Raman spectroscopy and machine learning-based optical probe for tuberculosis diagnosis via sputum.

Tuberculosis (TB) is a contagious disease that causes 1.5 million deaths per year globally. Early diagnosis of TB patients is critical to control its spread. However, standard TB diagnostic tests such as sputum culture take days to weeks to produce results. Here, we demonstrate a quick, portable, easy-to-use, and non-invasive optical sensor based on sputum samples for TB detection. The probe uses Raman spectroscopy to detect TB in a patient's sputum supernatant. We deploy a machine-learning algorithm, principal component analysis (PCA), on the acquired Raman data to enhance the detection sensitivity and specificity. On testing 112 potential TB patients, our results show that the developed probe's accuracy is 100% for true-positive and 93.4% for true-negative. Moreover, the probe correctly identifies patients on TB medication. We anticipate that our work will lead to a viable and rapid TB diagnostic platform.

Detection of rifampicin resistance of Mycobacterium tuberculosis using multiplex allele specific polymerase chain reaction (MAS-PCR) in Pakistan.

Drug resistance in tuberculosis (TB) is a major public health challenge in developing countries such as Pakistan. Multiplex allele specific polymerase chain reaction (MAS-PCR) is a DNA amplification method that could contribute to rapid detection and control of drug resistant tuberculosis (DR-TB) in Pakistan. The purpose of this study was to test the utility of MAS-PCR to detect resistance in Pakistan. Drug susceptibility testing (DST) was used to identify rifampicin resistant and susceptible clinical isolates from TB cases in Pakistan. MAS-PCR was used to detect the most frequent mutations in the gene rpoB among 213 resistant and 37 susceptible isolates. Among 213 clinical isolates, MAS-PCR identified mutation D435Y (Asp435Tyr) in 24 (11.3%) cases, H445Y (His445Tyr) in 14 (6.6%), S450L (Ser450Leu) in 124 (58.2%) and S450W (Ser450Trp) in 18 (8.4%) cases. MAS-PCR did not detect known mutations in 33 (15.5%) cases. Among 12 cases, a novel mutation at codon 434 (Met434Ile) and a common variant at codon 435 (Asp435Tyr) was detected in rpoB gene which is indicative of double mutation. In 4 isolates, a novel mutation at codon 432 (Gln432Pro) was identified. In an additional 4 isolates, mutations Met434Val and His445Asn were identified. Moreover, a mutation in rpoB (Leu452Pro) was found in 5 isolates. DNA sequencing confirmed the absence of mutations in rpoB in the 8 remaining isolates. MAS-PCR had 88.3% sensitivity and 100% specificity using DST as the reference, which suggested that this method could be implemented as an initial marker for screening of multi-drug resistant tuberculosis (MDR-TB) in Pakistan.

Mutation pattern in rifampicin resistance determining region of rpoB gene in multidrug-resistant Mycobacterium tuberculosis isolates from Pakistan.

The current study was undertaken to characterize the RRDR rpoB gene mutations among the rifampicin-resistant Mycobacterium tuberculosis (MTB) isolates from Pakistan. Rifampicin mutation patterns were analyzed by using PCR followed by rpoB gene sequencing. Among the 1080 referred TB cases, 63 (6%) were resistant against at least one first-line TB drug. Out of these 63 resistant isolates, 24 isolates (38%) were found to be resistant to isoniazid and rifampicin. Sequence analysis of multidrug-resistant tuberculosis (MDR-TB) isolates detected a single mutation in the RRDR region of the rpoB gene at codon 531, 516, 512, 528 and 533; however, 5 MDR-TB isolates lack any mutation in the RRDR region. A double mutation was observed in 1 MDR-TB isolate at codon 512 and 516 which are reported for the first time from Pakistan. Moreover, in 1 isolate a novel silent mutation was observed at codon 528. Further studies about these mutations may be helpful in the development of diagnostic tools for the detection of MTB in a high TB endemic area like Pakistan.