In this study, the diagnostic capacity of nucleotide MALDI-TOF MS assay to detect MTB in BALFs from presumed PTB patients was evaluated and compared with conventional methods used in our hospital. Our results revealed that the performance of nucleotide MALDI-TOF MS assay was significantly superior to that of smear microscopy, MGIT 960 culture and Xpert MTB/RIF test not only on overall BALF samples but also smear-negative ones. In the present study, the diagnostic sensitivity of nucleotide MALDI-TOF MS was 71.5% for BALFs from PTB patients, whereas those of smear, culture and Xpert MTB/RIF were 22.6%, 40.6%, and 40.8%, respectively. Together, about 50% and 30% patients benefited from nucleotide MALDI-TOF MS compared with smear and culture or Xpert MTB/RIF, respectively.
MALDI-TOF MS has been widely used in clinical microbiology detection for decades, with mature application for the identification of NTM based on proteins14–17. However, culture of mycobacteria was required if using protein-based MALDI-TOF MS assay, providing great advantages for fast-growing mycobacteria, while rapid detection of slow-growing mycobacteria, such as MTB, cannot be achieved. Nucleotide MALDI-TOF MS assay, using nucleic acids as substrates, can solve this above problem by targeting specific nucleotide polymorphisms. In 2017, a research study from Taiwan demonstrated that the detection limit of nucleotide MALDI-TOF MS assay on mycobacterial species identification was as low as 5 copies, with well-performed sensitivity and specificity of drug resistant detection18. Wu et al., have evaluated the prediction of MTB drug resistance with nucleotide MALDI-TOF MS, using phenotype drug resistance as reference and showed that it could be a promising tool for rapid detection of drug resistance 11. Nevertheless, the evaluation was conducted on the culture samples of MTB instead of clinical samples directly. Although it is featured with high throughput, speed, sensitivity, high accuracy and low cost, investigations on the application of nucleotide MALDI-TOF MS assay for PTB diagnosis in real world are still extremely limited and more research data are needed for support. To this end, we conducted this retrospective study to evaluate the diagnostic performance of nucleotide MALDI-TOF MS assay on BALFs from suspected PTB patients in Southwestern region of China with relatively large sample size. Li et al. and Yang et al. have published similar articles with very small sample size (37 and 52 BALFs respectively), likely resulting in big bias 19,20. The results of various methods in their study were generally higher than those obtained from our study. The influential factors among them may be various other than the sample size, such as study site, the experimental operators.
BALF is the most common used respiratory sample type for the diagnosis of PTB besides sputum, which is also preferred by clinicians due to its closer proximity to the lesion. Multiple studies have reported the capability of different molecular tests to diagnose PTB in BALFs, such as Xpert MTB/RIF assay, SAT-TB, LAMP, next-generation metagenome sequencing, nanopore sequencing, etc., with sensitivities raging from 45.18–94.4% 21–26. The comparatively lower results of the Xpert MTB/RIF assay in our study than those previously studies may be related to several factors, including study site, sample source, and the operator, suggesting that we need to improve the level of testing within our hospital. On the other hand, it is essential to emphasize the acquisition of high-quality specimens, since this often determines the final yield. Jiang et al., have demonstrated that attention to sputum quality can significantly increase bacteriological confirmation27. None the less, the very recent published study also showed a low sensitivity of Xpert MTB/RIF assay on BALFs to be 34.78%, lower than ours 28. We can also assess the impact of BALF quality on bacteriological confirmation in the future.
In our study, 14 cases were found to be nucleotide MALDI-TOF MS assay-negative but culture-positive. Finally, eight of them (57.1%) were clinically diagnosed with PTB and others were diagnosed with non-PTB. These false negatives may be due to insufficient nucleic acid or PCR inhibitors in BALFs29. On the other hand, 85 patients were found to be nucleotide MALDI-TOF MS assay-positive but culture-negative, 83 of which were diagnosed with PTB. This occurred might have several reasons. First, positive culture required live bacilli and the treatment history of patients might lead to negative cultures. Second, successful cultures require various factors including reagents, instruments, storage and expertise in operation30. In terms of the turnaround time of MGIT 960 culture and nucleotide MALDI-TOF MS assay, the detection time of results obtained from culture in our hospital ranged from 14 to 50 days where less than one day for nucleotide MALDI-TOF MS assay.
We here also analyzed the consistency of nucleotide MALDI-TOF MS assay and Xpert MTB/RIF for rifampicin sensitivity with a good agreement between the two assays. Rifampicin susceptibility culture results using MGIT 960 system, were all available for the six discordant samples. We observed that all samples discordant with culture results were RIF-resistant using nucleotide MALDI-TOF MS assay. Several reasons might be responsible for this inconsistence. MGIT 960 drug susceptibility testing was prone to yield false sensitive results for samples with minimal inhibitory concentrations (MICs) for rifampicin close to cut-off value31. Heteroresistance should also be considered as a factor that might influence the outcomes since nucleotide MALDI-TOF MS assay has ability to detect low-level heterogeneous drug resistance with the limit of 0.1%32. Certainly, technical errors of nucleotide MALDI-TOF MS assay leading to false resistance cannot be ruled out, after all, the results of the other two methods were consistent.
Our study has several limitations. First, it was a retrospectively study conducted at a single center. We couldn’t control several factors appeared in this study, such as not all of samples were conducted using culture or Xpert MTB/RIF assay. In order to obtain much more precise evaluation of performance of nucleotide MALDI-TOF MS assay, prospective and multicenter studies should be designed in future. Second, smear microscopy and nucleotide MALDI-TOF MS assay were performed on all of the samples, whereas MGIT 960 culture and Xpert MTB/RIF were not always done, thus resulting in some biases because of the missing data. Third, bronchoscopy was invasive and not acceptable to all patients, which was performed after carefully considered to be necessary to obtain BALFs, such as a high suspicion of PTB. This resulted in the majority of enrolled patients diagnosed as PTB, with bias in terms of specificity. Finally, in terms of the inconsistent samples for rifampicin susceptibility identified with nucleotide MALDI-TOF MS and Xpert MTB/RIF, we didn’t perform sequencing to further validate.