4.1.1. GenoType Line Probe Assays (LPA)

Molecular tests such as LPA are considered ideal for rapid diagnosis and can be used directly on diagnostic samples. LPA uses nuclei acid amplification techniques such as PCR and reverse hybridization to rapidly detect drug resistance mutations.

Two affordable LPA tests are INNO-LiPA Rif TB and GenoType MTBDRplus. INNO-LiPA Rif TB was introduced by Innogenetics and has been approved by the WHO, with high sensitivity and specificity for the identification of M. tuberculosis bacteria and drug resistance mutations [68].

GenoType MTBDR, introduced by Hain Lifescience, has a sensitivity and specificity of 99% and 100% for rifampicin resistance and 88.4% and 100% for isoniazid resistance. MTBDRplus, an improved version, has been validated by the WHO and has shown outstanding analytical efficacy for the identification of multidrug-resistant tuberculosis (MDR-TB).

The GenoType MTBDRplus Test Version 2.0, approved by the WHO in 2012, allows instant identification of mutations and has a high recognition rate of rifampicin resistance. These molecular tests have made significant improvements in the diagnosis and identification of drug resistance in tuberculosis [16,69].

A retrospective analysis investigated the effectiveness of the sample amplification tests in the detection of M. tuberculosis complex and the diagnosis of multidrug-resistant tuberculosis (MDR-TB). The laboratory at the Microbiology Department of the Bhopal Memorial Hospital and Research Center in Madhya Pradesh, India, examined sputum samples from patients with suspected tuberculosis. Of the 1294 acid-fast bacilli AFB-positive sputum samples tested with LPA, M. tuberculosis complex was detected in 94.04% of the samples but not identified in 5.9% of them [70].

Also, 5.1% of sputum samples were found to be negative for M. tuberculosis complex by LPA and culture. In a small percentage of AFB-positive samples, M. tuberculosis complex could not be identified by LPA, even if confirmed by culture. These results highlight the limitations of the LPA test in detecting M. tuberculosis in certain sputum samples and highlight the importance of using multiple diagnostic methods for a complete and accurate assessment of MDR-TB [70].

Another study from Nigeria analyzed a total of 67 gastric samples and 31 sputum samples to assess the presence of M. tuberculosis in children. The M. tuberculosis detection method by sandblasting microscopy (SM) was found to provide positive results in 3.0% of gastric samples and in 16.1% of sputum samples. In contrast, the use of polymerase chain amplification (LPA) detected M. tuberculosis in 41.8% of gastric samples and in 58.1% of sputum samples [71].

Comparing these results with other similar studies, it was observed that the LPA method had a higher yield in detecting M. tuberculosis in sputum samples and the SM method provided poorer results overall. In addition, it was found that the use of LPA enabled the detection of M. tuberculosis in sputum samples that were initially negative for the SM test. These findings suggest that the use of the LPA method may improve the diagnosis of pulmonary tuberculosis in children, providing a higher sensitivity in identifying M. tuberculosis in sputum samples [71].

Another study conducted in Central India looked at the 1528 sputum samples analyzed and found that 1294 were positive in the microscopic (smear) test and 234 were negative in this test. Of the 1294 LPA tests performed, 77 samples (5.9% of the total) did not show the specific band for the M. tuberculosis complex. Of the samples with the TUB band present (1217 samples), different types of drug resistance were identified. A total of 67 (5.1%) sputum samples were negative for M. tuberculosis complex by both LPA and culture [70].

The performance of the GenoType MTBDRplus assay is comparable to that of the GeneXpert MTB/RIF assay. A meta-analysis showed excellent sensitivity and specificity for the detection of resistance to isoniazid, rifampicin, and MDR-TB. However, the sensitivity of the test may vary, being higher in smear-positive cases and lower in smear-negative cases. Also, the test may have a high percentage of invalid results for direct smear-negative sputum samples [72].