Tuberculosis (TB) is the number one cause of death globally by an infectious agent after Covid-19 (WHO, 2023). Every year, TB kills more people than AIDS and malaria combined. Worldwide, 2 billion people have TB infection and 10 million new TB infections are reported every year. Of the new infections, 5-10% progress to pulmonary disease (Bloom et al., 2017). Two key pillars of an effective TB strategy are early diagnosis and effective treatment. Effective treatment depends on early diagnosis which not only identifies the tubercle bacilli before disease progression but also detects any drug-resistant strains that can be used to guide the treatment regimen (Nahid et al., 2006). We hereby present a rapid, simple, portable, inexpensive nucleic-acid based test for the simultaneous identification of Mycobacterium tuberculsosis and rifampicin resistance.

Diagnosis of TB is currently through phenotypic, immunologic, bacteriologic, radiologic, histologic, and molecular biology techniques. Phenotypic approaches such as pigment production, niacin, nitrate catalase, arylsulfatase, and potassium tellurite reduction; iron uptake, and tween 80 hydrolysis tests suffer from lack of specificity, inability to differentiate between mycobacterial species, and inability to identify drug-resistant strains. Immunologic tests include the Mantoux tuberculin skin test (TST) and the Interferon-γ release assays (QuanriFERON TB Gold or In-Tube and T-SPOT.TB) (Azadi et al., 2018; Yusoof et al., 2022) .

The TST is nonspecific for M tuberculosis, cannot differentiate between active and latent mycobacterial disease, can give positive results following BCG vaccination or exposure to atypical mycobacteria, can give false results arising from challenges in the administration and interpretation of the test and can give negative results in immunodeficient individuals, children or in individuals with military or extrapulmonary infection. The Interferon-γ release assays release Interferon-γ following the stimulation of patient lymphocytes with M tuberculosis specific antigens in vitro. The Interferon-γ release assays suffers from reduced accuracy due to challenges in the collection and transportation of blood specimens. This assay is also expensive, is not widely available, can detect latent infection more accurately but cannot differentiate it from active disease, and can yield negative or equivocal results in immunodeficient individuals (Yusoof et al., 2022).

Radiologic tests overcome some of the limitations of the phenotypic and immunologic tests and include chest X-rays and. Chest X-rays provide a cheaper and widely available method for TB diagnosis and are based on the detection of lesions suggestive of TB. However, chest X-rays do not provide a method for the definitive diagnosis of TB because pulmonary involvement can be caused by other infectious, granulomatous, or lymphoproliferative states. Chest x-rays also have moderate sensitivity and poor specificity. Chest computer-assisted tomography provide a higher sensitivity than chest X-ray but are more expensive than chest X-ray, generate more exposure to radiation than chest x-ray, are not always specific for TB, and their normal results do not always rule out ocular tuberculosis (Bhalla et al., 2015).

Bacteriologic methods such as culture are preferable since they are much more definitive, specific, and accurate than the phenotypic, immunologic, and radiologic methods. Culture, the gold standard for TB diagnosis, provides definitive proof of the viability of the microorganisms, allows identification of mycobacterial species, and also allows drug sensitivity testing. Culture has a high sensitivity of 95% and a specificity of 98%. However, culture is expensive to perform, cumbersome, and time consuming. Turnaround times take up to 8 weeks because the tuberculosis bacilli grow very slowly. Automated systems such as the Bactec TB System have simplified most of the culture work, producing faster results with higher sensitivity and efficiency compared to the Lowesntein Johnson (LJ) culture method. This system also can differentiate MTB from MOTT more rapidly and reduce the time taken for MTB susceptibility testing (Zhang et al., 2020).

Molecular biology methods overcome the limitations of the culture methods since they are faster with very short turnaround times, less cumbersome, and have reasonably high sensitivity and specificity. They also provide quick quantification of mycobacterial load in the sample, have a low risk of carryover and cross-contamination, have fewer biosafety requirements than culture, detect active lesions and dormant disease in latent infection, and some like the Gene Expert System can identify drug resistance (Nurwidya et al., 2018; Yusoof et al., 2022).

Current molecular biology methods for TB diagnosis include PCR tests, Nucleic Acid Hybridization Techniques, DNA sequencing, RFLP, SNP Analysis, PFGE, RAPD Analysis, Deletion Mapping, AFLP and Spoligotyping. However, they are expensive, and have a limited availability, detect DNA only hence prone to contamination, require stable electricity, highly trained scientists, and well-equipped laboratories. Molecular biology methods also have not been validated for ocular samples, have variable sensitivity and which is lower for non-respiratory samples, do not allow ruling out tuberculous etiology, and amplify nucleic acids from microorganisms which may be dormant or unviable (Nurwidya et al., 2018; Yusoof et al., 2022).  

Whereas culture and molecular biology methods are more useful, they are not practical options in many resource-limited settings where TB is most prevalent because of their cost and facility requirements.  As a result, most diagnostic techniques in these settings involve microscopy. Microscopy is based on acid-fast staining. The main advantages of microscopy are that it is fast, easy to perform, cheap, and widely available. However, microscopy has a low sensitivity since some non-Mtb bacteria are also acid-fast and a detection threshold of more than 5,000 bacili/mL is needed. Microscopy is also unable to differentiate pathogenic and non-pathogenic mycobacteria since both groups are acid-fast and morphologically similar and is unable to identify drug resistant strains. The sensitivity of microscopy is also variable and ranges from 20-70% with a specificity of 95-98% (Azadi et al., 2018).

As is evident, current diagnostic methods are hampered by high cost, inaccessibility, non-specificity, variable sensitivity, lack of portability and applicability in field or point of care (POC) settings, and high turnaround times. These factors hinder the effective and early diagnosis of tuberculosis hence are an obstacle to timely and effective treatment. This is of paramount importance especially since majority of TB infections are reported in regions known to suffer from high rates of poverty and marginalization (Azadi et al., 2018; Nurwidya et al., 2018; Yusoof et al., 2022).

There’s need for a test that provides all the benefits of culture but which is cheaper, easy to perform, has a faster turnround time, can detect resistance, is widely available, is accurate, sensitive, and specific, and which, like microscopy, does not entirely require well equipped laboratory, expensive facilities, stable electricity, and highly trained personnel. Such a diagnostic test needs to be portable so that it can be conducted in the field or in a point of care (POC) setting. It also should not be restricted or centralized to a regional or specialized facility but should be widely available (Wang et al., 2020).

Such an ideal test should not be constrained by the need for a constant and reliable electricity supply, air conditioning, skilled computer operators, routine maintenance, or expensive reagents and specific instruments.  Even though TB is curable, the emergence of multi drug resistance has hampered treatment efforts. Multidrug resistant strains of TB together with the AIDS epidemic have fuelled the rise in case numbers. A test that accurately and simultaneously detects TB together with multi drug resistance while meeting the above requirements will greatly enhance the early diagnosis and management of TB (Nandlal et al., 2022).

At KIBs, we have created a rapid, simple, portable, inexpensive nucleic-acid based test for the simultaneous identification of mTB and rifampicin resistance. The test is based on rapid extraction that doesn’t require electricity, highly trained personnel, or sophisticated laboratory equipment. The extraction technique is sensitive, rapid, reproducible and equipment-free and visualization is via lateral flow detection. Probes have been designed around 16srRNA and the 81-bp rifampicin resistance determining region (RRDR) on the rpoB gene.

Use of sputum and gastric lavage samples is often a bottleneck to effective and quick diagnosis.  Gastric lavage samples are invasive while sputum samples are not easy to collect in younger patients who are unable to expectorate or in individuals without productive sputum (Schoch et al., 2007; Theron et al., 2013). To resolve this problem, our test kit utilizes oral saliva swabs which are easy to collect and are non-invasive and non-viscous. Oral saliva swabs have previously been shown to produce high quality DNA for the diagnosis of pulmonary Tb (Wood et al., 2015; Mesman et al., 2019; Song et al., 2021). These swabs also do not produce aerosols hence protect the collector from infection (Song et al., 2021). We are hopeful that this novel diagnostic kit will revolutionize the daignosis and management of TB in respurce-deprived regions.