systems.

The long multiplication time of MTB has resulted in delay in culture results (10–14 days for liquid culture and 3–4 weeks for solid culture), limiting its clinical impact in sense of true patient care. These tests also require high-tech infrastructure and skilled personnel thereby amounting to huge running costs. Besides, there exists a lack of culture facilities and qualitative diagnostic labs in the public health sector in most developing countries. This makes patients more vulnerable leading to delay in diagnosis, treatment initiation and thereby increasing the chances of further disease transmission.

Subsequently, serology, an adjunctive test for TB diagnosis received wide acceptance in India and created a huge market in the Indian diagnostic sector. However, later clinical evidence proved the unacceptable diagnostic accuracy of these serological tests. Based on the critical assessment of these reports, the WHO issued a recommendation against the use of commercially available kits for TB diagnosis.

Taking note of this problem, the Indian government too issued a notice “banning the use of all serological-based tests for clinical diagnosis of TB”. Thus, in spite of a plethora of diagnostic tests available for TB, the drawbacks associated with these different types of tests have contributed to (1) under diagnosis or misdiagnosis of the disease, leading to increased morbidity and mortality; (2) to over diagnosis of the disease, leading to unnecessary treatment with attendant consequences to the patient (3) delayed diagnosis of drug resistance, leading to acquisition of additional resistance, morbidity and transmission. However, notable advances in TB diagnostic technologies have been made in the past several years; and one such molecular assay, Gene Xpert MTB/RIF (Cepheid Inc., Sunnyvale, CA, USA) has the potential for providing meaningful assistance in patient management and TB control.

Gene Xpert MTB/RIF, changing the paradigm for TB diagnostics
Xpert MTB/RIF, (CE-marked and FDA-approved) is the most recent, commercial and fully automated diagnostic molecular test; with a potential to facilitate effective patient management. This real time PCR assay is based on highly-specific detection of Mycobacterium tuberculosis DNA within the patient specimen.

Fluorescent probes based on a unique molecular beacon chemistry, aid the detection of changes in amplified DNA that would confer resistance to Rifampin, an important drug of the first-line treatment regimen, This assay involves a single specimen processing step, followed by automated molecular protocols such as genomic DNA extraction, real-time PCR amplification, and product detection within a closed cartridge; thus greatly reducing the chances of cross-contamination.

Also, the entire assay is completed within 90 minutes directly using an unprocessed clinical specimen, in a time equivalent to that required for smear microscopy. Importantly, the sample processing step offsets the infectious nature of the mycobacterium within the sample, reducing the biohazard risks for the user while handling specimens with a high mycobacterial load. This feature ensures the requirement of minimal training and also that the assay can be housed even in a non-conventional laboratory, as an on demand near patient technology which could be performed even in a doctor's office if necessary.

What makes Gene Xpert MTB/RIF successful?
Gene Xpert MTB/RIF has proven its instrumental robustness in different environmental conditions with highly variable temperatures; in sophisticated laboratories as well as on the field; demonstrating its feasibility for bedside applicability. But more importantly, its diagnostic accuracy has been unswerving across patient groups with capricious disease severity (in terms of mycobacterial load: smear negative and smear positivity). The assay is superior to conventional techniques, smear microscopy and culture. The assay detects an additional ~70% patients that are missed out by smear microscopy; and detects ~90% patients being positive by the gold standard, culture.

 Although, this new technology misses out on a few of the cases, this limitation is masked by its extremely short reporting time, determination of patients MDR-TB status and importantly potential to adapt on field bedside. This has been proven by a field-based feasibility in India, . Under this survey, Gene Xpert MTB/RIF reports were provided to the patients on the same day accurately diagnosing 90% patients with TB. Based on the results of these tests, appropriate therapy could be initiated for MDR-TB patients from the first day itself, unlike conventional testing which would take between three to eight weeks.

Can this cater to patients with extrapulmonary TB, HIV-TB and pediatrics?
Extrapulmonary TB constitutes almost 15% of all TB cases. This form of TB can be termed as “delayed” TB infection as it does not present any visual or clinical symptoms till the severity of the infection aggravates. But in a first-ever study on extrapulmonary TB using Gene Xpert MTB/RIF, ~80% patients with high clinical suspicion of TB were diagnosed accurately, many of which were negative by liquid culture, which is the gold standard. A similar study on both pulmonary (n=96) and extrapulmonary (n=50) specimens has been conducted at our laboratory, confirming a high diagnostic accuracy (pulmonary specimens, sensitivity: 80.8%, specificity: 83.6%; extra pulmonary specimens, sensitivity: 55.5%, specificity: 92.6%) (SRL Ltd., unpublished data).

In another group of TB patients (HIV-TB and pediatrics) with inability to expectorate sputum, this assay has proven to be a good alternative while using alternative specimen types. It has demonstrated higher diagnostic accuracy in comparison to microscopy (but equivalent to culture), while using alternative samples like gastric aspirates, urine, broncho-alveolar lavage, pus or lymph nodes.

Gene Xpert MTB/RIF automated molecular testing – Now a part of primary TB screening
But to say the least, the diagnostic arena is evolving at a lightning speed; assessing the potential of the same and realizing its high clinical impact on patient care, the WHO has revised TB case definitions, and now states that “A bacteriologically confirmed TB case is one from whom a biological specimen is positive by smear microscopy, culture or WHO-approved rapid diagnostics (such as Xpert MTB/RIF). All such cases should be notified, regardless of whether TB treatment has started.”

To add to this, molecular based- line probe assays further facilitate in optimizing patient treatment regimen especially in MDR-TB patients. The assay detects for resistance to important second-line anti-tubercular drugs (injectables, fluoroquinolones, ethambutol) thereby aiding rapid and accurate care for MDR-TB patients.